IMPROVING THE ANTI-TUMOUR EFFICACY OF

ALBENDAZOLE

Anahid Ehteda

A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy

Faculty of Medicine St. George Clinical School University of New South Wales

March 2011

ORIGINALITY STATEMENT

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation, and linguistic expression is acknowledged.

Signed ……………………………………………………

Date ……………………………………………………..

I

ABSTRACT

Albendazole (ABZ) is a benzamidazole derivative that binds to β-tubulin and inhibits the polymerisation of microtubules. ABZ has a remarkable activity against a variety of tumour cell lines including colorectal, liver, and ovarian cancer cells. In pre-clinical models, ABZ has been shown to have both anti-tumour and anti-angiogenic activities. Nevertheless, poor aqueous solubility of ABZ limits its absorption, and as a result, its efficacy. Therefore, the first aim of this project was to improve the aqueous solubility of ABZ. Using a combination of ionisation with acid and complexation with hydroxypropyl-β-cyclodextrin (HPβCD), a relatively high concentration of ABZ in solution was achieved. Comparison of pharmacokinetic profiles of ABZ/HPβCD with a conventional formulation of ABZ in hydroxypropyl methylcellulose (ABZ/HPMC) in nude mice revealed that complexation with HPβCD results in a significantly higher peak plasma concentration (Cmax) and area under the curve (AUC) of ABZ and its metabolites. Moreover, in mice-bearing human colorectal cancer cells, HCT-116, ABZ/HPβCD treatment led to a significant delay in tumour growth with increase in survival of animals as compared with vehicle and ABZ/HPMC treatments.

The second aim of this research was to improve the efficacy of ABZ using combination therapy. To this end, the interaction between ABZ and different chemotherapeutic agents were assessed using the Sulforhodamine B assay (SRB) and quantified by median effect analysis method. Among the tested agents, a synergistic anti-proliferative effect was observed with the combination of ABZ and 2- methoxyestradiol (2ME) in HCT-116 and DU145. Of interest, 2ME, a microtubule targeting agent binds to similar colchicine-binding site of β-tubulin as ABZ and inhibits microtubules polymerisation. Synergistic interaction of ABZ and 2ME was accompanied with the activation of extrinsic pathway of apoptosis.

In vivo, the combination of low concentration of ABZ with 2ME resulted in an increase in the survival rate of mice-bearing HCT-116 tumours. This effect was accompanied by a decrease in plasma and tumour vascular endothelial growth factor (VEGF) as well as a reduction in microvessel density. In addition, combination therapy led to a significant decrease in proliferation rate of the tumour and an increase

II

in apoptosis. Noticeably, high concentration of ABZ, in combination with 2ME, resulted in an antagonistic effect on tumour growth and survival of the animals.

Taken together, the solubility and anti-tumour efficacy of ABZ was highly increased by complexation with HPβCD, leading to the conclusion that the formulation may be suitable for parenteral administration. Moreover, combination of ABZ and 2ME has shown promising results in pre-clinical model. Additionally, the finding that the combination of two microtubule-binding agents that share the same binding site can act synergistically in vitro and in vivo may lead to the development of new therapeutic strategies in cancer treatment.

III

PUBLICATIONS ARISING FROM THIS THESIS

1. Anahid Ehteda, Peter Galettis, Krishna Pillai, David L. Morris. Combination of Albendazole and 2-Methoxyestradiol significantly improves the survival of mice-bearing human colorectal cancer tumor. (submitted for review)

2. Anahid Ehteda, Peter Galettis, Stephanie Wai Ling Chu, David L. Morris. Complexation of Albendazole with Hydroxypropyl-β-cyclodextrin improves its pharmacokinetic profile and antitumor efficacy in nude mice. (Submitted for review)

CONFERENCE POSTER PRESENTATIONS

1. Ehteda A., Galettis P., Morris DL. The Effect of Albendazole and 2- Methoxyestradiol alone and in Combination in Colorectal Cancer cell line. The St George Medical Symposium, Sydney, Australia 2009

2. Ehteda A., Galettis P., Morris DL. The Effect of Albendazole and 2- Methoxyestradiol as a single agent and Combination on the Survival of HCT- 116 Tumour-bearing Nude Mice. The St George Medical Symposium, Sydney, Australia 2010

IV

ACKNOWLEDGMENTS

There are a number of individuals I would like to acknowledge whose support has made this thesis possible. I would like to thank my supervisor Professor David Morris for his support and guidance during the course of my PhD study. I also wish to express my gratitude to my co-supervisor Dr. Peter Galettis for his patience and guidance. I am truly grateful.

I would like to thank my colleagues and friends, Samina Badar, Krishna Pillai, Peyman Mirarabshahi, Stephanie Chu, Yan Cai, Soheila Rahgozar, Peng Yao, Ahmed Mekkawy, Steven Lim, Peter Luk, and New Yien for their advice, support, and friendship.

I also wish to convey my gratitude to Professor George Murrell for allowing me to work in his lab. I would like to thank the staff and the students of the Department of Orthopaedics, Marina Zimmermann, Tiffany Rankin, Ai Wei, Twishi Gulati, Sylvia Chung, and Patrick Lam for providing such an enjoyable atmosphere to work in.

Finally, I express my deepest gratitude to my father, my lovely sister, Ayeh, and my dear brother, Aryo for their support, encouragement, and unconditional love on which I have always relied.

V

Dedicated in Loving Memory of My Mum

TABLE OF CONTENTS

Originality Statement ...... I Abstract ...... II Publications arising from this thesis ...... IV Conference Poster Presentations ...... IV Acknowledgments ...... V List of Figures ...... XI List of Tables ...... XIII Glossary of Abbreviations ...... XIV

Chapter 1. Literature Review 1.1. Microtubules ...... 1 1.1.1. Microtubule Structure ...... 1 1.1.2. Tubulin Isotypes ...... 4 1.1.3. Tubulin Post-Translational Modifications ...... 4 1.1.4. Microtubule Dynamics ...... 6 1.1.5. Microtubule Associated Proteins (MAPs) ...... 8 1.2. Microtubule-targeting Agents ...... 10 1.2.1. Microtubule De-stabilising Agents ...... 12 1.2.1.1. Colchicine...... 12 1.2.1.2. Vinca Alkaloides ...... 14 1.2.1.3. Other Microtubule De-stabilisers ...... 15 1.2.2. Microtubule Stabilising Agents ...... 20 1.2.2.1. Taxanes...... 20 1.2.2.2. Epothilones ...... 22 1.2.2.3. Other Microtubule-stabilising Agents ...... 23 1.2.3. Mechanism of action of microtubule-targeting agents ...... 27 1.2.3.1. Cell Death and Apoptosis ...... 27 1.2.3.2. Anti-vascular Action ...... 30 1.2.5. Toxicity of Microtubule-targeting Agents ...... 32 1.3. Albendazole ...... 33 1.3.1. Metabolism ...... 33 1.3.2. Bioavailability and Absorption ...... 34 1.3.3. Mode of Action ...... 35

VI

1.3.3.1. Cytotoxic Effect ...... 35 1.3.1.2. Apoptotic Effect ...... 36 1.3.1.3. Anti-angiogenic Effect ...... 36 1.3.4. Potential of Albendazole as an Anti-cancer Agent ...... 37 1.3.4.1. Pre-clinical Studies ...... 37 1.3.4.2. Clinical Studies ...... 37 1.3.5. Toxicity and Side Effects ...... 38 1.4. 2-Methoxyestradiol ...... 38 1.4.1. Metabolism and Bioavailability ...... 39 1.4.2. Mechanism of Action ...... 40 1.4.2.1. Cytotoxic Effect ...... 40 1.4.2.2. Apoptotic Effect ...... 41 1.4.2.3. Anti-angiogenic Effect ...... 42 1.4.3. Potential of 2-methoxyestradiol as an Anti-cancer agent ...... 43 1.4.3.1. Pre-clinical Studies ...... 43 1.4.3.2. Clinical Studies ...... 45 1.4.3.3. Other Therapeutic Indications of 2-Methoxyestradiol ...... 46 1.4.4. Toxicity and Side Effects ...... 46 1.5. Combination Therapy ...... 47 1.6. Drug Delivery Systems ...... 49 1.7. Aims and Hypothesis...... 52

Chapter 2. Materials and Methods 2.1. Materials ...... 55 2.1.1. Reagents...... 55 2.1.2. Assay Kits ...... 57 2.1.3. Antibodies ...... 57 2.2. Methods ...... 58 2.2.1. Drug Preparation ...... 58 2.2.1.1. In Vitro Experiment ...... 58 2.2.1.2. In Vivo Experiment ...... 58 2.2.1.2.1. ABZ formulations ...... 58 2.2.1.2.2. 2ME formulation ...... 58 2.2.2. Cell Culture...... 59 2.2.3. Cell viability assay ...... 59 2.2.4. The Sulforhodamine B (SRB) Assay ...... 59

VII

2.2.5. Cytotoxicity Assay ...... 60 2.2.5.1. Single agent treatment ...... 60 2.2.5.2. Combination treatment ...... 60 2.2.5.3. Drug Interaction Study ...... 61 2.2.6. Enzyme-Linked Immunosorbent Assays ...... 62 2.2.7. Western Blotting ...... 64 2.2.8. Tubulin Polymerisation Assay ...... 65 2.2.9. Caspase Activity ...... 65 2.2.10. Mice ...... 66 2.2.11. Evaluation of ABZ formulations on tumour growth ...... 66 2.2.12. Pharmacokinetics Study ...... 67 2.2.13. High Performance Liquid Chromatography (HPLC) ...... 67 2.2.13.1. Apparatus and Chromatographic Conditions ...... 67 2.2.13.2. HPLC Standards ...... 68 2.2.13.3. Quality Controls ...... 68 2.2.13.4. Validation of the method ...... 68 2.2.13.5. Plasma extraction ...... 68 2.2.14. Toxicity Evaluation ...... 69 2.2.15. In Vivo Combination Study ...... 69 2.2.15.1. Pilot Studies...... 69 2.2.15.2. Combination Therapy ...... 70 2.2.16. Immunohistochemistry ...... 70 2.2.16.1. Proliferation Rate ...... 71 2.2.16.2. Apoptosis ...... 71 2.2.16.3. Microvessel Density ...... 72 2.2.17. Statistical analysis ...... 72

Chapter 3. Comparison of Pharmacokinetics and Anti-tumour Efficacy of Two Formulations of Albendazole in Nude Mice 3.1. Introduction ...... 74 3.2. Results ...... 75 3.2.1. HPLC Analysis ...... 75 3.2.1.1. Standards and Calibration Curves ...... 75 3.2.1.2. Validation of the method ...... 77 3.2.1.3. Pharmacokinetics of ABZ/HPßCD and ABZ/HPMC ...... 79

VIII

3.2.2. Comparison of the effect of ABZ/ethanol, ABZ/HPβCD, and ABZ/HPMC on cell proliferation ...... 82 3.2.3. Anti-tumour efficacy ...... 84 3.2.4. Immunohistochemistry Analysis ...... 86 3.2.5. Tumour and Plasma VEGF ...... 88 3.3.6. Toxicity Evaluation ...... 90 3.4. Discussion ...... 91

Chapter 4. Compbination of Albendazole with Chemotherapeutic Agents 4.1. Introduction ...... 95 4.2. Results ...... 96 4.2.1. Interaction of ABZ with Anti-parasitic Agents ...... 96 4.2.1.1. Single Agent Effect ...... 96 4.2.1.2. Effect of Combination Treatment...... 97 4.2.1.2.1. Combination of ABZ with DEC and PZQ ...... 97 4.2.1.2.2. Combination of ABZ and IVE ...... 97 4.2.2. Interaction between ABZ and MTAs ...... 101 4.2.2.1. Single Agents Effect ...... 101 4.2.2.2. Effect of Combination Therapy ...... 104 4.2.2.2.1. Combination of ABZ and PTX ...... 104 4.2.2.2.2. Combination of ABZ and VBL ...... 104 4.2.2.2.3. Combination of ABZ and CLC ...... 107 4.2.2.2.4. Combination of ABZ and 2ME ...... 109 4.3. Discussion ...... 111

Chapter 5. The Effect of Albendazole and 2-Methoxyestradiol as a Single Agent and in Combination In vitro and In vivo 5.1. Introduction ...... 114 5.2 Results ...... 115 5.2.1. Interaction between ABZ and 2ME: Simultaneous versus Sequential Treatment ...... 115 5.2.2. Interaction between CLC, CA4, ABZ and 2ME ...... 119 5.2.2.1. Interaction between ABZ and CA4 ...... 121 5.2.2.2. Interaction between CLC and CA4 ...... 123

IX

5.2.2.3. Interaction between CLC and 2ME ...... 125 5.2.2.4. Interaction between 2ME and CA4 ...... 127 5.2.3. Interaction between ABZ and 2ME ...... 129 5.2.3.1. Effect of Combination of ABZ and 2ME on Microtubule--related Proteins 130 5.2.3.1.1. α-tubulin ...... 130 5.2.3.1.2. Acetylated α-tubulin ...... 132 5.2.3.2. Effect of Combination of ABZ and 2ME on Apoptosis--related Proteins ... 134 5.2.3.3. Effect of Combination of ABZ and 2ME on Angiogenesis-related Proteins ...... 138 5.3.3.3.1. Hypoxia Inducible Factor 1α (HIF-1α) ...... 138 5.2.3.3.2. Vascular Endothelia Growth Factor (VEGF) ...... 140 5.2.4. Comparison of the Effect of 2ME-ethanol and 2ME/CMC/HPßCD In Vitro ..... 143 5.2.5. Toxicity Evaluation of Simultaneous Administration of ABZ and 2ME Combination In Vivo ...... 145 5.2.6. Comparison of the Efficacy of Various Dose of ABZ and 2ME on Tumour Growth In Vivo ...... 145 5.2.7. Effect of the Combination of ABZ and 2ME on the Survival of Tumour Bearing Mice ...... 147 5.2.8. Effect of the Combination of ABZ and 2ME on Tumour Cell Proliferation ...... 150 5.2.9. Effect of the Combination of ABZ and 2ME on Tumour Angiogenesis ...... 152 5.2.10. Effect of the Combination of ABZ and 2ME on Apoptosis in Tumour cells..... 154 5.2.11. Effect of the Combination of ABZ and 2ME on Tumour and Plasma VEGF ... 156 5.3. Discussion ...... 159

Chapter 6. Summary and Future Direction 6.1. ABZ Formulation ...... 167 6.2. Combination Therapy ...... 168

References ...... 171

X

LIST OF FIGURES

1.1. Polymeisation of Microtubules ...... 3 1.2. Polymerisation dynamics and the GTP cap ...... 7 1.3. Binding sites of vinblastine, colchicine, and paclitaxel ...... 11 1.4. Two major apoptosis signaling pathways ...... 29 1.5. The structure of ABZ and its metabolites ...... 33 1.6. Pathway of 2ME formation ...... 40

3.1. Chromatogram of blank human plasma spiked with ABZSO, ABZSO2, and ABZ ..76

3.2. Standard Curves of ABZSO, ABZSO2 and ABZ using peak area ...... 76

3.3. Mean plasma concentration of ABZSO, ABZSO2, and ABZ ...... 80 3.4. Cytotoxic effect of ABZ/HPßCD, ABZ/HPMC and ABZ/EtOH ...... 83 3.5. Kaplan-Meier survival curve ...... 85 3.6. Immunohistochemical analysis of angiogenesis, tumour cell proliferation, and apoptosis ...... 87 3.7. Effect of vehicle, ABZ/HPMC, and ABZ/HPβCD on VEGF level in tumour tissue86 3.8. Plasma concentration of human VEGF in HCT-116 tumour-bearing mice ...... 89 3.9. Effect of various doses of ABZ/HPβCD on mice body weight ...... 90 4.1. Dose response curves for 72 hours PZQ, DEC and IVE treatment on HCT-116 and DU-145 ...... 98 4.2. Effect of the combination of ABZ plus PZQ and ABZ plus DEC on HCT-116 and DU145 cell lines ...... 99 4.3. Effect of the combination of ABZ and IVE on HCT-116 and DU-145 cell lines ... 100 4.4. Dose response curves for 72-hour PTX and VBL treatment on HCT-116 and DU145 cell lines ...... 102 4.5. Dose response curves for 72-hour CLC and 2ME treatment on HCT-116 and DU145 cell lines ...... 103 4.6. Effect of 72-hour simultaneous ABZ and PTX treatment for HCT-116 and DU145 ...... 105 4.7. Effect of 72-hour simultaneous ABZ and VBL treatment for HCT-116 and DU145 ...... 106 4.8. Effect of the combination of ABZ and CLC on HCT-116 and DU145 cell lines ... 108

XI

4.9. Effect of the combination of ABZ and 2ME on HCT-116 and DU145 cell lines ...... 110 5.1. Dose-response curves and cytotoxic interaction between ABZ and 2ME ...... 117 5.2. Dose response curves and cytotoxic interaction between ABZ and 2ME...... 118 5.3 Dose-response curves for 72-hour CA4 treatment on HCT-116 and DU145 ...... 120 5.4. Effect of the combination of ABZ and CA4 on HCT-116 and DU145 cell lines ... 122 5.5. Effect of the combination of CLC and CA4 on HCT-116 and DU145 cell lines ... 124 5.6. Effect of the combination of CLC and 2ME on HCT-116 and DU145 cell lines ... 126 5.7. Effect of the combination of 2ME and CA4 on HCT-116 and DU-145 cell lines .. 128 5.8. Effect of ABZ and 2ME on tubulin polymerisation in HCT-116 cells ...... 131 5.9. Effect of ABZ and 2ME on tubulin acetylation in HCT-116 cells...... 133 5.10. Effect of ABZ, 2ME, and their combintion on caspase activation ...... 135 5.11. Effect of ABZ, 2ME, and their combination on DR5 protein expression ...... 136 5.12. Effect of ABZ, 2ME, and their combination on P53 protein expression ...... 137 5.13. Effect of ABZ, 2ME, and their combination on HIF-1α expression in hypoxia ... 139 5.14. Effect of single-agent treatment on VEGF and cytotoxicity ...... 141 5.15. Effect of combination therapy on VEGF and cytotoxicity...... 142 5.16. Dose response curves of 2ME/CMC/HPβCD and 2ME/EtOH in HCT-116 and DU145 cells ...... 144 5.17. Effect of the various concentrations of ABZ and 2ME on HCT-116 xenograft tumour growth ...... 146 5.18. In vivo response of HCT-116 xenografts to high dose of ABZ in combination with 2ME ...... 148 5.19. In vivo response of HCT-116 xenografts to low dose of ABZ in combination with 2ME ...... 149 5.20. Effect of ABZ, 2ME and the combination of ABZ and 2ME on inhibition of tumour cell proliferation in HCT-116 xenograft mice ...... 151 5.21. Effect of ABZ, 2ME and their combination on angiogenesis in HCT-116 xenograft tumours ...... 153 5.22. Effect of ABZ, 2ME and the combination on apoptosis in HCT-116 xenograft tumours ...... 155 5.23. Effect of ABZ, 2ME and their combination on VEGF levels in subcutaneous HCT- 116 xenograft tumour ...... 157 5.24. VEGF levels in plasma of mice-bearing HCT-116 xenograft treated with ABZ, 2ME and their combination ...... 158

XII

LIST OF TABLES

1.1. Microtubule de-stabilising agents and their stages of clinical development ...... 17 1.2. Microtubule stabilising agents and their stages of clinical development ...... 25 1.3. Pre-clinical anti-tumour activity of 2ME ...... 44 2.1. List of reagents ...... 55 2.2. List of assay kits ...... 57 2.1. List of Antibodies ...... 57

3.1. Precision and accuracy of ABZ, ABZSO and ABZSO2 in human plasma ...... 76

3.2. Pharmacokinetic parameters of ABZ, ABZSO and ABZSO2 following intraperitoneal administration of ABZ/HPMC and ABZ/HPβCD ...... 81 3.3.Median survival analysis of animals that were treated with vehicle, 50 mg/kg ABZ/HPβCD, and 150 mg/kg ABZ/HPMC ...... 85

4.1. IC50 values of PTX, VBL, CLC, and 2ME for 72-hour drug treatment on HCT-116 and DU145 ...... 101 5.1. Percentage of cells that were affected by the combination of ABZ and 2ME following 24 hours treatment ...... 129 5.2. Median survival of animals that were treated with the vehicle, 50 mg/kg ABZ, 25 mg/kg 2ME and the combination of the two agents ...... 148 5.3. Median survival of animals that were treated the vehicle, 25 mg/kg ABZ, 25 mg/kg 2ME and the combination of the two agents ...... 149

XIII

GLOSSARY OF ABBREVIATIONS

2ME 2-Methoxyestradiol ABZ Albendazole ABZSO Albendazole Sulfone

ABZSO2 Albendazole Sulfoxide

ABZSO2-NH2 Albendazole 2-aminosulfone AFP Alpha-feto Protein Ang2 Angiopoietin-2 AUC Area Under the Curve bFGF Basic Fibroblast Growth Factor CA1-P Combretastatin A1 phosphate CA4 Combretastatin A4 CA4-P Combretastatin A4 phosphate CEA Carcinoembryonic Antigen CEPs Circulating Endothelial Precursors CI Combination index CLC Colchicine CMC Carboxymethyl Cellulose CYP1A2 Cytochrom P4501A2 CYP3A4 Cytochrome P4503A4 DEC Diethylcarbamazine DMSO Dimethyl Sulfoxide DR5 Death Receptor 5 EDTA Ethylenediaminetetraacetic Acid Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′- EGTA tetraacetic acid tetrasodium salt ELISA Enzyme-linked Immunosorbent Assay EROD Ethoxyresorufin O-Deethylase FBS Fetal bovine serum FDA Food and Drug Administration

XIV

FMO Flavin-containing Monooxygenase FAK Focal adhesion kinase GDP Guanosine Di Phosphate GST Gluthation S-transferase GTP Guanosine Three Phosphate

H2O2 Hydrogen Peroxide HATs Histone Acetyltransferases HDACs Histone Deacetylases HSP90 Heat Shock Protein 90 hEGF Human Epithelial Growth Factor HIF-1α Hypoxia-inducible Factor 1-α HPLC High Performance Liquid Chromatography HPMC Hydroxypropyl Methyl Cellulose HPβCD Hydroxypropyl-β-cyclodextrin HUVECs Human Umbilical Vein Endothelial Cells i.p. Intraperitoneally IVE Ivermectin mCRPC metastatic castration-resistant prostate cancer MDR multidrug resistance MROD Methoxyresorufin O-Demethylase MTAs Microtubule-targeted Agents MTD Maximum-tolerated Dose MTOC Microtubule Organising Centre NaOH Sodium hydroxide NCD Nanocrystalline NO Nitrogen Monoxide NOS Nitric Oxide Synthase NP-40 Nonidet P40 NSCLC Non-small cell lung carcinoma OD Optical Density PBS Phosphate Buffered Saline

XV

PDGF Platelet-derived growth factor Pgp P-glycoprotein PMSF Phenylmethanesulfonyl Fluoride PSA Prostate-specific Antigen PTX Paclitaxel PVDF polyvinylidene fluoride PZQ Praziquantel QC Quality Control RIPA Buffer Radioimmunoprecipitation lysis buffer RNS Reactive Nitrogen Species ROS Reactive Oxygen Species RPMI Roswell Park Memorial Institute s.c. Subcutaneously SEM Standard Error of Measurment SOD Superoxide Dismutase SRB Sulforhodamine B TBS Tris Bufferd Saline TCA Trichloroacetic acid TCP tubulin carboxypeptidases Treatment of Hormone-Refractory Metastatic Prostate TROPIC Cancer Previously Treated with a Taxotere-Containing Regimen TSP-1 Thrombospondin-1 TTL Tubulin Tyrosine Ligase Terminal Deoxynucleotidyl Transferase dUTP Nick end TUNEL Labeling TX Taxol uPA Urokinase-type Plasminogen Activator VBL Vinblastine VDAs Vascular Disrupting Agents VEGF Vascular Endothelial Growth Factor VEGFR2 Vascular Endothelia Growth Factor Receptor 2 VCR Vincristine

XVI

VDS Vindesine VFL Vinflunine VNB Vinorelbine

XVII

Chapter 1 LiteratureLiterature ReviewReview

1.1. MICROTUBULES

Microtubules, actin filaments and intermediate filaments are the main components of the cytoskeleton in eukaryotic cells 1. Microtubules are essential in maintenance of cell shape, intracellular trafficking, and cell motility. They are also critical in mitosis, playing a key role in mitotic spindle apparatus 2. Highly dynamic spindle microtubules are crucial in all stages of mitosis; in prometaphase, for correct attachment of chromosomes to the spindle at their kinetochore; in metaphase, for the movement of chromosomes to their position at the metaphase plate; and in anaphase and telophase for the separation of chromosomes after completion of metaphase-anaphase checkpoint 3, 4.

Microtubules are highly dynamic structures and the drugs that target microtubules exert their therapeutic effects by interfering with microtubule dynamics. Given the importance of microtubules in mitosis, it is not surprising that microtubule-targeting agents (MTAs) are amongst the most successful drugs in cancer treatment.

1.1.1. Microtubule Structure

Microtubules are cylindrical protein filaments present in all eukaryotes. The structural subunit of MTs is tubulin, a heterodimeric protein composed of α- and β-tubulin monomers. These monomers have similar masses of ~55 kDa and interact non- covalently to form tubulin heterodimers. Each tubulin monomer has three functional domains: the N-terminal domain containing the GTP-binding site; an intermediate domain; and the C-terminal domain, that forms the binding surface for many microtubule-associated proteins (MAPs) 5. In the tubulin dimer, GTP is always bound to α-tubulin. This GTP is stable and non-exchangeable, therefore the site is known as the N-site. In contrast, the nucleotide associated with β-tubulin at the E-site is exchangeable and can be hydrolysed to GDP shortly after assembly 6.

In most eukaryotes, tubulin gene family encodes multiple tubulin isoforms or isotypes 7.

For instance, eight isotypes of α-tubulin and seven isotypes of β-tubulin are expressed in 1 Chapter 1

humans at different subcellular distributions 8. In addition, both α- and β-tubulin undergo a variety of posttranslational modifications 9. Therefore, these factors contribute in the functional diversity of microtubules.

Microtubules are formed by the longitudinally assembly of protofilaments, each of which is composed of a chain of α- and β- tubulin heterodimers. The protofilaments arrange in parallel and form the cylindrical wall of microtubules. The orientation of heterodimers gives the microtubule a polarity, with a minus end capped with α-tubulin and a plus end capped with β- tubulin (figure 1.1) 10, 11.

In the cell, microtubules are nucleated in a region called “microtubule organising center” (MTOC) or centrosome and grow from their plus end outwards toward the cell membrane. While the plus ends explore the cytoplasmic space and interact with the cell cortex and other cellular structures, the minus end anchors in the MTOC 12.

Chapter 1 Chapter 2

Figure 1.1. Polymeisation of Microtubules. Heterodimers of α- and β- tubulin assemble to form a short microtubule nucleus These heterodimers are arranged in a head-to-tail fashion to form protofilaments which assemble longitudinally and form microtubules. Each microtubule has a plus (+) end, capped by β- tubulin and a minus end (-) capped by α- tubulin (Adapted from Jordan and Wilson, 2004) 3.

1 Chapter 3

1.1.2. Tubulin Isotypes

As mentioned above, α- and β-tubulin are encoded in multiple isotypes by different genes that are located on different chromosomes and have cell-specific expression patterns 13. These isotypes are distinguished by the last twenty amino acids in their carboxy-terminal tails 14. Alterations and mutations in tubulin isotypes, especially in β- tubulin (which contains the binding site of almost all MTAs), have been shown to affect the cell response to MTAs. For example, increased expression of the neuronal specific 15 class III β-tubulin (βIII) has been implicated in drug resistant cell lines . In non-small cell lung cancer cells (NSCLC), βIII-tubulin knockdown increases the efficacy of MTAs through suppression of microtubule dynamics 16. In addition, clinical evidence suggests that the expression of βIII tubulin is correlated to resistance to taxanes and vinca alkaloids in breast, ovarian and lung cancers. High expression of βIII is also a marker of poor prognosis in NSCLS 17 and advanced ovarian cancer patients that received a combination of platinum and paclitaxel 18.

Increased expression of βII, βIVa, βIVb, βV, and βVI isoforms of β-tubulin have been described in certain cancer cell lines which are resistant to MTAs . However, no clinical correlation between the level of these tubulin isotypres and resistance to MTAs has been reported 19. Similarly, mutations in both α- and β-tubulin are associated with resistant of tumour cell lines to taxanes, vinca alkaloids, epothilones, 2-methoxyestradiol (2ME) (Reviewed by Verrills and Kavallaris 15), peloruside and laulimalide 20. However, these mutations have not been reported in clinical samples.

1.1.3. Tubulin Post-Translational Modifications

Tubulins can undergo several post-translational modifications including tyrosination/detyrosination, acetylation/deacetylation, phosphorylation, poly- gluyamylation and poly-glycylation. These modifications occur upon incorporation of tubulin into the microtubule and mostly affect the C-terminal region of α/β-tubulin on the outer surface of microtubules 2, 21. Chapter 1 Chapter 4

Tyrosination/detyrosination cycle refers to the cleavage of tyrosine residue of α-tubulin from tubulin by tubulin carboxypeptidases (TCP), and its re-addition to the chain by tubulin tyrosine ligase (TTL) 22. During tumour growth, TTL is often inactive, suggesting that TTL activity may have a role in the regulation of tumour cells 23. Tubulin detyrosination has been also implicated in poor prognosis of breast cancer 24. Recent studies revealed that TTL suppression and resulting detyrosinated tubulin accumulation alter the microtubule dynamics by affecting its key regulator proteins, EB1 and Clip170 25, 26.

Acetylation of α-tubulin has been implicated in the regulation of microtubule stability and microtubule function 27. Acetylation and deacetylation are catalysed by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively. Among HDACs, HDAC6 is responsible for regulating tubulin acetylation. HDAC6 is exclusively localised in the cytoplasm where it interacts with microtubules. Overexpression of HDAC6 results in a global deacetylation of α-tubulin in vitro, and movement of chemotactic cells in vivo, suggesting that deacetylation regulates microtubule-dependent cell motility 28. Nevertheless, the role of acetylated tubulin seems to be subtle and is not crucial for cell survival. It has been shown that elimination of tubulin acetylation in Tetrahymena using site-directed mutagenesis has no observable outcome 29 and disruption of HDAC6 gene that leads to hyper-acetylated tubulin has no significant effect on cell proliferation and differentiation 30. Acetylation of α-tubulin has been shown to reduce the rate of depolymerisation of cytoplasmic microtubules by microtubule-depolymerising agents. Finally, increase in α-tubulin acetylation reduces the dynamics of microtubules and compromises their capacity to mediate the focal adhesion dynamics, a process required for rapid cell migration 31, 32.

Phosphorylation occurs on serine residue (Ser172) by Cdk1/cycline B complex that regulates mitosis entry 33. Several other kinases phosphorylate tubulin in vitro. However, the in vivo modification sites and relevance have not been identified 34. Polyglycylation and polyglutamylation are polymodifications that involved in the addition of polypeptide side chains of glycines and glutamates, respectively, to glutamate residue in the C-terminal of both α- and β-tubulin 35, 36. Polyglycylation has 1 Chapter 5

an important role in axonemal organisation and certain cell cycle events such as cytokinesis 37, 38. Polyglutamylation seems to regulate the interactions between microtubule-associated proteins (MAPs) in neuronal cells 39, 40.

1.1.4. Microtubule Dynamics

Microtubule dynamics play a critical role in several cellular processes such as intracellular trafficking, cell organisation and cell division. Microtubules display two types of non-equilibrium dynamics; dynamic instability and treadmilling. Dynamic instability is a process in which microtubules grow and shorten by a reversible addition and loss of α- and β-tubulin heterodimers at their minus and plus ends 21, 41. This highly prominent behaviour controls the distribution of microtubules and allows them to bind to specific structures like kinetochores 42.

Dynamic instability is characterised by four parameters: the rate of microtubule growth; the rate of microtubule shrinkage; the frequency of transition from the growth or pause to shrinkage (catastrophe); and the frequency of transition from shortening to pause or growth (rescue) 3, 43, 44.

Treadmilling is the second form of dynamic behaviour of microtubules which is particularly important in mitosis. Treadmilling is characterised by net growth of microtubules at one end and balanced net shortening at the opposite end 43, 45-47. Both dynamic instability and treadmilling can coexist in a microtubule population. The extent to which microtubules display each of these behaviours appears to be highly dependent on the condition that is prevalent 4.

Microtubule dynamics depend on the hydrolysis of GTP. Shortly after the addition of α/β-tubulin heterodimer to microtubules, GTP at E-site is hydrolised to GDP. Microtubule-containing GDP is extremely unstable and depolymerise rapidly. Nevertheless, a cap of GTP-tubulin at the microtubule-GDP end provides enough stability to sustain microtubule growth. This happens when the rate of subunits addition

is faster than hydrolysis of GTP of previously added subunit. Conversely, when the 1 Chapter 6

addition rate of heterodimers to microtubule is low, or when the GTP-tubulin cap is lost, the microtubules switch to a phase of shrinkage by a process called catastrophe (figure 1.2) 3, 42, 48.

Figure 1.2. Polymerisation dynamics and the GTP cap. Microtubule dynamics depend on the hydrolysis of GTP. Shortly after the addition of α/β-tubulin heterodimer to microtubules, GTP is hydrolised to GDP. Microtubules containing GDP are depolymerised rapidly. When the rate of subunit addition is faster than GTP hydrolysis, a cap of GTP-tubulin sustains microtubule growth. In contrast, when the rate of addition of heterodimers to microtubule is low, or if GTP- tubulin cap is lost, the microtubules switch to a phase of shrinkage by a process called catastrophe (Adapted from Calligaris et. al.) 49.

Chapter 1 Chapter 7

1.1.5. Microtubule Associated Proteins (MAPs)

Dynamic behaviour of microtubules is tightly regulated by a balance between the activity of microtubule-stabilising and microtubule–destabilising proteins which bind along the microtubules or tubulin dimers, and regulate microtubule assembly and disassembly 50. Changes in the expression levels of these proteins are associated with aggressiveness of a variety of human cancers and a determinant factor in their sensitivity to MTAs 51.

Microtubule are stabilised by I) inhibiting the catastrophe, which results in persistent microtubule growth, II) rescuing a depolymerising microtubule, thereby decreasing shortening phase, and III) reducing shrinkage speed. Conversely, Microtubule de- stabilisers induce catastrophes, inhibit rescues, and increase shrinkage speed 44.

One of the most extensively studied MAPs is Tau. Tau is primarily expressed in neurons where its phosphorylated form binds to tubulin and promotes polymerisation and stabilisation of axonal microtubules. Abnormal phosphorylation of tau is correlated with various neurodegenerative disorders such as Alzheimer’s disease 52. Tau is also expressed in non-neuronal tissues including breast cancer cells. In preclinical models of breast cancer, low expression of Tau is associated with increased sensitivity to paclitaxel 53. However, clinical studies did not confirm predictive value of Tau protein in patients with breast cancer receiving paclitaxel 54-56. Correlation of the high Tau levels with resistance to paclitaxel has been reported only in estrogen receptor-positive breast cancers, suggesting that Tau may interact with estrogen and affect the sensitivity to paclitaxel 57, 58. In patients with non-muscle invasive bladder cancer, high expression of Tau is correlated with resistant to paclitaxel 59.

MAP2 is found in terminally differentiated neurons and plays a pivotal role in dendrite development 60. In non-neuronal cells, the expression of MAP2 leads to the formation of 61 stable microtubule bundles . MAP2 is thought to be an ancillary marker in skin 1 Chapter 8

tumours with neuroendocrine origin 62. The expression of MAP2 in metastatic melanoma cells results in the stabilisation of microtubule, cell cycle arrest in G2/M phase, and tumour suppression both in vitro and in vivo 63, 64. MAP2 expression is also associated with sensitivity to MTAs. In docetaxel-sensitive pancreatic carcinoma, MAP2 levels are higher in comparison with paclitaxel-refractory pancreatic cancers 65, and in breast cancer cell lines, elevation of MAP2 leads to increased sensitivity to paclitaxel 66.

MAP4 is another microtubule-stabilising protein that specifically promote rescue events 67. In culture cells, reduction of MAP4 decreases the growth of microtubules 68. It has been shown that phosphorylation of MAP4 completely eradicates its microtubule stabilising activity 69, 70, and overexpression of mutant form of MAP4 that mimic phosphorylation, results in perturbations in cell progression into and through mitosis 70. Phosphorylation of MAP4 is also correlated with a decrease in PTX sensitivity in taxol- resistant ovarian cancer cell lines 71. In contrast, the expression of non-phosphorylated form of MAP4 is increased in vinblastine-resistant cells 72.

Stathmin (aslo known as oncoprotein 18) that originally identified as an oncoprotein is a member of microtubule-destabilising proteins 51. Stathmin has a critical role in the regulation of mitosis and acts as a catastrophe promoter and a tubulin-sequestering enzyme, depending on the pH and the region of the protein involved 73. Overexpression of stathmin has been reported in a variety of human cancers 74 and in many of these malignancies, high levels of stathmin is correlated with poor prognosis 59, 75, 76. Suppression of stathmin inhibits in vitro cell proliferation and migration, as well as in vivo tumourigenicity 77. It has also been reported that inhibition of stathmin sensitises retinoblastoma 78 and breast cancer cells 79 to low doses of vincristine and PTX. Such effect was not observed in chemotherapeutic agents that do not target microtubules. Stathmin also alters the drug binding to tubulin and arrest the cells at G2/M phase of cell cycle 79. A recent study showed that suppression of stathmin led to microtubule stabilisation, inhibition of hypoxia inducible factor 1α (HIF-1α) protein accumulation and suppression of vascular endothelial growth factor (VEGF) 80. Chapter 1 Chapter 9

Survivin is an apoptosis inhibitor that controls microtubule stability and assembly of mitotic spindle and regulates microtubule dynamics 81. While survivin is overexpressed in almost all types of tumours 82, normal cells from the same organs do not express survivin 83. Survivin has been implicated in PTX resistance, as PTX-resistant ovarian cancer cells display defective mitotic response to PTX 84.

Kinesins are microtubule motor proteins and potential targets in cancer therapy. Eg5 is a kinesin that is critical in the formation of spindle during mitosis 85. Overexpression of Eg5 correlates with poor prognosis in patients with bladder cancer. It also represents a prognostic factor in predicting early intravesical recurrence in non-muscle invasive bladder carcinoma patients 86. Inhibitors of Eg5 suppress tumour growth by inducing mitotic arrest 87. These inhibitors are effective in both PTX-resistant and PTX-sensitive cell lines 51.

1.2. MICROTUBULE-TARGETING AGENTS

Microtubule-targeting agents (MTAs) are one of the most promising classes of drugs in cancer therapy. Among these compounds are taxanes and vinca alkaloids which are successfully used for the treatment of various human cancers. Many MTAs are being evaluated in clinical trials and several new compounds are under investigation and development.

Three well-established binding-sites on β-tubulin are taxane binding site, vinca binding site and colchicine binding site 88 (figure 1.3). MTAs exert their therapeutic effect through induction of mitotic arrest that can lead to cell death 13. Traditionally, MTAs are classified into two major groups. One group is microtubule-stabilising agents that polymerises microtubules and increases microtubule polymer mass. The second group that is known as microtubule-destabilising agents, inhibits the polymerisation of microtubules and reduces microtubule polymer mass 1, 3, 89. Nevertheless, these effects can only be observed when the drugs are given and maintained at very high 1 Chapter 10

concentrations. At clinically relevant concentrations, all MTAs suppress the dynamics of microtubules without changing the microtubule polymer mass 3, 90.

Figure 1.3. Binding sites of vinblastine, colchicine, and paclitaxel. Vinblastine binds to high- affinity sites at microtubule plus end. Colchicine forms a complex with tubulin dimers and co- polymerises into microtubule lattice. Paclitaxel binds along the interior surface of the microtubule (Adapted from Jordan and Wilson 3)

Chapter 1 Chapter 11

1.2.1. Microtubule De-stabilising Agents

1.2.1.1. Colchicine

Colchicine (CLC), a naturally derived compound, was isolated from meadow saffron Colchicum autumnale in 1820. CLC is one of the first identified MTAs and its binding site and mechanism of action has been extensively investigated. CLC has been widely used to elucidate the function and properties of microtubules. Indeed, tubulin was initially purified based on its affinity to CLC 91-93.

CLC has anti-inflammatory and anti-mitotic properties and has been used to treat gout, pseudogout, autoinflammatory diseases, Mediterrenian fever, liver cirrhosis, and amyloidosis 94-96. CLC inhibits microtubules polymerisation at concentrations well below the concentration of free tubulin in solution, indicating that it inhibits the polymerisation of microtubules by binding to the microtubule ends 97. It has been shown that free CLC does not bind to the microtubules unless it first binds to the soluble tubulin and forms tubulin-colchicine complex with tubulin dimer 98. This reversible pre- equilibrium complex then binds to microtubule ends and reduces the rate of tubulin addition to microtubules. The complex binds more tightly to its tubulin neighbors than tubulin itself and decreases the rate of tubulin dissociation 95, 99.

It has been shown that low concentrations of CLC-tubulin complex suppress microtubule dynamic instability without altering the polymer mass. The complex decreases the rate and the extent of growing and shortening phases, reduces the catastrophe frequency, and enhances the rescue frequency 98, 99. Low concentrations of CLC block cell division in prophase, whereas those cells in metaphase and anaphase stages of cell cycle complete mitosis 100. As a result of the mitotic block, cells undergo abnormal mitosis characterised by partial or complete absence of spindle, nuclear envelope breakdown, condensed chromosomes, and undivided centromeres 97.

Although colchicine is a potent anti-mitotic agent, its application in cancer is hampered by its toxicity, as doses which are necessary for anti-tumour effect are highly toxic to Chapter 1 Chapter the normal cells 49. Despite this, development of colchicine-domain binders as potential 12

anti-cancer agents has recently generated much interest (Table 1.1). These agents form the largest family of low molecular weight drugs with anti-vascular activity at their non- toxic concentrations 101, 102. They cause a rapid and selective shutdown of tumour vasculature, leading to necrosis of tumour cells. Several vascular disrupting agents (VDAs) such as combretastatins are currently being evaluated in preclinical models or in clinical trials.

Combretastatins, isolated from Cape Bushwillow tree Combretum caffrum 103, are structurally related to colchicine 102. Combretastatins cause a rapid but reversible vascular shut-down in a variety of animal tumour models at doses well below their maximum tolerated doses 104-107. Combretastatin A4 phosphate (CA4-P) is the most widely studied VDAs. Following administration, it is rapidly converted to CA4 and CA4 binds to tubulin. Another VDA from this group is combretastatin A1-P (CA1-P), which is a sodium phosphate prodrug of CA1 and is more potent than CA4-P 108. Combretastatins mediate their effect on tumour vasculature mainly through alteration the morphology and function of endothelial cytoskeleton. Disruption of interphase microtubules by CA4-P leads to re-organisation of actin cytoskeleton and an increase in the permeability of tumour vasculature 109, 110. Another combretastatin analogue is AVE- 8062 with more powerful effect on tumour growth and tumour blood flow stasis compared with CA-4-P 111, 112.

EPC2407 is another VDA with potent anti-tumour activity in pre-clinical models. It induces tumour cell apoptosis and selectively suppresses the growth of proliferating cells, including multidrug-resistant cells (MDR). EPC2407 is highly effective in mouse tumour models, producing tumour necrosis at doses that correspond to only 25% of its maximum tolerated dose (MTD). In combination treatment, EPC2407 significantly enhanced the anti-tumour activity of cisplatin, resulting in complete regression of tumours in animals 113, 114.

ABT-751 (E7010) is an orally bioavailable sulfonamide with anti-tumour activity against wide variety of tumour cells including those resistant to current chemotherapies 115, 116

. Recent studies suggest that ABT-751 causes morphological changes and disrupt 1 Chapter tumour vasculature 117. 13

MPC-6827 (Azixa) is another small molecule VDA with cytotoxic effects in a variety of tumour cells at low nanomolar concentrations. It has been demonstrated that the activity of MPC-6827 is unaffected by overexpression of MDR pumps in cell culture. This property may explain the 14-fold greater exposure of the drug in mouse brain relative to plasma, as MDR pumps play an important role in the maintaining of blood-brain barrier 118, 119.

NPI-2358 is a synthetic analog of NPI-2350, a natural product isolated from Aspergillus sp fungus. NPI-2358 has potent in vitro activity against variety of human tumour cell lines including those with MDR profiles 120.

1.2.1.2. Vinca Alkaloides

Vinca alkaloids were originally isolated from the leaves of the Madagascar periwinkle, Catharanthus rosea over 40 years ago 3. Since the discovery of their biological properties including anti-leukemic effect and granulocytopenia as a result of bone marrow suppression 121, 122, they have been used for the treatment of solid tumours and haematological malignancies (Table 1.1)

The binding site of vinca alkaloids is on both the β-subunit of tubulin dimer and microtubule itself 123. Vinblastine (VBL), a member of vinca family, has been shown to bind to β-tubulin in a rapid and reversible manner and induces conformational changes in microtubule 124, 125. The binding affinity of VBL to tubulin at microtubule ends is extremely high 126, 127. It has been reported that binding of one or two VBL molecules to microtubule plus end results in a 50% decrease in dynamic instability and treadmilling 3.

Similar to CLC, the mode of action of vinca alkaloids largely depends on the drug concentration. At relatively high concentrations, they depolymerise microtubules, disrupt mitotic spindles, and block the dividing cells in mitosis. At clinically relevant concentrations, they suppress microtubule dynamics, block mitosis, and induce apoptosis without depolymerising microtubules 128-130. Chapter 1 Chapter 14

Due to the clinical efficacy of VBL and vincristine (VCR), the naturally occurring members of vinca alkaloids, various semi-synthetic analogues including vinorelbine, vindesine and vinflunine have been developed 3. Vinblastine and vincristine exhibit great activity against haematological malignancies. However, side effects such as neurotoxicity and myelosupression limit their applications 131.

Vinorelbine (VNB), a semi-synthetic analogue of vinca is less neurotoxic than vincristine and vinblastine, and more rapidly absorbed and metabolised by human hepatocyte compared with VBL and VCR 132, 133. Similar to other members of vinca family, vinorelbine induces mitotic arrest and apoptosis in melanoma cell lines 134. Vinorelbine is used as a single agent or in combination with other chemotherapeutics for the treatment of various malignancies including leukemia and lymphoma 135, 136.

Vinflunine (VFL, Javlor) is another member of the vinca family which is more potent than VBL, VCR, and VNB. VFL exhibited a marked anti-tumour activity in vivo with tumour regression being observed in human renal and NSCLC xenograft 137, 138. Compared to other vincas, VFL has less affinity to tubulin 139, 140, which results in the formation of smaller and fewer spiral filaments, effects that may be correlated with its reduced neurotoxicity 141, 142. VFL has shown anti-angiogenic and vascular-disrupting activities in in vitro models 143 along with anti-metastatic effects, as reported in two in vivo models 144, 145.

Vindesine (VDS) is a semi-synthetic analogue of VBL with higher in vitro activity compared with VCR and VBL 146, and higher heamatological toxicity compared with VCR 147. VDS is being used in combination regimens for treatment of leukemia, lymphoma, and NSCLC 148-150.

1.2.1.3. Other Microtubule De-stabilisers

Dolastatins are naturally occurring peptides that were isolated from the East Indian Sea Hare Dolabella auricularia 151. Dolastatin-10 is the most potent derivative among dolastatins. It inhibits microtubule polymerisation by binding to tubulin near the Chapter 1 Chapter exchangeable nucleotide and vinca binding site 123. Dolastatin 10 entered clinical trial 15

for the treatment of various tumours including pancreatic, kidney, prostate and liver cancer, as well as haematological malignancies. However, these trials were not encouraging as summarised in two recent reviews 152, 153. TZT-1027 (Soblidotin) is synthesised from dolastatin 10 with greater anti-tumour activity and reduced toxicity compared with its parent compound 154. TZT-1027 has anti-angiogenic effect at lower doses 155 and disrupts tumour vasculatures at higher concentrations, leading to extensive necrosis of tumour 156.

Eribulin is a synthetic macrocyclic analogue of the marine natural macrolide halichondrin B with a unique mechanism of action 157. Eribulin binds to the microtubule ends and inhibits polymerisation of purified tubulin. It also induces tubulin aggregates that compete with soluble tubulin for addition to the plus ends of the microtubule 158. Preclinical studies show that eribulin is active against taxane-resistant cell lines 159 and several human tumour xenografts including melanoma, ovarian, breast, and colon cancer 160. In clinical trials, eribulin improved the overall survival of heavily pretreated patients with breast cancer that led to its approval by the FDA for the treatment of metastatic breast cancer 161.

Indibulin (D-24851) is a synthetic compound which was identified in a cell-based screening assay to discover cytotoxic agents. The binding site of indibulin is distinct from CLC and vinca alkaloids domains 162. Interestingly, neurotoxicity that is normally associated with MTAs is not observed with indibulin 162. The reason is that indibulin is able to discriminate between mature neuronal microtubules and less-modified tubulins present in immature neuronal or non-neuronal microtubules, whereas vincristine and colchicine act on both types of tubulin. This effect has been attributed to inaccessibility of mature tubulin to the drug due to the posttranslational modifications 163. Chapter 1 Chapter 16

Table 1.1. Microtubule de-stabilising agents and their stages of clinical development

Binding Drug Therapeutic uses Stage of References domain Clinical Development Colchicine Colchicine Non-neoplastic diseases (gout, familial Appears to have 98 domain Mediterranean fever) failed trials due to the toxicity Combretastatins Potential vascular targeting agents; trials 36 phase I-III 164-168 (CA-1-P, CA-4-P, with advanced solid tumours as a single trials AVE8062) agent or combined with radioimmunotherapy and other chemotherapeutics 2-methoxyestradiol Trials with advanced solid tumours as a 6 phase I-II trials 169-175, EntreMed Inc. single agent or combined with immunotherapy and other chemotherapeutics ENMD-1198 Advanced tumours 1 phase I trials 176 , EntreMed Inc. EPC2407 Potential vascular targeting agent; trials 2 phase I-II trials Clinicaltrials.gov.org with advanced solid tumours ABT-751 Solid tumours including colorectal, renal, 12 phase I-II 177-182 breast cancers, NSCLC, neuroblastoma, trials CRPC, and relapsed ALL MPC-6827 Refractory solid tumours, refractory brain 6 phase I-II trials 183 metastases, glioblastoma, metastatic melanoma, 17 Chapter 1

Table 1.1. Continued.

Binding Drug Therapeutic uses Stage of Clinical References domain Development Colchicine NPI-2358 Potential vascular targeting agent; trials with 2 phase I-II trials 184 Domain lymphoma and advanced solid tumours including NSCLC 185-188 DMXAA Potential vascular targeting agent; trials with 11 phase I-III trials dvanced and refractory solid tumours including NSCLC and prostate cancer as a single agent or in combination with other chemotherapeutics Vinca Domain Vinblastine Hodgkin’s disease, testicular germ-cell cancer; In clinical use; 268 189-191 trials with other tumours including glioma, phase I-IV trials in metastatic melanoma, hodgkin’s disease, and progress bladder in combination with other chemotherapeutics Vincristine Leukemia, lymphomas; ; trials with other In clinical use; 700 192-195 tumours including multiple myeloma, non- phase I-IV trials Hodgkin lymphoma, rhabdomyosarcoma lymphoblastic leukemia, neuroblastoma in combination with other chemotherapeutics Vinorelbine Trials with solid tumours including NSCLC, 269 phase I-IV trials 196-199 metastatic breast cancer, squamous cell carcinoma of the head and neck as a single agent or in combination therapy

18 Chapter 1

Table 1.1. Continued.

Binding Drug Therapeutic uses Stage of Clinical References domain Development Vinca Domain Vindesine ALL, chronic myelogenous leukemia, lung, In clinical use; 36 phase 200-202 breast, and colorectal cancers. Trials with I-IV trials NSCLC, lymphoblastic leukemia, neuroblastoma, anaplastic large cell lymphoma, and recurrent or disseminated squamous cell carcinoma of the uterine cervix in combination with radiotherapy and other chemotherapeutics Vinflunine Trials with solid tumours including urothelial 18 phase I-III trials 203-207 cancer of the bladder, metastatic breast cancer, NSCLC, CRPC and gastric cancer as monotherapy or combined with other chemotherapeutic agents Other biding Eribulin mesylate Metastatic breast cancer, Squamous cell In clinical use 208-210, sites carcinoma of head and neck, Advanced solid 45 Phase I-III trials clinicaltrials.gov.org tumours

Indibulin Advanced solid tumours 6 phase I trials 211, 212 TZT-1027 Trials with advanced solid tumours including 3 phase I-II trials 213-215 NSCLC and soft tissue sarcoma as a single agent or in combination therapy 19 Chapter 1

1.2.2. Microtubule Stabilising Agents

1.2.2.1. Taxanes

Paclitaxel (taxol; PTX) is a natural product isolated from the bark of the yew tree in 1967 216. PTX was first approved for clinical use in 1995 and today it is widely used to treat ovarian cancer, breast cancer, NSCLC and Kaposi’s sarcoma 3. While taxanes have a weak binding affinity to soluble tubulin, they bind strongly to tubulin along the length of the microtubules 217. PTX diffuses through small openings in the microtubules and binds to its binding site in the β-subunit, on the inner surface of microtubules. Upon binding, PTX induces a conformational change in tubulin that increases its affinity to neighboring tubulin molecule, results in stabilisation and polymerisation of microtubule 3, 89, 218.

In spite of the promising results in patients, some issues have limited the application of PTX. Due to its lack of water solubility, PTX is administered intravenously in a formulation containing polythoxylated castor oil (Cremophor) and dehydrated ethanol. The use of this solvent is associated with the risk of hypersensitivity reactions 219, 220. Additionally, Cremophor can alter the pharmacokinetic and efficacy of PTX. Cremophor micelles entrap PTX, prevent its accumulation in erythrocytes thereby reducing the fraction of free PTX 221. In addition, cremophor has been shown to nullify the in vitro antiangiogenic effect of taxanes 222. Another disadvantage of PTX is that the drug is a substrate for the drug effluex pump P-glycoprotein (Pgp), a well characterised ATP-binding cassette transporter (ABC transporter) pump, that is involved in drug resistance 223.

To overcome these drawbacks, novel analogues and formulations have been developed (Table 1.2). Docetaxel (Taxotere), is a semi-synthetic analogue of PTX with higher cytotoxicity and improved water solubility 15, 89. In addition, docetaxel has been shown to have a longer retention time in cancer cells 224. Docetaxel is currently used to treat a range of solid tumours including breast cancer, prostate cancer and NSCLC 190. Chapter 1Chapter

20

Abraxane (Abraxis BioScience) is a nanoparticle albumin-bound PTX, that has been developed to reduce the side effects associated with cremophor formulation. In a phase III clinical trial comparing abraxane and docetaxel therapy in patients with metastatic breast cancer, abraxane displayed an improved safety profile and enhanced efficacy compared with docetaxel 225. Abraxane treatment is also associated with a longer progression-free survival of patients with metastatic breast cancer as compared with docetaxel treatment 226. Abraxane has also shown activity against other human cancers including prostate and gynecological tumours 227, 228.

Tesetaxel (DJ-927) is a novel oral semi-synthetic taxane with high solubility and lack of neurotoxicity. Tesetaxel is more potent than taxol and docetaxel in various tumour cell lines and unlike most taxanes, its cytotoxicity is not affected by Pgp expression level in tumour cells 229.

Cabazitaxel (XRP6258) is another semisynthetic taxoid with a poor affinity for Pgp 230 and greater blood-brain barrier penetration compared with PTX and docetaxel 231. In a phase III TROPIC (Treatment of Hormone-Refractory Metastatic Prostate Cancer Previously Treated with a Taxotere-Containing Regimen) trial, cabazitaxel demonstrated survival benefit in metastatic castration-resistant prostate cancer (mCRPC). On this basis, the FDA and the European Medicines Agency approved cabazitaxel for the treatment of patients with mCRPC who have previously been treated with docetaxel 232.

Ortataxel (SB-T-101131, IDN5109, BAY59-8862) is a taxane derived molecule with improved solubility and tolerability. Ortataxel has higher anti-tumour activity compared to PTX and has the ability to overcome MDR 233. Ortataxel is currently being evaluated in phase I and II clinical trial for treatment of solid tumours.

Milataxel (MAC-321, TL00139) is a docetaxel analogue that retains activity against Pgp-expressing cells. It has been shown that malitaxel partially or completely inhibits tumour growth in three tumour models that were resistant to PTX due to the overexpression of P-gp 234. Malitaxel has recently entered phase II clinical trials for the treatment of colorectal cancer and NSCLC.

Another taxane analogue is larotaxel (RPR 109881A) with a broad spectrum of Chapter 1Chapter activity and improved toxicity profile compared to PTX and docetaxel. Larotaxel not 21

only overcomes the MDR mechanism, but also crosses the blood-brain barrier 235. Larotaxel is currently being evaluated in trials in combination with other chemotherapeutic drugs for the treatment of advanced pancreatic and breast cancers.

Taxoprexin (DHA-paclitaxel) is a PTX fatty acid conjugate with no effect on polymerisation of microtubules. In M109 mouse tumour model, taxoprexin has been reported to be less toxic than PTX, and cured all tumour-bearing animals, whereas paclitaxel cured none. This effect was attributed to the alteration of pharmacokinetics of PTX by the fatty acid, that resulted in increased area under the curve (AUC) of the drug in the tumour and decreased AUC in normal cells 236. Taxoprexin is currently under investigation in trials for the treatment of advanced malignancies (clinicaltrials.gov).

1.2.2.2. Epothilones

Epothilone A and B were discovered as antifungal agents in 1986 and as metabolites of the myxobacterium, Sorangium cellulosum in 1987 237. Epothilones compete with PTX for binding to tubulin and displace [3H]-PTX from microtubules, suggesting that they share the same binding site as taxanes 237, 238. Despite this, electron crystallography analysis has shown that epothilones interact with β-tubulin through a unique and independent molecular interaction 239. Epothilones seem to be active against taxane-resistant tumours 240, as they are not substrates for Pgp efflux pump 190, 241. They are also effective against PTX-resisatant cells resulted from mutant β- tubulin 242.

Epothilone B (patupilone, EPO906) has been evaluated in clinical trials against a wide variety of malignancies 243 including taxane resistant tumours. Due to its ability to cross the blood-brain barrier, epothilone B has activity in patients with progressive or recurrent brain metastases from NSCLC 244, 245.

Ixabepilone (BMS-247550) is a semi-synthetic analogue of epothilone B. Ixabepilone was approved by the FDA for the treatment of metastatic and locally advanced breast cancer in patients whose tumours are refractory or resistant to anthracyclines, taxanes, and capecitabine 147, 246. A recent study showed that ixabepilone in combination with capecitabine significantly improved progression- 1Chapter 22

free survival of patients with metastatic breast cancer in comparison with capecitabine as a single agent 247.

Sagopilone (ZK-EPO, ZK219477) is the first fully-synthetic epothilone that retains activity against MDR tumours and is not recognised by ABC transporters 248. In preclinical models, sagopilone demonstrated significant anti-tumour activity against a wide range of human cancer cell lines 249. Similar to epothilone B, sagopilone is able to cross the blood brain barrier. In animal models of glioblastoma, sagopilone suppressed the growth of orthotopically implanted human glioma and CNS metastasis 250. However, in a recent phase II trials, no evidence of anti-tumour activity of sagopilone against recurrent glioblastoma was observed 251.

Epothilone D (KOS-862) is highly active in taxane-resistant cell lines 252 and has shown superior in vivo efficacy compared with epothilone B 253. In a phase II trials, epothilone D demonstrated anti-cancer activity in pretreated patients with NSCLC and breast cancer 254.

Dehydelone (KOS-1584) is a novel analogue of epothilone D with improved pharmacokinetic properties 255. Dehydelone is approximately 3-12 times more potent than epothilone D with increased tissue penetration and reduced exposure to CNS 255, 256.

1.2.2.3. Other Microtubule-stabilising Agents

Discodermolide, a taxane domain-binder, was isolated from the seaweed, Discodermia dissolute. While discodermolide is more active than PTX against various MDR cell lines 241, 257, 258, its ability to overcome Pgp mediated resistance is less pronounced than epothilones 257, 259. Discodermolide has been reported to have a synergistic interaction with PTX in vitro 258. It was postulated that the two agents synergistically suppressed the dynamics of microtubules 3 and that PTX and discodermolide induced a conformational change in microtubule that led to the alteration in binding of the other drug 260. Due to the severe pulmonary toxicity in three patients in phase I clinical trial, concerns about the safety of discodermolide has been raised 261. Chapter 1Chapter

23

Eleutherobin is another microtubule stabilising agent whivh was isolated from soft coral species Eleutherobia in 1997 262. Although eleutherobin has a potent cytotoxic activity against various cell lines in vitro 263, it has no advantages over PTX, as it is a Pgp substrate 262, 264, 265 and has less activity against PTX-sensitive cell lines compared with PTX 259.

Sarcodyctins, isolated from the Mediterranean stoloniferan coral Sarcodyction roseum 266, are active against Pgp-overexpressing cell lines but like eleutherobin, have lower cytotoxicity compared with PTX 259.

Laulimalide, isolated from chocolate sponge, Cacospongia mycofijiensis in 1988 267, binds to a distinct binding site on microtubules from other microtubule-polymerising agents 268. Although laulimalide is less potent than PTX against some cancer cell lines such as SKOV3, it is 100 times more effective than PTX in MDR cells that overexpress Pgp 267, 269.

The above candidates may have been discontinued, as they are not listed on clinicaltrials.gov.

Chapter 1Chapter

24

Table 1.2. Microtubule stabilising agents and their stages of clinical development

Binding Drug Therapeutic uses Stage of Clinical references domain Development Taxane Site Paclitaxel Breast and ovarian cancers, kaposi’s sarcoma; trials with In clinical use 270-272, numerous other tumours as a single agent and in 1668 trials; 56 completed clinicaltrials.gov combination with other chemotherapeutics phase I-IV trials Abraxane Metastatic breast cancer; trials with other tumours as a In clinical use 273, single agent and in combination with other 70 phase I-IV trials clinicaltrials.gov chemotherapeutics Docetaxel Breast, ovarian, hormone refractory prostate cancers; trials In clinical use 274-276, with other tumours as a single agent and in combination 1349 trials; 47 completed clinicaltrials.gov with other chemotherapeutics phase I-IV trials Tesetaxel Solid tumours including gastric, bladder, breast, and Phase I-III trials 277, 278, colorectal cancers clinicaltrials.gov Cabazitaxel mCRPC, squamous cell carcinoma, breast cancer, NSCLC, In clinical use 232, 279-281, ; trials with NCSLC and mCRPC tumours as a single 16 phase I-III trials clinicaltrials.gov agent and in combination with other chemotherapeutics Ortataxel Refractory non-hodgkin's lymphoma, advanced renal cell 7 phase I-II trials clinicaltrials.gov carcinoma, NSCLC Milataxel NSCLC, malignant mesothelioma, and colorectal cancer 3 phase I-II trials 282, 283, clinicaltrials.gov Larotaxel locally advanced/metastatic urothelial tract or bladder 8 phase I-III trials 282-285, cancer, Metastatic breast cancer, NSCLC, advanced clinicaltrials.gov

pancreatic cancer

25 Chapter 1

Table 1.2. Continued

Binding Drug Therapeutic uses Stage of Clinical references domain Development Taxoprexin Metastatic kidney, colorectal, pancreatic, prostate, 9 phase I-III trials 286-291, clinicaltrials.gov and melanoma cancers, advanced lung and liver cancers, advanced skin and eye melanoma, gastric and oesophageal adenocarcinoma Other biding Epothilone B Advanced prostate cancer, CNS malignancies, 71 phase I-III trials 286-290, clinicaltrials.gov sites melanoma, solid tumours including NSCLS, breast, gallbladder, kidney, ovarian, colorectal cancers Ixabepilone Metastatic or locally advanced breast cancer; In clinical use 147, 247, 291, 292, trials with other tumours as a single agent and in 111 phase I-III trials clinicaltrials.gov combination with other chemotherapeutics Sagopilone Metastatic melanoma and breast cancer, refractory 14 Phase I-II trials 293-297 solid tumours, malignant glioma, brain metastasis, ovarian cancer, advanced prostate cancer, and NSCLC Epothilone D NSCLC, metastatic colorectal, prostate and breast 6 Phase I-II trials 254, 298, 299 cancers Dehydelone NSCLC 1 phase IIIb/IV trials Clinicaltrials.gov

26 Chapter 1

1.2.3. Mechanism of action of microtubule-targeting agents

1.2.3.1. Cell Death and Apoptosis

It is well known that disruption of the mitotic spindle by MTAs activates spindle- assembly checkpoint, which in turn induces a prolonged mitotic arrest and eventually leads to cell death 300, 301. Nevertheless, the molecular mechanisms by which MTAs induce cell death have not been fully elucidated. Several studies have suggested that the process is caspase-dependent as caspase inhibitors can inhibit or delay cell death 302, 303. Caspases are cysteine proteases which are the main executors of apoptosis. Apoptosis, the most common form of cell death, is a tightly regulated pathway and its dysregulation is implicated in many pathological conditions including cancer. Apoptosis can be induced by two pathways: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway (figure 1.4). The extrinsic pathway operates through death receptors on the cell surface and the intrinsic pathway is controlled by Bcl2 family. The intrinsic pathway becomes activated in response to cellular insults and chemotherapeutic agents including MTAs, and its activation leads to the disruption of mitochondrial membrane 304, 305. It has been shown that the suppression of microtubule dynamics and alteration of mitochondria parameters such as production of reactive oxygen species, reduction in mitochondrial respiration rate, and change in mitochondria membrane, are concomitant events that lead to apoptosis 306, 307. MTAs modulate the expression and activity of Bcl-2 protein family by: I) hyperphosphorylation of the anti-apoptotic members of the family such as Bcl2 and Bcl-xl, thereby down-regulating or inactivating these proteins 308-311, and II) up-regulation of pro-apoptotic members of the family such as Bad, Bax, Bad, and Bim 312-314. These modulations lead to mitochondria permeabilisation, release of pro-apoptotic proteins into the cytosol, and activation of the intrinsic pathway of apoptosis.

The p53 protein and its effector p21 may also be affected by MTAs. It has been reported that upregulation and nuclear translocation of p53 is associated with cell cycle arrest in G1 phase induced by MTAs 315-317. In response to microtubule Chapter 1 Chapter dynamic suppression, p53 which is sequestered in cytoplasm by microtubules, 27

translocates to the nucleus 315, 316. P53 can also translocate to mitochondria in response to apoptotic signals and directly activates pro-apoptotic proteins and forming inhibitory complexes with anti-apoptotic proteins 318-320.

The extrinsic pathway is not a common pathway induced by MTAs. However, few studies reported its involvement in MTA-induced cell death. For instance, 2ME up- regulates death receptor 5 (DR5) in a variety of cell lines including breast, cervical, glioma, and prostate cancer cells, thereby activates the extrinsic pathway of apoptosis 321. PTX has also been shown to induce apoptosis through the activation of the extrinsic pathway 322, 323.

Mitotic catastrophe is another mode of cell death induced by MTAs. It is caused by aberrant mitosis and characterised by the formation of multinucleated and giant cells containing uncondensed chromosomes 324. PTX treatment has been shown to induce aberrant metaphase in which sister chromatids fail to segregate properly. This leads to prolonged metaphase and cell death by mitotic catastrophe 325, 326. Docetaxel has also been reported to induce mitotic catastrophe in breast cancer cells 327. Among microtubule-depolymerising agent, CA4 induces mitotic catastrophe in lymphocytic leukemia and lung cancer cell lines and arrest cells in metaphase 328, 329.

MTAs are also able to induce premature or accelerated senescence as a result of terminal cell cycle arrest 330-332. Senescence is characterised by inability of cells to replicate even in the presence of mitogen stimiulation, alteration of cell phenotype (flattened, enlarged, granular morphology), arrest at interphase cell-cycle (either G1 or G2), and finally a senescence-associated β-galactosidase activity producing blue staining when react with X-gal at pH 6 332. Discodermolide has been shown to induce senescence in A549, a NSCLC cell line. This effect is accompanied by sustained activation of Erk1/2, and two markers of senescence, p66Shc and PAI-I 333. Disorazole, a microtubule-depolymerising agent also induces senescence in A549, HCT-116, a human colon cancer cell line, H1299, a lung cancer cell line, and UPCI:SCC103, an oral squamous carcinoma cell line 334. Chapter 1 Chapter

28

Figure 1.4. Two major apoptosis signaling pathways. Death receptors can activate the intrinsic pathway of apoptosis by caspase-8-mediated cleavage of the pro-apoptotic BCL2 superfamily member, BID. BID interacts with the pro-apoptotic BAX and BAK, which cause release of mitochondrial cytochrome c and activation of caspase-9 and -3. This amplifies apoptosis via the extrinsic pathway of apoptosis. Conversely, DNA damage can induce upregulation of some death receptors, such as FAS and death receptor 5 (DR5), through both p53-dependent and p53-independent mechanisms. This upregulation increases cellular sensitivity to death-receptor ligands. In some cell types, death-receptor engagement of the cell-extrinsic pathway is sufficient for apoptotic death. In other cell types, commitment to apoptosis is mediated through cell-intrinsic pathway, which leads to amplification of the death-receptor. Adapted from Ashkenazi et. al. 335 Chapter 1 Chapter

29

1.2.3.2. Anti-vascular Action

The tumour vasculature is a promising target for cancer therapy. Two strategies have been used to inhibit vascular function. The first approach is anti-angiogenic therapy which aims at preventing neo-vascularisation in tumour 102, 336, and the second approach is using VDAs, that aims at rapid and selective shut-down of the established vasculature of tumour 337.

Anti-angiogenic compounds are classified into two categories: direct angiogenesis inhibitors which target endothelial cells; and indirect angiogenesis inhibitors that either inhibit tumour cells from producing angiogenic factors such as VEGF and basic fibroblast growth factor (bFGF), or block the receptor of an angiogenic factor in endothelial cells 338, 339.

Most MTAs have been shown to inhibit several endothelial cell functions involved in angiogenesis at their low concentrations 340-344. Some MTAs can inhibit angiogenesis by suppressing the proliferation of endothelial cells and inducing a slight de-polymerisation in their microtubules 341, 342, whereas others inhibit angiogenesis without affecting cell proliferation or microtubule organisation 340, 343, 345, 346.

Inhibition of angiogenesis by MTAs could also be related to their effect on thrombospondin-1 (TSP-1), a potent endogenous inhibitor of angiogenesis in endothelial and cancer cells 347, 348. MTAs impair the mobilisation and viability of the circulating endothelial precursors (CEPs) 349, thereby inducing the expression and secretion of TSP-1 347. In addition, in vitro studies revealed that MTAs inhibit the migration and motility of endothelial cells by damaging the reorientation of MTOC 343. Further, low concentrations of some MTAs such as PTX and VFL significantly disrupt the mitochondrial functions in endothelial cells, resulting in slowing down the cell cycle progression 346, 339.

VEGF has a predominant role in angiogenesis 350 and its down-regulation is associated with inhibition of angiogenesis and migration of endothelial cells 351-353. Docetaxel has been reported to block the activation of VEGF signaling pathways downstream of VEGF signaling via VEGF receptor 2 (VEGFR2), resulting in Chapter 1 Chapter inhibition of paxillin phosphorylation and αvβ3 integrin activation through the 30

inhibition of focal adhesion kinase (FAK) phosphorylation. FAK has a key role in the dynamic reorganisation of the cytoskeletal network that precedes cell migration and its phosphorylation provides sites for interaction with other focal adhesion- associated proteins. Docetaxel also prevents the activation of endothelial nitric oxide synthase (eNOS) through the inhibition of Akt phosphorylation. Finally, docetaxel causes the ubiquitination and proteasomal degradation of the cytoplasmic chaperon, heat shock protein 90 (HSP90). HSP90 has a key role in tumourigenesis and angiogenesis, as several proteins involved in proliferation, cell cycle control, angiogenesis, and apoptosis are HSP90 client proteins. Among these proteins is tubulin which has been shown to be dissociated from HSP90 by docetaxel 354.

In addition to their direct effect on vascular endothelial cells, MTAs also inhibit tumour cells from producing angiogenic factors such as VEGF. In a murine model of breast cancer, anti-angiogenic effect of docetaxel was reported to be associated with VEGF suppression 355. Similarly, vincristine and docetaxel directly down-regulate VEGF expression in leukaemia cell lines 352. This down-regulation could be due to the inhibition HIF-1α 353, 356. In hypoxia, HIF-1α translocates into the nucleus and induces the expression of several angiogenic factors including VEGF, platelet- derived growth factor (PDGF), angiopoietin-2 (Ang2) and NOS among many others. HIF-1α also activates the transcription of the genes that are involved in invasion, glucose metabolism, and cell survival 357, 358. Recent studies suggest that MTAs share the ability to reduce HIF-1α levels, suggesting that their effect on HIF-1α levels might be due to the microtubule disruption 356. Down-regulation of HIF-1α is associated with disruption of interphase microtubule rather than mitotic arrest, indicating that HIF-1α suppression proceeds mitotic arrest and contributes to the anti-tumour efficacy of MTAs 353, 356.

Besides their anti-angiogenic properties, some MTAs including colchicine, can directly damage pre-existing blood vessels within the tumours. Colchicine was the first VDA to show anti-vascular effect 359. Vinblastine and vincristine 360, 361 have also been reported to disrupt tumour vasculature in animal models at their MTD. In contrast, combretastatins 105, 107, 362, TZT1027 363, 364, and ABT-751 365 effectively disrupt tumour microvasculature at doses well below their MTDs. Chapter 1 Chapter

31

VDAs cause rapid and extensive damage to tumour vasculature which leads to a decrease in tumour blood flow, an increase in vascular permeability, collapse of blood vessels, and necrosis of tumour cells 102, 339. These anti-vascular effects are accompanied by alteration of endothelial cells morphology, disruption of junctions between the cells, and increase in vascular permeability 109. VDAs also induce apoptosis in endothelial cells 366, 367 by arresting the cell cycle at G2/M phase 368. Indeed, endothelial cells are more sensitive to the cytotoxic effect of vascular- disrupting MTAs compared with other cell types 110 with proliferating endothelial cells being more sensitive than quiescent cells 105. This could be due to the fact that vascular-disrupting MTAs can accumulate in endothelial cells at nearly five times higher concentrations than in other cells 110, 341, 369. Notably, VDAs seem to disrupt tumour vessels without affecting normal tissues 370. This selectivity has been attributed to immature and fragile nature of tumour vasculatures which makes them more susceptible to VDAs action 371.

1.2.5. Toxicity of Microtubule-targeting Agents

The main toxicity of MTAs is the high rate of neuropathy associated with the use of these compounds 372. This dose-limiting toxicity manifests as a painful peripheral axonal neuropathy and is not currently manageable 373. Other manifestations of neuropathy are constipation, intestinal paralysis and urinary retention which result from toxicity in the autonomic nervous system 190. Central neurotoxicities including headache, dizziness and mental depression may also occur 90

Haematological toxicity which is frequently refered as myelosuppression 374, 375 is another adverse effect of MTAs that result from the inhibition of rapidly dividing hematopoietic cells. Neutropenia occurs shortly after treatment and is particularly observed when MTAs are combined with other chemotherapeutic agents 203, 376, 377. This side effect is one of the most frequent dose-limiting toxicities reported in clinical trials 213, 378. Chapter 1 Chapter

32

1.3. ALBENDAZOLE

Albendazole (ABZ) [1 methyl(5-proylthio)1H-BZ-2yl carbamate], a benzimidazole (BZ) antiparasitic agent, was first approved for human use in 1982 379. ABZ was initially used for the treatment of intestinal nematodes, but later found applications in systemic infections such as cysticercosis and echinococcosis. Recently, ABZ is being tested in the global programme for the elimination of lymphatic filariasis 380.

Figure 1.5. The structure of ABZ and its metabolites

1.3.1. Metabolism

Following administration, ABZ rapidly metabolises to its active metabolite, albendazole sulfoxide (ABZSO) and inactive metabolite albendazole sulfone

(ABZSO2) (figure 1.5). Intestinal and hepatic metabolism of ABZ take place by two diverse microsomal enzymatic systems, flavin-containing monooxygenase (FMO) and cytochrome P4503A4 (CYP3A4) 381. Both FMO and CYP3A4 participate in

ABZ sulfoxidation, while cytochrome P4501A2 (CYP1A2) is responsible for ABZ 1 Chapter 33

sulfonation 382. ABZSO has two enantiomers which can be separated by HPLC; (+) ABZSO is related to FMO activity, whereas (-) ABZSO is produced by the P450 system 381. Inhibition of either enzyme may alter the concentrations of ABZ and ABZSO in vivo. Such drug interaction could also influence the chemotherapeutic effect of ABZ, since it is not known which (or both) enantiomer is responsible for ABZ activity 383.

ABZ and its metabolites significantly induce ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities which act as cytochrome P4501A1 and cytochrome P4501A2 substrates, respectively. In HepG2 cells, high concentrations of ABZ cause a marked increase in P4501A whereas little or no increase in EROD and MROD activities were detected. This suggests that high concentrations of ABZ or its metabolites have an inhibitory effect on induction of cytochrome P450 enzymes 384 .

1.3.2. Bioavailability and Absorption

Poor water solubility of ABZ (~0.2 μg/ml at pH 7.4) results in a decrease in its absorption from gastrointestinal system and consequently, reduction of its efficacy and bioavailability. The actual bioavailability of ABZSO is directly related to its short-term presence in human plasma. ABZSO has a half-life of 9 hours and its protein binding is 70%. The concentration of ABZSO in cerebrospinal fluid and brain tissue have been shown to be 50% and 40% of plasma levels, respectively 385.

In order to increase the bioavailability of ABZ, many approaches have been proposed. It has been demonstrated that ionization in an acid medium could increase the solubility of ABZ. However, this dissolution profile is not enough to prepare high concentrations of ABZ. Alternatively, addition of surfactants such as co-solvent agents and bile salts can enhance the absorption of ABZ. Yet, many of these excipients can irritate the linings of digesting system 386. Another method for improving ABZ solubility is elaboration of solid dispersions with polyvinylpyrrolidine 387. Limiting factors for such products are the use of organic co- solvents and the high concentrations of the complexing agents 388. Recently, it has

been reported that hydroxypropyl β-cyclodextrin (HPβCD) and L-tartaric acid 1 Chapter

34

significantly increase the bioavailability and larvicidal activity of ABZ compared to its commercial suspension 389.

Due to its lipophilicity, ABZ can cross plasma membrane by passive diffusion 390. Nevertheless, the existence of other uptake mechanisms such as ABC drug efflux transporters cannot be excluded. ABC-transporters are transmembrane proteins that utilise the energy of ATP hydrolysis to perform certain biological processes including translocation of various substrates across membranes. They are also responsible for several non-transport-related processes such as translation of RNA and DNA repair 391, 392. ABC transporters are associated with tumour resistance, cystic fibrosis, bacterial multidrug resistance, and a range of other inherited human diseases 393. Merino et. al. studied the interaction of ABZ and its metabolites with breast cancer resistance protein (BCRP/ ABCG2) and MRP2 (ABCC2) using MDCKII cells transduced with human BCRP1, MDR1, MRP2 and murine Bcrp1 cDNAs. These drug efflux transporters affect the bioavailability of many drugs and extrude a wide range of xenotoxins from intestine, liver, and other organs. They demonstrated that ABZ is a BCRP and Bcrp1 substrate 394. Therefore, the use of BCRP/Bcrp1 inhibitors may improve the oral bioavailibility of ABZ and reduce its intestinal elimination and hepatobilary secretion.

1.3.3. Mode of Action

1.3.3.1. Cytotoxic Effect

ABZ exerts its therapeutic effect in helminths through binding to the colchicine binding site on β-tubulin and therefore, inhibiting the polymerisation of microtubule 395, 396. In ovarian cancer cell lines, 1A9 and 1A9PTX22, ABZ has been shown to depolymerise microtubules in a dose-dependent manner 397.

ABZ inhibits cell proliferation at threshold concentrations of 0.1-0.25 μM with a maximum anti-proliferative effect being reached between 1-10 μM. The effect of ABZ on cell cycle depends upon the concentration of the drug. At 0.5 μM, ABZ treatment leads to cell cycle arrest at G0/G1, possibly due to the inhibition of DNA Chapter 1 Chapter

35

synthesis, and at 1 μM, it causes cell accumulation in the G2/M phase of cell cycle, probably owing to its effect on the microtubules 398, 399.

1.3.1.2. Apoptotic Effect

In animal models, it has been shown that ABZ generates oxidative stress by producing reactive oxygen and nitrogen species (ROS and RNS, respectively). This effect is through the suppression of gluthation S-transferase (GST) activity, an enzyme which is required for ROS neutralization. As anaerobic or facultative aerobic organisms, parasites have none or very low antioxidant defense. Therefore, ROS and RNS could represent a powerful defense mechanism against parasites. ABZ treatment increases the activity of catalase and superoxide dismutase (SOD), as well as the levels of serum nitrogen monoxide (NO). Enhanced NO, leads to the formation of peroxynitrite, a potent RNS that causes tissue damage during pathological changes. High activity of SOD results in an increase in hydrogen 400, 401 peroxide (H2O2) levels in host cells that ultimately leads to apoptosis . However, this effect has not been studied in cancer cells or in animals bearing xenograft tumour.

In tumour cells, ABZ markedly down-regulates the expression of Mcl-1 protein 402, a Bcl-2 related protein whose over-expression confers resistance to apoptosis-inducing agents in ALL 403. Moreover, ABZ treatment increases the release of cytochrome c and bax protein level, and reduces the levels of Bcl-2 protein 402.

1.3.1.3. Anti-angiogenic Effect

In vitro, ABZ suppresses the expression of HIF-1α protein 404. ABZ treatment also inhibits VEGF-stimulated cell proliferation, migration, tube formation, and permeability in endothelial cells. It also down-regulates VEGFR2 in endothelial cells. The inhibitory effect of ABZ on neovascularization has also been observed in mice retina 405.

In OVCAR-3 tumour-bearing mice, intraperitoneal administration of ABZ has been demonstrated to reduce VEGF levels in ascites fluid and plasma 406. Furthermore, Chapter 1 Chapter ABZ treatment decreased vascular density as compared with control group 406. In a 36

more recent study, a single dose of ABZ was shown to down-regulate the expression of both HIF-1α mRNA and protein in OVCAR-3 tumour. This effect was accompanied by VEGF suppression in the tumour, suggesting that VEGF reduction was mediated by the HIF-1α suppression 404.

1.3.4. Potential of Albendazole as an Anti-cancer Agent

1.3.4.1. Pre-clinical Studies

Oral administration of ABZ at a concentration of 300 mg/kg significantly suppresses the growth of subcutaneous SKHEP-1 in mice 398. In a xenograft model of peritoneal carcinomatosis, regional treatment with ABZ markedly reduced peritoneal tumour growth 399. In nude mice bearing OVCAR-3 the formation of malignant ascites was profoundly suppressed by intraperitoneal administration of ABZ 406.

1.3.4.2. Clinical Studies

In a pilot study with a small number of patients (n=7), oral administration of 10 mg/kg/day ABZ for 28 days led to stabilisation of carcinoembryonic antigen (CEA) and alpha-feto protein (AFP) levels in patients with colorectal cancer and hepatocellular carcinoma, respectively. ABZ was reported to be well tolerated and no significant changes in haematological, liver, and kidney function tests were observed 407. In a recent phase I clinical trial, ABZ was given orally at doses up to 2400 mg/day. MTD was determined to be 2400 mg/day with myelosupression being the main dose-limiting toxicity. Although 12% of patients had a stable tumour marker during the treatment, no objective response was observed 408. Nevertheless, improving bioavailability through re-formulation and/or combination of ABZ with other anti-proliferative or anti-angiogenic agents may lead to a superior pharmacokinetic profile and efficacy. Chapter 1 Chapter

37

1.3.5. Toxicity and Side Effects

At doses used for intestinal helminths treatment, all reported side effects seem to be mild and self-limiting, and none has been life threatening. Gastrointestinal symptoms occur with a frequency of >1%. One of the most frequent side effects is abnormality in liver function with a frequency of about 20% of cases which could be the consequence of idiosyncratic reactions to the drug 409. Temporary alopecia is other side effect that has been reported to occur in 5% of treated cases. ABZ has also some impact on bone marrow. As mentioned before, since these tissues have high cell turnover, they are more sensitive to MTAs. ABZ is known to be teratogenic, therefore, there has been a debate over the risks of administration of the drug to pregnant women. However, no adverse effect has been reported in any pregnancy when exposed in the first semester 410.

Other reported side effects are headache, dizziness, hypersensitivity, leucopenia, and pancytopenia.

1.4. 2-METHOXYESTRADIOL

2ME is an endogenous metabolite of estradiol and 17β-estradiol, with anti-tumour and anti-angiogenic properties. It has been proven that esterogenic compounds interact with microtubules and interfere with cell division. These agents inhibit microtubule polymerisation at high concentrations by binding to colchicine domain 411. Estradiol, the main estrogenic hormone, also binds to microtubules and induces mitotic arrest 412, 413. However, the effect of 2ME on mitosis is more pronounced than estradiol. Indeed, it has been proposed that 2ME is responsible for anti-mitotic effect of estradiol 413, 414. The level of 2ME in human serum ranged from 30 pM in adult males to 30 nM in pregnant women 415, that are much lower than that required for tubulin depolymeriseation. 2ME has a very low affinity towards estrogen receptor (<0.1% compared with estradiol affinity) and does not show estrogenic activity 416. Chapter 1 Chapter

38

1.4.1. Metabolism and Bioavailability

2ME is formed by sequential hydroxylation of 17β-estradiol at a number of sites by cytochrome P450 isozymes CYP1A1, CYP1A2 and CYP3A 417, followed by Ο- methylation by catechol-O-methyltransferase enzyme 418, an enzyme that is found in large amounts in various tissues including liver, kidney, uterus, breast, placenta as well as lymphocytes and erythrocytes 419. Oxidation of estradiol at position 17 yields estrone. While 2-hydroxyestrone and 4-hydroxyestrone are formed by hydroxylation of A-ring of estrone, 16α-hydroxyestrone and estriol are produced by the metabolism of its D-ring. 2-hydroxyestradiol is then methylated to 2ME and 2-methoxyesterone 416 (figure 1.6).

2ME has a half-life of approximately half an hour in rodents. This short half-life is due to sulphation and glucuronation of 2ME at position 3 or 7 with rapid excretion 420. In contrast, the half-life of 2ME in humans has been reported to be 1-2 days 169. The reason for this relatively long half-life in human is not clear, but may result from differences between species in enterohepatic recirculation processes 417. Another factor that may have contributed to the prolonged half-life of 2ME is its high binding affinity to plasma proteins, as this property is associated with slow distribution and elimination of a drug 421.

Following administration, 2ME is extensively oxidised to 2-methoxyestrone that is relatively inactive. In fact, the growth inhibitory effect of 2-methoxyestrone is approximately 1% of 2ME 419. In clinical trial, the concentration of 2- methoxyestrone has been shown to be 10-fold higher than 2ME 170.

2ME is a highly lipophilic compound with very low aqueous solubility 422 (approximately 1.8 μg/ml H2O at 37ºC) . This problem together with its extensive first pass metabolism led to the modification of the steroid structure to develop analogues with more desirable properties. Modification of A ring 423, modification of hydroxyl group at position 17, preparation of water-soluble prodrugs 424, and encapsulation of 2ME within biocompatible and biodegradable polyelectrolytes 425 have been shown to improve metabolic stability, bioavailability, and anti-tumour efficacy of 2ME. Modification of 2ME at positions 3 and 17 led to the development Chapter 1 Chapter

39

of ENMD-1198 with improved metabolic stability. ENMD-1198 is currently in a phase I clinical trial in patients with refractory solid tumors 426.

Figure 1.6. Pathway of 2ME formation 427

1.4.2. Mechanism of Action

1.4.2.1. Cytotoxic Effect

Similar to other estrogenic compounds, 2ME binds at or near the colchicine domain and inhibits the polymerisation of tubulin. 2ME is active against variety of tumour cell lines including lymphoblast 428, 429, melanoma 423 lung 430, colon 423, ovarian 423, breast 416, and prostate cancer 423. IC50 (the concentration of the drug resulting in 50% reduction in cell viability) values of 2ME for most cell lines were reported to be below 1 μM. The noteworthy feature of 2ME is that it does not exhibit cytotoxicity Chapter 1 Chapter

40

in quiescent endothelial cells 418 or normal mammary epithelial cells 431, and that it only targets cells that are actively proliferating.

There is a moderate correlation between the IC50 value and inhibition of the polymerisation of tubulin, as in a panel of cell lines, the IC50s have been reported to be approximately one to two orders of magnitudes lower than those required for inhibiting of tubulin polymerisation 418, 432. 2ME induces G2/M cell cycle arrest as a result of de-polymerisation of tubulin, and G1 arrest as a consequence of the suppression of DNA synthesis 433, 434.

1.4.2.2. Apoptotic Effect

2ME induces apoptosis through diverse mechanisms. 2ME activates both extrinsic and intrinsic pathways of apoptosis. In human colorectal 435 and lung cancer cells 436 that harbour wild-type p53, 2ME increases the expression of p53 and p21 WAFI/CIPI which induces apoptosis associated with p53. Conversely, in neuroectodermal brain tumour cell lines, 2ME induces apoptosis through disruption of NFκB pathway without altering p53 protein levels or changing the proteins involved in intrinsic apoptosis pathway 437. 2ME also exerts its effect through Akt-dependent process and reactive oxygen species (ROS). In leukemia cells, 2ME has been shown to induce oxidative injury, which results in attenuation of Akt, activation of JNK, down- regulation of Mcl-1 and ultimately, mitochondrial injury and apoptosis 438. 2ME has also been reported to phosphorylate Bcl-2 and Bcl-xL in a variety of cell lines, thereby attenuating their anti-apoptotic activity 431, 439.

In addition to intrinsic or mitochondrial pathway, extrinsic or receptor-mediated pathway of apoptosis has also been implicated in cytotoxic effect of 2ME. This pathway involves the activation of death receptors such as TNF receptor, Fas, death receptor 4 and 5 (DR4 and DR5). Interaction of these receptors with their respective ligands results in the activation of signal transduction cascade initiated by the recruitment of DR-associated molecules and subsequent activation of caspase 8 440. In various human cancer cell lines including breast, cervical, prostate, glioma, and primary endothelial cells, 2ME up-regulates the expression of DR5 protein levels and activates the downstream caspase 8 and caspase 3 321. In bovine vascular Chapter 1 Chapter endothelial cells, 2ME treatment leads to the formation of NO within the plasma 41

membrane resulting in senescence and apoptosis 441. Senescence is a gradual process in which cells become refractory to proliferation stimuli and lose their ability for replication 442. Enhancement of NO that coincides with the suppression of superoxide dismutase, as reported in leukaemia cells, may lead to peroxynitrite generation and triggers free radical damage and apoptosis 438.

1.4.2.3. Anti-angiogenic Effect

2ME has been shown to inhibit the expression, nuclear accumulation, and transcriptional activity of HIF-1α. This effect seems to occur post-transcriptionaly as 2ME treatment does not affect HIF-1α mRNA 353. 2ME also inhibits VEGF expression in both hypoxia and normoxia conditions in a dose-dependent manner 353. Further, 2ME partially inhibit the upregulation of VEGFR2 induced by serum deprivation in endothelial cells 443.

Besides its inhibitory effect on the proteins involved in angiogenesis, 2ME affects various steps of angiogenesis including migration, tube formation and invasion through a collagen matrix 444. In endothelial cells, 2ME suppresses the activity of bFGF-mediated stimulation of urokinase-type plasminogen activator (uPA). uPA is a tumour-associated proteolytic factor which contributes to metastasis 445.

In animal models, 2ME targets both tumour cells and endothelial cells compartment of tumour. In a mice-bearing human breast cancer cells, a concomitant reduction in tumour volume and vascularisation has been reported following 2ME treatment 446. In an orthotopic brain tumour model, 2ME-treated rats represented a significant reduction in tumour volume which was accompanied by a decrease in HIF levels 447.

In addition to tumour models, anti-angiogenic effect of 2ME has been reported in non-tumour tissues. In the mouse corneal micropocket assay, 2ME inhibited VEGF- and bFGF-induced neovascularisation by 54% and 39%, respectively 446. In the chick chorioallantoic assay, 2ME treatment results in the suppression of bFGF-induced angiogenesis 448. Chapter 1 Chapter

42

1.4.3. Potential of 2-methoxyestradiol as an Anti-cancer agent

1.4.3.1. Pre-clinical Studies

Preclinical studies (table 3.1) show that 2ME suppresses tumour growth and angiogenesis in a wide variety of human tumour xenograft including head and neck carcinoma 449, breast cancer 353, 450, sarcoma and B16 melanoma 419, and prostate cancer 451. In addition to primary tumours, 2ME is effective in controlling metastasis. Oral administration of low (25 mg/kg/day) and high (150-200 mg/kg/day) doses of 2ME reduce experimental lung metastasis by 70% and 90%, respectively 418. Similar results were obtained in mice model of lung metastasis originated from pancreatic cancer in which, a 60% reduction in the number of lung colonies was reported after 2ME treatment 452.

Chapter 1 Chapter

43

Table 1.3. Pre-clinical anti-tumour activity of 2ME

Tumour Tumour Inoculation Dose Vehicle Treatment Effect Ref cells Period Gastric cell 107 cells, i.p1. injection 200 mg/kg i.p. PBS2 Day 0,3,6 60-70%↓ metastasis foci in abdomen, 453 significant ↑ in survival UM-SCC-11A (head and 5x106 cells, s.c4. 150 mg/kg, i.p. Empty liposomes Days 8-12, 15- ~ 70% ↓ tumour volume, ~ 60% ↓ 449 neck squamous cells) 19, 22-24 tumour vessel formation MCF-7/ DOX 107 cells, s.c.3 30 mg/kg, orally propylene Twice a week for ~ 50% ↓ in tumour volume 454 (doxorubicin-resistant glycol:tetrahydrofuran two weeks breast cancer cells) 4T1 ( Breast cancer cells) 103 cells into mammary 10, 25, 50 mg/kg/day, 95% Ethanol Day 0-16 ~ 68% ↓ tumour volume (50 mg/kg) 450 fat pad and femur s.c. significant inhibition of bone metastasis (50mg/kg) A549 ( NSCLC ) 2x106 cells, tail vein 50 mg/kg, orally 20% DMSO4 in olive oil Day 5-21 ~ 42.3% ↓ lung colonies 455 Androgen dependent ? cells, 25 mg/kg, i.p. Olive oil 3 days ~ 47% ↓ tumour volume (2ME), 456 prostate cancer cells s.c. ~ 80% ↓ tumour volume (2ME+androgen withdrawl) MCF-7/ DOX 107 cells, s.c. 2-ME 30 mg/ kg, i.p. propylene Twice a week for ~ 95% ↓ tumour volume 457 (doxorubicin-resistant doxorubicin 5mg/kg glycol:tetrahydrofuran two weeks breast cancer cells) i.p. MIA PaCa-2 3x106 cells, tail vein 50 mg/kg, orally 2% DMSO in olive oil Day 3-21 60% ↓ lung metastasis colonies 452 (pancreatic tumour) Hela-S3 cell, cervical 3-4x106 cell, s.c. 75 mg/kg, orally DMSO in olive oil 15 days 34% ↓ tumour volume 458 carcinoma HEC-1-A (endometrial 106 cells, s.c. 100 mg/kg, orally Olive oil 18 days No difference in tumour volume between 459 carcinoma cells) control and treated group Met-A-Sarcoma cells 106 cells, s.c. 100 mg/kg, orally Olive oil Day 0-12 76% ↓ tumour weight 419 MCF-7 106 cells in 50 mg/kg, orally 10% DMSO & Day 0-16 92% of 2ME treated mice developed 460 (breast cancer cells) mammary fat pad 90% peanut oil tumours c.f. 33% of vehicle treated mice MDA-MB-435 106 cells s.c. 75 mg/kg orally 0.5% CMC5 Day 14-31 No effect on tumour volume or weight 460 (breast cancer cells) MDA-MB-435 106 cells in Increasing doses 15–150 10% DMSO & 18 days 47% ↑ in tumour volume and 43% ↑ 460 (breast cancer cells) mammary fat pad mg/kg/day i.p. 90% peanut oil tumour weight at 150 mg/kg dose Multiple myeloma 3x107 cells, s.c. 100 mg/kg/day, orally 0.5% CMC 11 days 35-40% ↓ tumour volume 460

1intraperitoneally, 2 Phosphate Buffered Saline, 3 subcutaneously, 4 Dimethyl Solfuxide, 5 Carboxy-methyl cellulose 44 Chapter 1

1.4.3.2. Clinical Studies

Promising results from pre-clinical studies led to a phase I clinical trial for the treatment of hormone refractory prostate cancer. Administration of 2ME (Panzem®) at a dose of 1.2 g/day resulted in stable disease as shown by prostate specific antigen (PSA) levels. Moreover, the majority of patients experienced stable levels of angiogenic factors bFGF and VEGF. Nevertheless, no measurable disease response was observed 174. In other trial, patients with solid tumours were administered 2ME at doses ranging from 400 to 3000 mg bid. Similar to the previous study, no partial response was reported. However, in one patient with ovarian cancer, a very promising result was observed 169. In a more recent trial, patients with metastatic breast cancer were treated 2ME at doses up to 1000 mg/day. Although there was no objective response, prolonged stable disease was reported in two patients and a complete response in one patient with bone metastasis that led to recalcification of lytic lesions 170. In a phase II clinical trials, daily oral administration of 1 g 2ME to patients with relapsed and plateau phase multiple myeloma led to a minor response and prolonged stable disease 173.

Pharmacokinetic studies revealed a large inter-patient variability, as well as extremely low plasma concentration 169. The latter may results from extensive first pass metabolism, as 80-90% of administered 2ME is converted to 2-methoxyestrone 174. Inter-patient variability has been attributed to poor bioavailability of 2ME as a result of its dissolution rate and limited absorption 461.

The continued development of 2ME led to its re-formulation in a nanocrystal colloidal dispersion (NCD). Treatment of patients with relapsed platinum resistent or refractory epithelial ovarian cancer with NCD formulation of 2ME resulted in a modest anti-tumour activity, with two patients experiencing clinical benefit for more than one year. However, despite improved bioavaiability, inter-patient variability was still a major problem 172.

As mentioned earlier, ENMD-1198 is a novel analogue of 2ME with increased cytotoxicity and anti-angiogenic activity, and improved metabolic stability and pharmacokinetics profiles 426. In a phase I clinical trial, ENMD-1198 was shown to 1Chapter 45

have a higher bioavailability compared to Panzem®. In addition, it prolonged stabilisation of disease in patients with pancreas, prostate and ovarian cancer 176.

1.4.3.3. Other Therapeutic Indications of 2-Methoxyestradiol

2ME has been shown to suppress the development of collagen type-II-induced arthritis through inhibition of angiogenesis and by diminishing the pro-inflammatory mediator, NO 462. In rat models of collagen-induced arthritis (CIA), 2ME treatment significantly delayed the onset of the disease and reduced the severity of clinical CIA 463. Additionally, due to its anti-mitogenic effect in human airway smooth muscle cells, 2ME is effective in treating airway wall structural changes in asthma that occurs in response to persistent inflammation 433. In mice model of allergic airway inflammation with subepithelial fibrosis, 2ME treatment resulted in a profound reduction in cellular infilteration of perivascular and peribronchial lung tissues, as well as a decrease in airway fibrosis 464.

1.4.4. Toxicity and Side Effects

In animal models, administration of therapeutic doses of 2ME does not induce hair loss and gastrointestinal disorders, or a decrease in circulating leukocytes in bone marrow and thymus 446, 462. A significant reduction in plasma cholesterol has been reported in several studies 465, 466. Body weight loss has been observed when 2ME is given at a dose of 150 mg/kg 446. However, the weight loss is not accompanied by other clinical signs of toxicity 418.

In clinical trials, 2ME (Panzem®) was well tolerated and consistent with pre-clinical studies, myelosupression and other haematological toxicities associated with MTAs were not observed 467. The most common side effects of 2ME were hot flashes, thrombosis and fluid retention 427. There were no dose-limiting toxicities, and MTD was not reached 169. For nanocrystalline (NCD) formulation of 2ME, reported adverse effects were fatigue, nausea, diarrhea, neuropathy, edema, and dyspnea 172. MTD of NCD formulation was determined to be 1g every 6 hours. Reported MTD for ENMD-1198 analogue was 425 mg/m2/day at which the main side effect was neutropenia. Similar to 2ME, ENMD-1198 did not induce myelosupression 176. 1Chapter 46

1.5. COMBINATION THERAPY

Combination therapy for the treatment or prevention of diseases is widely used in oncology and other areas of medicine. The main goals of combination therapy are to enhance efficacy, reduce dosage and toxicity, and reduce or delay the development of drug resistance 468.

Three possible interactions have been defined when drugs are used in combination. Pharmacodynamic interaction between drugs occurs when the agents cooperatively act on the same target 469. Drugs may also interact pharmacokinetically if an agent potentiates the absorption and distribution, and reduce the metabolism and excretion of the other agent 470. Another type of drug interaction is known as coalistic combination in which all involved drugs are individually inactive but become activated when used in combination 471-473.

During the past decades, attempts have been made to quantitatively measure the effect of drugs as a single agent and in combination. Several methods have been developed and extensively used for drug-combination analysis. By definition, synergy is "an effect which is greater than additive effect" and antagonism is "an effect that is less than additive effect" 474. However, in a review article published in 1995, 13 different methods for determination of synergy based on different principles were listed 471. Currently, the most widely used method is the median- effect analysis introduced by Chou and Talalay 475. This method was developed based on physiochemical principle of the mass-action law with mathematical principle of induction and deduction 470.

The median-effect equation is:

m fa § D · ¨ ¸ fu © Dm ¹

that defines the relationship between the dose and the effect of a given drug. fa is the fraction that is affected or inhibited; fu is the unaffected fraction; D is the drug dose;

Dm is the dose required to produce a 50% growth inhibition; and m is the coefficient signifying the shape of the dose-effect curve (m = 1, > 1, < 1 indicate hyperbolic, Chapter 1Chapter sigmoidal and flat sigmoidal dose effect curves, respectively). By using this 47

equation, dose and effect become interchangeable as the dose for any given effect can be determined if m and Dm values are known. The parameters of potency (Dm) and curve shape (m) are also defined.

Using the following equation that is derived from median-effect equation, the dose- response curve can be plotted.

§ f · ¨ a ¸ log¨ ¸ log uu log DmDm m © fu ¹

The median-effect equation for single drug can be extended to multiple drugs. Therefore, for the multiple drug-effect system the equation is:

(fa)1,2 / (fu)1,2 = (fa)1 / (fu)1 + (fa)2 / (fu)2 = (D)1 / (Dm)1 + (D)2 / (Dm)2

In order to quantify synergism or antagonism, combination index (CI) for two drugs is calculated using CI equation:

D D CI 1  2 Dx 1 Dx 2 where CI<1,=1, and >1 indicate synergism, additivity and antagonism, respectively.

In the above equation (D)1 and (D)2 are the doses of each drug as a single agent with x% growth inhibitory effect when they are used in combination, and (Dx)1 and (Dx)2 are the expected doses of each drug as a single agent that also results in a growth inhibition of x%. These doses are calculated from the median-effect equation based on the data obtained from dose-response curves of single drugs. In addition, the CI equation can be expanded to include more than two drugs using the following equation:

n n D j CI x ¦ j 1 Dx j

Median effect analysis is valid when all drugs in combination are active. If one drug

has no activity but it potentiates the cytotoxicity of another drug, the interaction is 1Chapter

48

known as enhancement or potentiation or augmentation and the median effect analysis cannot be applied as there will be no Dm and m to calculate the combination index 468, 470.

1.6. DRUG DELIVERY SYSTEMS

One of the main obstacles in early development of drugs is their low bioavailability. Bioavailability is defined as rate and extend to which a drug becomes available at the site of action. Two determinant factors in bioavailability of a drug are its aqueous solubility, which makes a drug pharmacologically active, and its permeability, that allows a drug to pass through biological membranes 476, 477.

Many of the pharmacological properties of drugs can be improved by alteration of their formulations. The key characteristics of any formulation are solubility and stability. In other words, the solvent should be able to solubilise the compound at the desired concentration and at the same time, maintain the chemical stability of the drug 478. Depending on the physicochemical properties of a drug and the biological target it intended to interact, various solubilising excipients can be employed. These include water-soluble organic solvents (polyethylene glycol, ethanol, glycrine, DMSO), water-insoluble lipids (castor oil, sesame oil, soybean oil), non-ionic surfactants (cremophor EL, polyethylene glycol, polysorbates), organic liquids (beeswax, oleic acids), phospholipids (distearoylphosphatidylglycerol, hydrogenated soy phosphatidylcholine), cyclodexrins (α-cyclodextrin, β-cyclodextrin, γ- cyclodextrin, hydroxypropyl-β-cyclodextrin, sulfobutylether- β-cyclodextrin), and pH modifiers 478.

The ideal drug formulation is an aqueous solution similar to the physiological fluids such as saline or 5% dextrose with a neutral pH. If the drug cannot be solubilised in saline, water, or dextrose at pH ׽ 7, the next choice is to increase the solubility by modifying the pH or adding a cosolvent 478. If this method is not sufficient, water- soluble organic solvent can be utilised alone or in combination with pH modification 479. If the drug cannot be solubilised by pH modification or in cosolvents, the next choice is to increase solubility by complexation. Chapter 1Chapter

49

The preferred complexing agents in drug delivery are cyclodextrins and their derivatives. Cyclodextrins (CDs) have been extensively used in pharmaceutical research and there are currently over 30 CD-containing pharmaceutical products worldwide 480, 481. CDs are cyclic (α-1,4)-linked oligosaccharides of glucopyranose units with a hydrophilic outer surface and a hydrophobic inner cavity 482. This structure allows them to interact with a large variety of guest compounds to form non-covalent inclusion complexes. α-, β-, and γ-CD are the most common CDs and their toxicity profile has been extensively investigated (reviewed by Antlsperger and Schmid, 1996 483). The toxicity of CDs is largely depends on the route of their administration. For instance, oral administration of β-CD induces limited toxicity in animals 484, 485, whereas its subcutaneous administration results in nephrotoxicity, a decrease in body and liver weight, an increase in kidney weight, and proximal tubular nephrosis and cellular vacuolation 486. Parenteral administration have similar effect on the kidney proximal tubules 487. Derivatives such as 2-hydroxypropyl-β-CD (HPβCD; Encapsin®) and sulphobutylether-β-CD (SEβCD; Captisol®) have therefore been synthesised to produce a less toxic and more water-soluble entities 488, 489.

Complexation with CDs results in improved solubility, dissolution, and permeability of the drug molecule. Furthermore, CDs increase the stability of compound by encapsulating the drug molecule and protecting it from degradation 490. Presence of drug/CD complex at the epithelial surface enhance the availability of dissolved drug molecules, especially for lipophilic drugs that have poor aqueous solubility 491. Studies have revealed that CDs increase oral bioavailability of the FDA’s Class II (poor solubility, high permeability) drugs. However, they may hamper bioavailability of Class III (high solubility, poor permeability) and Class I drugs (high solubility, high permeability) 480. Several studies suggested that the solubilisation of low water-solubilised drugs could be enhanced by the combination pH modification and cyclodextrin complexation 492, 493. This technique has been successfully employed for the solubilising nonsteroidal anti-inflammatory drugs (NSAIDs) 494-496.

Other promising method for drug delivery is nanotechnology. Nanotechnology is the science of engineering systems and materials on a molecular scale 497, and the idea is

to administer the nanoparticles into the bloodstream and allow them to pass through 1Chapter

50

the leaky blood vessels of tumours and release the drug inside the tumour 498. As a result, nanoparticles preferentially accumulate in tumours through the enhanced permeability and retention effect (EPR) 499 which can be translated into higher anti- tumour efficacy and lower toxicity. Nanoparticle drug delivery platform falls into the following categories: liposomes, nanoparticle albumin-bound technology (nab), metal nanoparticles, dendrimers, polymeric nanoparticles, and molecular targeted nanoparticles 497.

Liposomes are one of the first nanoparticle that were applied in medicine 500. They are spherical vesicles composed of a single or multiple bilayered membrane structures containing natural or synthetic lipids 501. Liposomal drug formulations improve the biodistribution and pharmacokinetics of a drug. For example, pegylated glycerol (PEG) doxorubicin increase intratumoural drug concentration while restricting the distribution of the drug in the plasma and normal tissues such as heart 502.

Nab technology utilises albumin as a carrier for the delivery of lipophilic chemotherapeutics. Albumin binds to glycoprotein receptor 60 (gp60) 503, thereby activating caveoli 504 that transports albumin across the cells to the interstitial space. Nab-paclitaxel is the first commercial drug based on the nab technology that has been developed 505 and approved for the treatment of breast cancer 506.

Metal nanoparticles consist of inert metals such as titanium or gold, which have been used for control released of chemotherapeutic agents 507. Although biocompatible, a significant fraction of the metal particles can be accumulated in the body after repeated administration, leading to toxicity 497.

Dendrimers are synthesised from synthetic or natural elements such as sugars, nucleotides, or amino acids. The cavities in dendrimers cores could be loaded with drugs by hydrogen bonds, hydrophobic interactions, or chemical linkages 508.

Polymeric nanoparticles have been extensively investigated as drug carriers 509. Synthesised from biocompatible and biodegradable polymers, these nanoparticles have been formulated to encapsulate hydrophobic and hydrophilic small drug molecules or macromolecules such as nucleic acids and proteins 510. Chapter 1Chapter

51

Molecular targeted nanoparticles have also shown promising clinical results that have eluded conventional chemotherapy 511-515. These include trastuzumab for the treatment of human epidermal growth factor receptor 2 (HER-2)-positive breast cancer 511, imatinib for the treatment of chronic myelogenous leukemia 515, and bevazicumb for the treatment of colorectal and renal cancers 512, 513. Currently, several molecular targeted nanoparticles such as BIND-014, a nanoparticle of docetaxel with controlled-released property, are under investigation in clinical trials 516.

1.7. AIMS AND HYPOTHESIS

The concept that ABZ represented a potent cytotoxic and anti-angiogenic agent with an excellent safety profile, underpinned its development for cancer treatment. This project was therefore designed to improve the efficacy of ABZ as an anti-cancer agent. To achieve this goal, two approaches were employed. The first approach was to re-formulate ABZ with the aim of improving its aqueous solubility and therefore, its pharmacokinetic profile and efficacy. The second approach was to develop a novel therapeutic strategy using combination therapy.

ABZ is a molecule with basic functional groups which is amenable to ionisation in acidic medium. Additionally, it possesses heterocyclic aromantic rings that could be readily complexed with HPβCD. Therefore, it is hypothesised that these two properties of the molecule, if utilised correctly will solubilise the compound. Thus, the first aim of this thesis is to investigate the effect of the combination of ionisation and complexation on the solubility and anti-tumour efficacy of ABZ.

Combination therapy is the main strategy to improve the efficacy of chemotherapeutic regimens. The traditional approach for introducing new agents into cancer therapy is to add the drug to an established cytotoxic agent. MTAs has been shown to have synergistic interaction with one another 3. Therefore, the interactions between ABZ and three MTAs that differ in their binding sites on β- tubulin were examined. Moreover, the combinations of ABZ with three antiparasitic agents, ivermectin, diethylcarbamazine and praziquantel, were evaluated. These 517-519 compounds synergise with ABZ in the treatment of nemathodes . 1Chapter

52

Among the above compounds, a synergistic interaction between ABZ and 2ME was observed. Therefore, the mechanisms underlying the synergistic interaction between the two agents in in vitro and in vivo models were investigated. Chapter 1Chapter

53

Chapter 2 Materials and Methods

2.1. MATERIALS

2.1.1. Reagents

Table 2.1.List of reagents

Reagent Supplier

2-Hydroxypropyl beta Cyclodextrin CTD. Inc, Florida, USA

2-Methoxyestradiol Sigma-Aldrich, Australia

3,3′-Diaminobenzidine tetrahydrochloride tablet Sigma-Aldrich, Australia

Acetonitrile Sigma-Aldrich, Australia

Acrylamide/ Bis solution 37.5:1 (30% w/v) Bio-Rad, USA

Albendazole Sigma-Aldrich, Australia

Albendazole Sulfone GlaxoSmithKline

Albendazole Sulfoxide GaxoSmithKline.

Albumin from Bovine Serum – Fatty acid free Sigma-Aldrich, Australia

Ammonium persulfate Sigma-Aldrich, Australia

Bovine serum albumin Sigma-Aldrich, Australia

Bromophenol Blue Sigma-Aldrich, Australia

Carboxymethyl methyl cellulose (CMC) Sigma-Aldrich, Australia

Citric Acid Sigma-Aldrich, Australia

Colchicine Sigma-Aldrich, Australia

Combretastatine A4 Sigma-Aldrich, Australia

Diethylcarbamazine Sigma-Aldrich, Australia

EGM Bulletkit Lonza, Australia Pty Ltd

Ethanol (HPLC Grade) Sigma-Aldrich, Australia ETHYLENE GLYCOL-BIS(2-AMINOETHYLETHER)- Sigma-Aldrich, Australia N,N,N′,N′-TETRA ACETIC ACID Fetal Bovine Serum (FBS) Sigma-Aldrich, Australia

Formalin Sigma-Aldrich, Australia

2Chapter 55

Table 2.1.Continued

Reagent Supplier Sodium Chloride Solution (0.9%) Baxter, USA Sodium Citrate Sigma-Aldrich, Australia Sodium dodecylsulfate (SDS) Sigma-Aldrich, Australia Sodium Hydroxide Sigma-Aldrich, Australia Streptomycin Gibco, Invitrogen, Australia Sulforhodamine B (SRB) Sigma-Aldrich, Australia Sulforic acid Sigma-Aldrich, Australia Tetramethylethylenediamine Sigma-Aldrich, Australia Ticholoroacetic acid Sigma-Aldrich, Australia Tris base Sigma-Aldrich, Australia Trypan Blue Sigma-Aldrich, Australia Trypsin-EDTA Solution Gibco, Invitrogen, Australia Tween 20 Sigma-Aldrich, Australia Western Lightning enhanced chemiluminescence GE Healthcare, UK X-Ray Film GE Healthcare, UK Xylene Sigma-Aldrich, Australia

Chapter 2Chapter

56

2.1.2. Assay Kits

Table 2.2. List of assay kits

Assay Kits Supplier

Caspase 3 colorimetric Assay Kit R & D System, USA

Caspase 8 colorimetric Assay Kit R & D System, USA

Caspase 9 colorimetric Assay Kit R & D System, USA

Human VEGF Duo Set Kit R & D System, USA

Human VEGF Quantikine ELISA Kit R & D System, USA

Protein Assay Kit Bio-Rad, USA

TUNEL Assay Kit Calbiochem, Australia

2.1.3. Antibodies

Table 2.3. List of Antibodies

Antibody Isotype Clone No. Supplier

Acetylated α - tubulin Mouse IgG2b 6-11B-1 Sigma-Aldrich, Australia Anti-mouse IgG/HRP - DAKO, Australia

Anti-rat IgG/HRP - DAKO, Australia

CD31 Rat IgG2a κ MEC-13.3 BD Bioscience, Australia

Death Receptor 5 Goat IgG R & D System, USA

Donkey-anti goat Ab - Santa Cruz Biotechnology, USA

HIF-1α Rabbit IgG H-206 Santa Cruz Biotechnology, USA

Ki67 Mouse IgG1 κ MIB-1 DAKO, Australia

P53 Rabbit IgG C-11 Santa Cruz Biotechnology, USA

VEGF Mouse IgG2a C-1 Santa Cruz Biotechnology, USA

α-Tubuin Mouse IgG1 DMA1 Sigma-Aldrich, Australia β-actin Mouse IgG1 AC-15 Sigma-Aldrich, Australia Chapter 2Chapter

57

2.2. METHODS

2.2.1. Drug Preparation

2.2.1.1. In Vitro Experiment

ABZ, 2ME, colchicine (CLC), and diethylcarbamazine (DEC) were stored at room temperature. PTX and ivermectin (IVE) were stored at 4ºC and CA4, vinblastine (VBL) and praziquantel (PZQ) were stored at -20ºC. All drugs were solubilised in absolute ethanol.

2.2.1.2. In Vivo Experiment

2.2.1.2.1. ABZ formulations

0.5 ml glacial acetic acid was added to 30 mg ABZ and the mixture was vortexed vigorously until ABZ completely dissolved. The solution was then added to 13.5 ml hydroxypropyl-β-cyclodextrin (HPβCD) (25% w/v in dH2O) and the pH was adjusted to 7.4 by addition of sodium hydroxide (NaOH). The solution was homogenous and no precipitation was observed.

To prepare the suspension formulation, ABZ suspended in sterile 0.9% sodium chloride containing 0.5% (w/v) hydroxypropyl methylcellulose (HPMC), yielding a final concentrations of 2 and 3 mg/ml. The suspension was then stirred for 24 hours at room temperature. ABZ/HPMC was vigorously shaken before the administration to the mice. For efficacy experiment, ABZ/HPMC was freshly prepared every second day.

2.2.1.2.2. 2ME formulation

2ME was solubilised in 25% HPβCD containing 0.5% (w/v) carboxymethyl cellulose (CMC) at a concentration of 1 mg/ml. After 3-day storage at 4ºC, 2ME was dissolved and no precipitation was observed. Chapter 2Chapter

58

Prior to evaluating in vivo, cytotoxicity of the formulation was compared to 2ME solubilized in ethanol and evaluated against HCT-116 colorectal cancer cells and DU145 prostate cancer cell line. In addition, the toxicity of the drug-free CMC/HPβCD solutions (vehicle) was assessed.

2.2.2. Cell Culture

HCT-116 and DU145 cells were maintained in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and 50 units/ml streptomycin. HUVECs (Invitrogen) were grown in a 1:1 mixture of M199 and Endothelial Cell Growth Medium supplemented (ECM) with bovine brain Extract, heparin, human epithelial growth factor (hEGF), Hydrocortisone, Gentamicin, Amphotericin B and 10% fetal bovine serum (FBS). The cells were incubated at

37°C in a 5% CO2 humidified incubator.

2.2.3. Cell viability assay

The percentage of viable cells was assessed prior to each experiment using 0.4% (w/v) trypan blue dye exclusion. After trypsinisation, cells were mixed with an equal volume of trypan blue solution and counted by hemocytometer. The percentage of viable cells was determined as follows:

number of viable cells (cell which did not stain) % viability total number of cells

For all in vitro and in vivo experiments, cells with >90% viability were used.

2.2.4. The Sulforhodamine B (SRB) Assay

Cell proliferation assay was performed in 96-well microtiter plates using the Sulforhodamine B (SRB) colorimetric assay 520. At the end of the treatment period, cells were fixed with 10% ice-cold trichloroacetic acid (TCA) for 30 minutes on ice followed by five washes with tap water. Cells were then stained for 15 minutes with

0.4% (w/v) SRB dissolved in 1% acetic acid, and washed five times with 1% acetic 2Chapter

59

acid. The plates were allowed to dry, SRB dye was solubilised in 10 mM Tris base (pH 7.4), and optical density (OD) value was determined at 570 nm using microplate reader. OD was calculated by subtracting the blank value from the absorbance of each well (blank is the mean OD of the background control wells). Cell viability was calculated using the formula:

OD of treated cells u100 OD of control

The IC50 value represented the concentration of the drug that is required to inhibit the proliferation of 50% of the cells compared with non-treated cells.

2.2.5. Cytotoxicity Assay

2.2.5.1. Single agent treatment

To determine the effect of the drugs on cell proliferation, HCT-116 and DU-145 cells were seeded at a density of 2500 and 3500 cells/well, respectively, in 96-well plates in triplicate. After 24 hours incubation, the medium was replaced with drug containing medium. Cytotoxic drugs were ABZ (concentration range 0.01-10 μM), PTX (concentration range 0.5-10 nM), VBL (concentration range 0.5-10 nM), 2ME (concentration range 0.01-100 μM), CLC (concentration range 1-50 nM) and CA4 (concentration range 0.05-1 nM), DEC (concentration range 0.1-100 μM), PZQ (concentration range 0.1-100 μM), and IVE (concentration range 0.1-100 μM). Since the stock solution of the drugs were prepared in ethanol, all cells including untreated control were incubated in medium containing 1% ethanol to ensure that ethanol would not mask the effect of the drugs. Cells were incubated with the drugs for 72 hours and then subjected to SRB assay. The IC50 values were calculated using GraphPad prism software.

2.2.5.2. Combination treatment

To assess the combined effect of ABZ with other compounds, cells were plated in

96-well plates as described above. Each drug was assayed alone and in combination 2Chapter

60

at a non-constant ratio. All cells including untreated control were incubated in medium containing 2% ethanol to ensure that ethanol would not confound the growth inhibition.

Combination of ABZ and 2ME was performed using the following schedules:

1. Simultaneous treatment for 24 hours 2. Simultaneous treatment for 72 hours 3. Treatment with ABZ for 24 hours, followed by 2ME treatment for further 24 hours 4. Treatment with ABZ for 24 hours, followed by 2ME treatment for further 48 hours 5. Treatment with 2ME for 24 hours, followed by ABZ treatment for further 24 hours 6. Treatment with 2ME for 24 hours, followed by ABZ treatment for further 48 hours. For other drugs in combination, 72 hours simultaneous treatment was carried out.

2.2.5.3. Drug Interaction Study

The interaction between drugs in combination was determined using CalcuSyn software from Biosoft (Cambridge, UK).

Following proliferation assay, the fraction of growth inhibition, defined as fraction affected (ƒa), was determined using the following formula:

ƒa = 1 - (Absorbance treated wells/Absorbance untreated wells)

The ƒa values of 0, 0.25, 0.5, 0.75 and 1 correspond to cell viability of 100%, 75%,

50%, 25% and 0%. ). ƒa and drug doses were then entered into CalcuSyn program that automatically calculates the combination index (CI) using Chou and Talalay method 475, 521. Briefly, Dm is the drug concentration required to produce the median effect, which is analogous to the IC50. Log ƒa / ƒu (ƒu : fraction unaffected) was plotted against log (D/Dm) and from the resulting median effect curves, the x- intercept and the slope (m) were calculated for individual drugs and for the Chapter 2Chapter combination. These parameters were used to calculate the concentration of drugs and 61

the combination to produce different levels of cytotoxicity (ƒa) using the following equation:

1 m ª f a º DD m « » ¬ 1 f a ¼

(DX) was then used to calculate the combination index (CI). CI is a parameter that indicates whether the doses of two drugs required to produce a given effect are greater than, (CI>1), equal to (CI=1) or less than (CI<1) the doses that are required if the interaction of the two drugs is additive. Therefore, CI<1, CI>1 and CI=1 indicates synergism, antagonism and additive effect, respectively.

CalcuSyn automatically calculates the CI of each combination using the following formula:

D D CI 1  2 Dx 1 Dx 2

where (D)1 and (D)2 are doses of the drugs 1 and 2 in combination that produce a given effect, and (DX)1 and (DX)2 are the doses of drug 1 and drug 2, respectively, which also produce the same effect when used individually. α is equal to 0 or 1, depends on whether the drugs are assumed to be mutually exclusive (have the same or similar mechanism of action) or mutually non-exclusive (have totally independent mode of action). However, it has been proven that there is no significant difference between the CI values calculated in the two methods. Therefore, the last version of CalcuSyn has been designed to calculate the CI using mutually non-exclusive equation.

2.2.6. Enzyme-Linked Immunosorbent Assays

HCT-116 were plated at a density of 14000 cells/well in 96-well plates and incubated in RPMI medium supplemented with 10% FBS. After 24 hours incubation, cells were treated with various concentrations of ABZ, 2ME and their combination in serum-free medium and incubated for 16 hours. The plates were then incubated for an additional 6 hours in incubator in normoxia, or placed in hypoxia chamber 2Chapter 62

(Billups-Rothenburg, Del Mar, CA) in 1% O2, 5% CO2, and 94% N2. The supernatants were then collected and stored at -80 ºC and the cells were subjected to SRB assay.

The concentration of VEGF in conditioned medium was determined using Human VEGF Duo Set Kit. Briefly, the wells were coated with capture antibody (1 μg/ml) in phosphate buffered saline (PBS; pH 7.4) by incubation overnight at room temperature. After five washes with wash buffer (0.05% tween 20 in PBS), the wells were blocked with 2% BSA in PBS and the plate was incubated for 1 hour at room temperature. After a wash step, standards and samples were added to each wells and the plate was incubated at room temperature for 2 hours, followed by three washes. Detection antibody (100 ng/ml) was then added to each well and the plate incubated for 2 hours at room temperature. The plate was washed and Streptavidine-HRP was added to each well. The plate was incubated at room temperature for 20 minutes, and substrate solution was then added to each well. The plate was incubated at room temperature for 20 minutes and the reaction was stopped using 2N sulfuric acid. The optical density was measured at 450 nm by microplate reader.

To measure the concentrations of VEGF in mice plasma, Human VEGF Quantikine kit from R&D Diagnostics was used according to the manufacturer’s instructions. Briefly, plasma samples and standards were diluted with assay diluent (1:1) in each well. The plate was incubated at room temperature for 2 hours, followed by 3 washes with wash buffer (buffered surfactant). After aspiration of wash buffer, 200 μl of VEGF conjugate (polyclonal antibody against VEGF conjugated to horseradish peroxidase) was added to each well. After 2 hours incubation at room temperature, the plate was washed three times and 200 μl of substrate solution (hydrogen peroxide and tetramethylbenzidine as chromogen) was added to each well. The plate was incubated in the dark at room temperature and for 25 minutes and then stop solution (2N sulfuric acid) was added to each well. The optical density was determined using a microplate reader at 450 nm. Chapter 2Chapter

63

2.2.7. Western Blotting

5 × 106 HCT-116 cells were seeded in 75 cm2 culture flasks and cultured for 24 hours. Cells were then treated with ABZ, 2ME or both simultaneously for 24 hours. At the end of the treatment period, the culture medium was discarded and the cells were washed twice with ice-cold PBS. The cells were lysed using 100 μl RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Trizma base pH 7.5) supplemented with 10% protease inhibitor cocktail. After 10 minutes incubation on ice, the cells were scrapped and transferred to a microfuge tube and stored at -80ºC. Prior to performing the protein assay, the tubes were centrifuged at 10,000 g at 4ºC for 10 minutes and the supernatant containing proteins were transferred to a separate tube.

To generate the lysate from tumour tissue, 100 mg tissue was cut into small pieces and homogenized using tissue homogeniser in RIPA buffer containing 10% protease inhibitor cocktail. The samples were then centrifuged at 20,000g at 4ºC for 20 minutes. Supernatants containing protein were transferred to a new tube and stored at -80ºC.

Protein concentrations were quantified using Bio-Rad protein assay kit as per manufacturer’s instruction. 50 μg proteins were mixed with 4× protein loading buffer and heated for 5 minutes at 95ºC. Samples were resolved on 10% (HIF-1α), and 12% gels (VEGF, α-tubulin, acetylated α-tubulin, DR5, P53) and electrophoresed using running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) for 2 hours at 80V. Proteins were then transferred to a polyvinylidene fluoride membrane (PVDF) using transfer buffer (25 mM Tris, 20% methanol) overnight at 30V at 4ºC, or for 2 hours at 60V. The membranes were then stained with 0.5% ponceau S solution to confirm transfer efficiency and equal loading of the proteins. The membranes were then blocked in 5% (w/v) skim milk or BSA (acetylated α- tubulin) in tris-buffer saline containing 0.05% Tween 20 (TBST) for 1 hour at room temperature, followed by incubation with primary antibody overnight at 4ºC (acetylated α-tubulin and DR5) or for 2 hours at room temperature. After four washes with TBST, the membrane was incubated with secondary antibody in TBST for 1 hour. The membranes were then washed six times with TBST and the bands Chapter 2Chapter were visualised by an enhanced chemiluminescence detection kit. The blot was then 64

stripped using Seppro western blot stripping buffer and re-probed with β-actin.

2.2.8. Tubulin Polymerisation Assay

Quantitative tubulin polymerisation assay was performed as previously described 522, 523. HCT-116 cells were plated at a density of 6×105 cells in six-well plates and incubated for 24 hours. The cells were treated with ABZ, 2ME and the combination of the two agents. After 24 hours incubation with the drugs, the wells were washed twice with warm PBS and the cells were lysed at 37ºC for 5 minutes in dark using

480 μl of hypotonic buffer (20 mM Tris, pH 6.8, 1 mM MgCl2, 2 mM EGTA, 0.5% Nonidet P-40, 2 mM PMSF, 10% (v/v) Protease inhibitor Cocktaile). The wells were then scraped and the lysates were transferred to microfuge tubes. Samples were centrifuged at 20,000 g for 10 minutes at room temperature. The supernatants containing soluble tubulin (cytosolic) were transferred to a new tube and the pellets (cytoskeletal or polymerised tubulin) were resuspended and in 480 μl of hypotonic buffer. Samples were mixed with 4× sample buffer (45% glycerol [v/v], 9.2% SDS [w/v], 0.3 M Tris pH 6.8 [v/v], 0.04%, bromophenol blue [w/v], 20% β- mercaptoethanol [v/v]), sonicated using Microson Ultrasonic Homogeniser and heated for 5 minutes at 95ºC. 15 μl of each sample was analysed on a 12% resolving gel. Electro-transfer and immunoblotting were performed as described in section 4.3.2.7. Membranes were incubated with anti-α-tubulin or acetylated anti-α-tubulin antibody followed by probing with anti-mouse HRP-linked secondary antibody. To quantify tubulin polymerisation, percentage of polymerized tubulin (%P) was calculated by dividing the densitometric value of polymerized tubulin by total tubulin content (polymerized fraction + soluble fraction).

2.2.9. Caspase Activity

To detect the activation of caspase 3, caspase 8, and caspase 9 in HCT-116 cells, colorimetric assay kits were used according to the manufacturer’s instructions. Cells (3 × 106) were incubated with vehicle, ABZ, 2ME or the combination for 24 hours, then ice cold cell lysis buffer (supplied with the kits) was added to the cells (25 μl 6 per 10 cells), and the mixture was incubated on ice for 10 minutes. Cells were then 2Chapter

65

centrifuged at 10,000 g for 1 minute at 4°C and the supernatant was transferred to a separate microfuge tube. Protein concentration in the supernatant was quantified using a Bio-Rad protein assay kit. The supernatants containing 100 μg of protein were transferred to a 96-well microplate, and lysis buffer was added to each well to make a total volume of 50 μl. Fifty microliters of 2× reaction buffer containing 1,4- DTT at a final concentration of 10 mM and caspase substrates (DEVD-pNA for caspase-3, IETD- pNA for caspase-8, and LEHD-pNA for caspase 9) were added to each well of the microplate. The microplate was incubated at 37°C for 4 hours, and the cleavage of the chomophore pNA from the caspase substrates was measured by reading the absorbance in each well at 405 nm in a microtiter plate reader. Negative controls were generated by omitting the substrates. Each sample was tested in duplicate, and the assays were repeated three times.

2.2.10. Mice

Ten-week-old female nude mice obtained from The Animal Resources Centre (Perth, Australia) and housed in a pathogen free environment for one week before the commencement of experiments. All experiments were conducted according to protocols approved by the Animal Experimentation Ethics Committee of the University of New South Wales. At the end of the experiment, mice were euthanised by an overdose of Lethabarb.

2.2.11. Evaluation of ABZ formulations on tumour growth

HCT-116 cells were harvested using 1% trypsin-EDTA and a single-cell suspension of 2x106 cells in 0.1 ml of matrigel were injected subcutaneously into the flank of the animals. When the tumours had grown to approximately 100 mm3, mice were randomised into three groups of 8 animals. Group 1 received ABZ/HPβCD vehicle, group 2 were treated with 50 mg/kg ABZ/HPβCD and group 3 were administered 150 mg/kg ABZ/HPMC. All groups received the drugs intraperitoneally every second day. Tumour volume was measured every three days using the formula: (shortest diameter)2 × longest diameter × 0.5. Animals were euthanised when the 3 tumour size reached 1000 mm . 2Chapter

66

2.2.12. Pharmacokinetics Study

Seventy-two mice were divided into fourteen groups of 5 animals and one group of two animals. Six groups received a single dose of ABZ/HPβCD. A similar groups of mice were administered ABZ/HPMC. Of the two remaining groups, one received the vehicle of ABZ/HPβCD and the other received the vehicle of ABZ/ HPMC. Two mice received no treatment (blank). The drugs were given intraperitoneally at a dose of 50 mg/kg. Animals were euthanised at ½, 1, 2, 4, 8, and 24 hours post drug administration and vehicle groups were euthanised two hours after treatment. Blood samples were taken by cardiac puncture and the samples were centrifuged at 5000 g for 10 minutes at 4qC. Plasma samples were stored at -80qC for HPLC analysis.

2.2.13. High Performance Liquid Chromatography (HPLC)

2.2.13.1. Apparatus and Chromatographic Conditions

The Shimadzu HPLC system consisted of a LC-20AD Pump, SIL-20A autosampler,

CBM-20A system controller and a RF-10AXL fluorescence detector. The chromatography was carried out using a Phenomenex Luna C18 column (3 μm, 150 x 3 mm). Mobile phase A contained of 15% acetonitrile, 0.01 M phosphoric acid, 5 mM tetrabutylammonium hydrogen sulfate and 85% water (v/v/w/v). Mobile phase B consisted of 30% acetonitrile, 0.01 M phosphoric acid, 5 mM tetrabutylammonium hydrogen sulfate and 70% water (v/v/w/v). A gradient mobile phase initiated with 100% mobile phase A using a linear gradient to mobile phase B at 3.01 min. The gradient remained constant for 4 minutes at which time, it was changed linearly to mobile phase A at 7.01 min until the end of the run. The run time for each sample was 15 min and the flow rate was 1.0 ml/min. Under these conditions, retention times of ABZ-SO, ABZ-SO2 and ABZ were 6.1, 9.3, and 10.6 min respectively. All data were recorded and analysed using Class-VP Chromatography Data System Software. Chapter 2Chapter

67

2.2.13.2. HPLC Standards

Stock solutions of ABZ, ABZSO and ABZSO2 were prepared in acidified methanol. The concentrations of the stock solutions were 100 μg/ml, 500 μg/ml and 1 mg/ml for ABZ-SO, 10 μg/ml, 100 μg/ml and 1 mg/ml for ABZSO2 and 10 μg/ml and 100 μg/ml for ABZ. Working standard solutions were prepared in human plasma at the concentrations of 0.1, 0.5, 1, 10, 25 and 50 μg/ml for ABZSO, 0.05, 0.5, 1, 5, 10, 25 and 50 μg/ml for ABZSO2 and 0.01, 0.05, 0.1, 0.2, 0.5, 1, and 2 μg/ml for ABZ. Samples with concentrations greater than the highest standards were diluted or re- injected at lower volume. Calibration curves were prepared with each set of sample.

2.2.13.3. Quality Controls

To ensure that the method is accurate and precise, quality control samples (QCs) for intra- and inter- day assays were analysed with each run. The QC samples were prepared in human plasma at concentrations of 0.2, 2.5 and 40 μg/ml for ABZSO,

0.1, 2.5 and 40 μg/ml for ABZSO2 and 0.02, 0.15 and 1.5 μg/ml for ABZ.

Precision and accuracy were evaluated by analysing the standard curves and three samples at each concentration level (low, medium and high) for each metabolite. In order to assess the precision of the method, the coefficient of variation of each tested concentration was calculated. To determine the accuracy of the method, the measured concentration of each QC was compared with its nominal values.

2.2.13.4. Validation of the method

The precision of the method was evaluated using the coefficient of variation for each sample (CV). The accuracy was calculated by comparison between the actual value of the QCs and the values that were determined by the method.

2.2.13.5. Plasma extraction

To 150 μl of plasma, 100 μl of sodium metabisulfate and 3 ml ethyl acetate were added. After a brief vortex, the samples were mixed for 20 minutes and the tubes Chapter 2Chapter were then centrifuged at 1800 rpm for 5 minutes. Supernatants were transferred to 68

new tubes and evaporated to dryness using thermo savant rotary vacuum chamber consisting of SC210A Speed Vac Plus, RVT4140 Refrigerated Vapor Trap and VLP200 ValuPump (Thermo Electron Corporation, Melbourne, Australia). The extracts were resuspended in 200 μl of mobile phase containing 15% acetonitrile, 0.01 M phosphoric acid and 5 mM tetrabutylammonium hydrogen sulfate.

2.2.14. Toxicity Evaluation

In an initial pilot study, five mice were administered with 5, 10, 25, 40 and 50 mg/kg ABZ/HPβCD for 28 consecutive days. In addition, two animals received the drug- free vehicles of 50 and 60 mg/kg ABZ/HPβCD. The body weight and general clinical status of the animals were recorded daily and considered as a surrogate for evaluation of general wellbeing.

2.2.15. In Vivo Combination Study

2.2.15.1. Pilot Studies

A pilot study was carried to evaluate the possible toxicity of the combination of ABZ and 2ME when administered simultaneously. There were two groups of 4 animals. Group 1 received the combination of 50 mg/ kg ABZ and 25 mg/kg 2ME and group 1 were given the combination of the two vehicles.

Another initial experiment using various doses of ABZ and 2ME was performed to determine the most effective combination doses. Four different combinations were evaluated as followed:

1. Vehicle 2. 50 mg/ kg ABZ + 25 mg/kg 2ME 3. 50 mg/ kg ABZ + 10 mg/kg 2ME 4. 25 mg/ kg ABZ + 25 mg/kg 2ME 5. 25 mg/ kg ABZ + 10 mg/kg 2ME

Chapter 2Chapter

69

There were 4 animals in each group. HCT-116 cells (2 × 106) in 100 μl matrigel were inoculated subcutaneously. When the tumour size reached 100 mm3, animals were treated with ABZ on day 1 followed by 2ME 24 h later. After 21 days treatment, the mice were euthanized by an overdose of Lethabarb.

2.2.15.2. Combination Therapy

HCT-116 cells (2 × 106) in 100 μl matrigel were inoculated subcutaneously into the hind legs of 10-week-old female nude mice (n=60). All animals developed palpable tumours by day 6 after tumour cell injection. When the subcutaneous tumours had reached 100mm3, mice were randomly assigned to the various treatment groups as followed:

1. Vehicle 2. 50 mg/kg ABZ 3. 25 mg/kg ABZ 4. 25 mg/kg 2ME 5. 50 mg/kg ABZ+25 mg/kg 2ME 6. 25 mg/kg ABZ+25 mg/kg 2ME

Animals were administered drugs intraperitoneally every second day. For combination experiment, ABZ was administered on day 1 followed by 2ME 24 h later (day 2). To assess the effect of individual drugs, animals were treated with ABZ on day 1 and the vehicle of 2ME on day 2, or ABZ vehicle on day 1 and 2ME on day 2. Control animals received the vehicles. Mice were euthanised when the tumour size reached 1000 mm3 and the tumours were excised and processed by immunohistochemistry for CD31 (microvessel density), Ki67 (proliferation rate) and apoptosis (TUNEL).

2.2.16. Immunohistochemistry

For all immunohistochemistry experiments, 6 samples from each treatment group and 5 slides from each sample were prepared. The average number of positive cells/area was calculated from 5 fields of each section. Chapter 2Chapter

70

2.2.16.1. Proliferation Rate

Tumour tissues were excised, fixed in 10% formalin, embedded in paraffin and 5 μm sections were prepared. The sections were de-paraffinised in xylene and immersed in graded ethanol (100%, 90%, 80% and 70%). After rehydration with dH2O, sections were boiled in 0.01 M sodiun citrate buffer (pH 6) for antigen retrieval, washed in tris buffer saline (TBS) and blocked by 3% H2O2 for endogenous peroxidase activity. Sections were then washed with TBS and non-specific binding sites were blocked by 10% skim milk in TBS. The slides were rinsed in TBS and incubated with mouse anti-human Ki67 antibody (1:50 dilution in TBS). After 2 hours incubation at 37°C, the sections were washed with TBS and incubated with rabbit anti-mouse/HRP (1:100 dilution in TBS) for 45 minutes at room temperature. Sections were washed and then incubated with 3,3′-Diaminobenzidine tetrahydrochloride tablet, dissolved in TBS containing 0.03% H2O2, followed by counterstaining with hematoxylin. The slides were then rinsed with tap water and mounted with glycerol gelatin. For negative control, dH2O was substituted for primary antibody.

2.2.16.2. Apoptosis

Apoptosis was detected in tumour tissues using a colorimetric TUNEL assay kit. Paraffin-embedded sections were de-paraffinized and rehydrated as described in section 2.2.17.1. The specimens were permeabilised by proteinase K, and washed with TBS. Endogenous peroxidase activity was blocked by incubating the sections with 3% H2O2. Sections were washed and incubated with TdT equilibrium buffer for 30 minutes. The slides were then incubated with TdT labeling reaction mixture for 1.5 hour at 37°C, followed by incubation with stop solution for 5 minutes at room temperature. After rinsing with TBS, the slides were covered with blocking buffer for 10 minutes and incubated with conjugate for 10 minutes. Sections were stained with DAB, washed and counterstained with methyl green. Sections were then dehydrated and mounted using permount. Positive control was generated by covering the specimen with 1 μg/μl DNase I in TBS/1 mM MgSO4 for 20 minutes following proteinase K treatment. For negative control, the exact procedure was done with the omission of TdT reaction mixture. Chapter 2Chapter

71

2.2.16.3. Microvessel Density

For CD31 staining, tumour tissues were snap frozen in liquid nitrogen and frozen sections (5 μm) were prepared and then fixed in cold acetone for 10 minutes at - 20ºC. After washing with TBS, endogenous peroxidase activity was blocked by 3%

H2O2 in TBS for 10 minutes. The sections were washed and incubated with 10% skim milk in TBS for 15 minutes. Slides were then rinsed and incubated with rat anti-mouse CD31 antibody (1:25 dilution in TBS) for 1 hour. Sections were washed again and rabbit anti-rat IgG/HRP (1:100 dilution in TBS) was applied. Following 45 minutes incubation and washing, sections were stained with DAB substrate solution, washed with TBS and counterstained with hematoxilyn. After rinsing the slides with tap water, they were mounted and CD31-positive vessels were analysed at ×200 magnification.

2.2.17. Statistical analysis

Data are presented as the mean ± SEM. All statistical analyses were performed using the GraphPad Prism software package version 5.0 (GraphPad Software Inc., San Diego, CA, USA). The survival days were determined by the Kaplan-Meier method and compared by the log-rank test. P values < 0.05 were considered to be significant. Differences between groups were evaluated using Student’s t-test and one-way ANOVA.

Chapter 2Chapter

72

Chapter 3

ComparisonComparison ofof PharmacokineticsPharmacokinetics aandnd AAntinti-ttumourumour EEfficacyfficacy ofof TwoTwo FFormulationsormulations ooff AAlbendazolelbendazole iinn NNudeude MiceMice

3.1. INTRODUCTION

ABZ has very low water solubility (0.2 μg/ml), allowing its preparation only as a suspension 524. It has been shown that the oral administration of suspension formulation displays a large difference in absorption and elimination in humans 525. This discrepancy is mainly due to the low aqueous solubility of the drug. Furthermore, the absorption of ABZ through gastrointestinal tract has been reported to be less than 5% 526. This property represents a problem in treating systemic diseases, as high doses and continuous administration of the drug is required to reach optimal plasma concentrations 527, 528.

In recent years, numerous attempts have been made to improve ABZ formulation and thereby enhancing its efficacy. These include the use of soybean oil emulsion 529, surfactants 530, liposomes 531, polyvinylpyrrolidone 532, 533 ionization by acids 386, 534, 535, complexation with cyclodextrins 524, 536 or a combination of these methods 388, 537, 538.

Cyclodextrins (CDs) are cyclic oligosaccharides composed of α-1-4-linked glucose units 539. The most common applications of CDs are to increase the solubility, safety, and the stability of the drugs 540. In aqueous solutions, CDs form inclusion complex with lipophilic drugs by taking up the drug molecule into their lipophilic inner cavity 539. Hydroxypropyl-β-cyclodextrin (HPβCD) is a derivative of natural β-cyclodextrin (β-CD) with improved water solubility and safety compared with β-CD 541. In a study carried out by our group, we showed that the complexation with HPβCD increased the cytotoxic effect of ABZ against ovarian cancer cells 542.

ABZ is a basic drug with a pka values of 2.8 and 10.28 526. Therefore, in acidic medium ABZ is in ionised form. Theoretically, drugs with a basic group in their structure can be solubilised in acidic medium providing that the pKa of the drug is above the pKa of the formulation 478. On the other hand, heterocyclic and aromatic rings in the chemical structure of ABZ make it an ideal candidate for complexation with HPβCD. Several studies have shown that the combination of pH adjustment and complexation with CDs improves the solubility of the drugs substantially 492, 493. Chapter 3Chapter

74

The aim of this chapter is to compare the anti-tumour efficacy of ABZ solution in HPβCD/acetic acid with ABZ suspension in hydroxypropyl methyl cellulose (HPMC) in human colorectal carcinoma xenograft. Additionally, the pharmacokinetics behaviour of the two formulations is also evaluated.

3.2. RESULTS

3.2.1. HPLC Analysis

3.2.1.1. Standards and Calibration Curves

The retention times for ABZSO, ABZSO2 and ABZ were 6.1, 9.3 and 10.6 minutes, respectively (figure 3.1) and no interfering peak was present in blank plasma. The accuracy of the assay was determined by calibration curves of ABZSO, ABZSO2 and ABZ. Peak areas of ABZ and its metabolites were found to increase linearly at drug concentrations between 0.5-50, 0.5-50, and 0.1-2 for ABZSO, ABZSO2 and ABZ, respectively. Linear regression of ABZSO, ABZSO2 and ABZ were 0.9992, 0.9991 and 0.9841, respectively (figure 3.2).

Chapter 3Chapter

75

Figure 3.1. Chromatogram of blank human plasma spiked with ABZSO (6.1 minutes),

ABZSO2 (9.3 minutes), and ABZ (10.6 minutes)

8000 ABZ r=0.9841 ABZSO r=0.9992

ABZSO2 r=0.9991 6000

4000 Area (x1000) Area

2000

0

0 10 20 30 40 50 Concentration (Pg/ml)

Figure 3.2. Standard Curves of ABZSO, ABZSO2 and ABZ using peak area

Chapter 3Chapter

76

3.2.1.2. Validation of the method

Low QCs of ABZ and its metabolite were undetectable in plasma, indicating that the low QCs were below the limit of quantification (LOQ).

Intra-day and inter-day accuracy and precision for ABZ and its metabolites are summarised in table 3.2. The intra-day precision ranged from 3.573% to 6.085% and the inter-day precision ranged from 4.363% to 11.627%. Intra-day and inter-day accuracy were within the 15% from the true values (Table 3.1). Chapter 3Chapter

77

Table 3.1. Precision and accuracy of ABZ, ABZSO and ABZSO2 in human plasma

Concentration ABZ ABZSO ABZSO2 (μg/ml) 0.02 0.15 1.5 0.2 2.5 40 0.1 2.5 40 Intra-day run Mean (n=3) 0.162 1.362 2.557 39.286 2.519 41.055 SD 0.009 0.049 0.156 2.024 0.113 1.944 CV (%) 5.613 3.573 6.085 5.152 4.485 4.735 Accuracy (%) 107.778 90.8 102.293 98.216 100.773 102.638 Inter-day run Mean (n=3) 0.174 1.583 2.493 40.693 2.623 41.74 SD 0.007 0.150 0.017 2.538 0.305 1.821 CV (%) 4.244 9.502 6.787 6.238 11.627 4.363 Accuracy (%) 113.333 105.556 99.733 101.733 104.933 104.35 78 Chapter 3

3.2.1.3. Pharmacokinetics of ABZ/HPßCD and ABZ/HPMC

Figure 3.3. shows the mean concentration-time profiles of ABZ and its metabolites following the administration of 50 mg/kg ABZ/HPMC and ABZ/HPßCD. The pharmacokinetic parameters for ABZ/HPMC and ABZ/HPβCD are summarised in Table 3.2.

Following intraperitoneal administration of the drugs, the peak plasma concentration

(Cmax) of ABZ/HPβCD and ABZ/HPMC were 13.7 μg/ml and 0.36 μg/ml, respectively, indicating a 38-fold higher concentration of the parent drug in mice that were treated with ABZ/HPßCD compared to the animals that received the equivalent dose of ABZ-HPMC. ABZ was cleared from plasma within 2 hours post administration, demonstrating the rapid metabolism of the parent drug to its active metabolite (ABZSO). The highest concentrations of the ABZSO for both formulations were detected at 1 hour following administration (Tmax). The peak plasma concentrations of ABZSO were 32.7 and 1.8 μg/ml for ABZ/HPβCD and ABZ/HPMC, respectively, which gradually declined after 7 hours and reached undetectable levels within 24 hours.

Area under the curve (AUC; area under the concentration-time curve) of the parent drug and its metabolites within 24 hours post-administration was calculated using prism software. A higher AUC value was obtained after treatment with ABZ/HPβCD compared to ABZ/HPMC. Animals that were treated with ABZ/HPβCD had an approximately 2.5-, 7- and 4-fold increase in AUC values of ABZ, ABZSO and

ABZSO2, respectively, compared to mice that received ABZ/HPMC.

3Chapter

7979

A.

ABZSO 40

g/ml) 30 ABZ/HPECD P ABZ/HPMC 20

10 Concentration ( Concentration 0

12 4 8 24 B. Ti me (h)

ABZSO 8 2

g/ml) 6 ABZ/HPECD P ABZ/HPMC 4

2

Concentration ( 0

12 4 8 24 C. Ti me (h)

ABZ 20

g/ml) 15 ABZ/HPECD P ABZ/HPMC 10

5

Concentration ( 0

12 4 8 24 Ti me (h)

Figure 3.3. Mean plasma concentration of ABZSO (A), ABZSO2 (B), and ABZ (C) following the administration of ABZ/HPMC and ABZ/HPβCD. Error bars indicate SEM. Chapter 3Chapter

8080

Table 3.2. Pharmacokinetic parameters of ABZ, ABZSO and ABZSO2 following intraperitoneal administration of ABZ/HPMC and ABZ/HPβCD.

ABZ ABZSO ABZSO2

HPßCD HPMC HPßCD HPMC HPßCD HPMC Cmax (μg/ml) 11.38 0.36 32.73 1.84 4.97 0.45

Tmax (h) 0.5 0.5 1 1 2 1 AUC 0-24 (μg/mlxh) 18.71 8.122 105.7 14.39 15.73 4.128

81 Chapter 3

3.2.2. Comparison of the effect of ABZ/ethanol, ABZ/HPβCD, and ABZ/HPMC on cell proliferation

Anti-proliferative effect of ABZ formulated in HPβCD and HPMC and ABZ dissolved in ethanol (ABZ/EtOH) were tested against HCT-116, DU145, and HUVECs cell lines. Cells were incubated with various concentrations of ABZ/HPβCD and ABZ/HPMC ranging from 0.01 to 100 μM. To ascertain that HPβCD and HPMC had no effect on cell viability, cytotoxicity of drug-free vehicles were also evaluated. The stock solution of ABZ/EtOH was 1 mM and the concentrations of 0.01 to 10 μM were used. Since the concentrations greater than 1% ethanol are toxic to cells, the highest concentration of ABZ/EtOH that was used in this experiment was 10 μM.

All tested formulations represented a dose-dependent toxicity against HCT-116, DU- 145, and HUVECs cell lines (Figure 3.4.). The IC50 values of ABZ/HPβCD were slightly lower than those of ABZ/EtOH in HCT-116 cell line (Figure 3.4.A), and HUVECs (0.37 μM and 0.26 μM, respectively). However, the differences were not statistically significant (P > 0.05). Both ABZ/EtOH and ABZ/HPβCD had an IC50 of 0.6 μM against DU145 cells.

IC50 values of ABZ were 0.6 μM for ABZ/HPβCD and 5.5 μM for ABZ/HPMC against DU145 cells (Figure 3.4.B), indicating a 9.5-fold increase in cytotoxicity of ABZ/HPβCD compared to ABZ/HPMC. Similar results were obtained with HCT- 116 cells (Figure 3.4.A) and HUVECs (Figure 3.4.C). In comparison with ABZ/HPMC, an approximately 13- and 11-fold increase in cell death after treatment with ABZ/HPβCD was observed in HCT-116 cells and HUVECs, respectively. The IC50 values of ABZ against HCT-116 and HUVECs were 0.37 μM and 0.51 μM for ABZ/HPβCD, and 4.7 μM and 3.1 μM for ABZ/HPMC, respectively. Treatment of the cells with drug-free vehicles at concentrations corresponding to the drugs had no cytotoxic effect.

3Chapter 82

A. 100

75 ABZ/HPECD ABZ/HPECD vehicle 50 ABZ/HPMC ABZ/HPMC vehicle

% of control 25 ABZ/EtOH

0

10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Dose (M)

B. 100

75 ABZ/HPECD ABZ/HPECD vehicle 50 ABZ/HPMC ABZ/HPMC vehicle

% of control 25 ABZ/EtOH

0

10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Dose (M)

C.

100

75 ABZ/HPECD ABZ/HPECD vehicle 50 ABZ/HPMC ABZ/HPMC vehicle % of control 25 ABZ/EtOH

0

10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Dose (M)

Figure 3.4. Cytotoxic effect of ABZ/HPßCD, ABZ/HPMC and ABZ/EtOH. HCT-116 (A), DU145 (B), and HUVECs (C). Cells were exposed to the indicated concentrations of the drugs for 72 hours. Results from three separate experiments are represented as mean ± SEM. Chapter 3Chapter

83

3.2.3. Anti-tumour efficacy

To determine whether increased plasma concentration of ABZ/HPβCD also led to increased anti-tumour efficacy, the effect of ABZ/HPβCD with ABZ/HPMC therapy on survival of mice-bearing HCT-116 tumour was compared. According to previous studies carried out in our lab, alternate-day dosing with 150 mg/kg ABZ/HPMC proved to be the most effective dosing schedule 398, 543. Therefore, for anti-tumour efficacy experiment, the effect of 150 mg/kg ABZ/HPMC was compared with 50 mg/kg ABZ/HPβCD.

2x106 HCT-116 cells were injected subcutaneously into the flank of the animals. When the tumours had grown to approximately 100 mm3, mice were randomised into three groups of 8 animals. Group 1 received ABZ/HPβCD vehicle, group 2 were treated with 50 mg/kg ABZ/HPβCD and group 3 were administered 150 mg/kg ABZ/HPMC. All groups received the drugs intraperitoneally every second day.

As depicted in figure 3.5, treatment with both ABZ/HPβCD and ABZ/HPMC prolonged the survival compared with that in control mice, with 50 mg/kg ABZ/HPβCD being superior to 150 mg/kg ABZ/HPMC. ABZ/HPMC treatment showed a median survival time of 31 days as opposed to mice from control group, which had a survival time of 23 days (p = 0.02). Treatment with ABZ/HPβCD resulted in a significant prolongation of survival (41.5 days), compared with ABZ/HPMC therapy (p=0.03), and control (p=0.001) (Table 3.3).

Chapter 3Chapter

84

100 Control 80 50 mg/kg ABZ/HPECD 150 mg/kg ABZ/HPMC 60

40

Percent survival Percent 20

0 10 20 30 40 50 Time (day)

Figure 3.5. Kaplan-Meier survival curve. Mice were inoculated with 2x106 HCT-116 cells. When the tumour size reached approximately 100 mm3, animals were treated with vehicle, 50 mg/kg ABZ/HPβCD, and 150 mg/kg ABZ/HPMC. Mice were euthanised when the tumour size reached 1000 mm3. Time is expressed in days since start of treatment.

Median Survival (Day) Log-Rank Control 23 - ABZ/ HPβCD 41.5 0.001 ABZ/HPMC 31 0.023

Table 3.3. Median survival analysis of animals that were treated with vehicle, 50 mg/kg ABZ/HPβCD, and 150 mg/kg ABZ/HPMC. The median survival was calculated using Kaplan-Meier statistics. The log-rank P-value is the comparison between each treatment group and untreated mice.

Chapter 3Chapter

85

3.2.4. Immunohistochemistry Analysis

To determine whether ABZ/HPβCD and ABZ/HMPC have direct anti-proliferative effects on tumour, immunohistochemistry analysis of Ki67 was carried out. As shown in figure 3.6.A, both formulations inhibited tumour cell proliferation as assessed by the decrease in Ki67-positive cells. However, treatment with ABZ/HPβCD resulted in a statistically significant decrease in proliferation rate compared with ABZ/HPMC (P < 0.05).

Microvessel density in the tumours was assessed by staining tumour sections for CD31 (Figure 3.6.B). The mean CD31-positive area in tumours derived from ABZ/HPβCD- and ABZ/HPMC-treated groups were significantly smaller than in tumours derived from vehicle-treated group (6.83×103 pixels2, 2.37×103 pixels2 and 4.63×103 pixels2, respectively, P <0.05).

Apoptosis was detected in tumour tissue by using TUNEL assay (Figure 3.6.C). While in tumour tissues obtained from animals treated with vehicle, only a few TUNEL-positive cells were detected, treatment with ABZ/HPβCD remarkably increased TUNEL-positive cells (from 1.2% to 7.9% positive cells). Therapy with ABZ/HPMC also enhanced the percentage of TUNEL-positive cells in tumour tissue samples (7%). Statistical analysis showed that both ABZ/HPβCD and ABZ/HPMC- treated tumours had a significantly higher percentage of TUNEL-positive cells compare to the vehicle-treated group (p<0.05). However, no difference in the degree of apoptosis was observed between the animals treated with ABZ/HPβCD and the mice that received ABZ/HPMC (P>0.05).

Chapter 3Chapter

86

100 10 10 80 8 8 60 6 6

40 pixels) 3 4 4 (10

% Ki67 Positive Ki67 % 20 2 2 % of Apoptotic Cells Apoptotic of %

0 Mean CD31-positive Area Vehicle ABZ/HPMC ABZ/HPECD 0 0 Vehicle ABZ/HPMC ABZ/HPECD Vehicle ABZ/HPMC ABZ/HPECD

Figure 3.6. Immunohistochemical analysis of angiogenesis, tumour cell proliferation and apoptosis. Tumour sections were stained with an anti-Ki67 antibody (Ki67) to detect proliferating tumour cells (A) and with anti-CD31 antibody (CD31) to detect vessel associated endothelial cells (B). Apoptotic cells were detected by TUNEL assay (C). Columns represent mean and bars represent SEM.

Chapter 3Chapter

87

3.2.5. Tumour and Plasma VEGF

The concentrations of VEGF in tumour and plasma from the treatment groups were compared using western blot and ELISA assay, respectively. Therapy with ABZ/HPβCD profoundly decreased VEGF levels in tumours (Figure 3.7). Similarly, mice that were treated with ABZ/HPβCD had lower plasma VEGF was significantly compared with control and HPMC-treated groups (p<0.05) (Figure 3.8).

1.0

0.8

0.6

0.4

Relative Intensity 0.2

0.0 Control ABZ/HPMC ABZ/HPECD

Figure 3.7. Effect of vehicle, (1-3), ABZ/HPMC (4-6) and ABZ/HPβCD (7-9) on VEGF level in tumour tissue. Following euthanasia, tumours were excised and protein lysates obtained from three randomly selected xenograft tumours were electrophoresed and immunoblotted with an anti-VEGF antibody.

3Chapter 88

25

20

15

10 VEGF (pg/ml ) 5

0 Control ABZ/HPMC ABZ/HPECD

Figure 3.8. Plasma concentration of human VEGF in HCT-116 tumour-bearing mice. Mice were treated with the vehicle, ABZ/HPβCD and ABZ/HPMC. Following euthanasia, blood samples were collected by cardiac puncture and plasma samples were subjected to VEGF ELISA assay. Columns represent mean VEGF concentration, bars represent SEM.

Chapter 3Chapter

89

3.3.6. Toxicity Evaluation

Vehicle containing more than 0.5 ml/kg acetic acid and/or 1 ml/kg sodium hydroxide was found to be toxic to the animals. Thus, the highest concentration of the drug that was administered to the animals was 50 mg/kg. Following a single intraperitoneal dose of 60 mg/kg ABZ/HPβCD, mice lost mobility and were euthanised within two hours. Other treatment regimens had no adverse effect on animal wellbeing. However, following the second dose of 50, 40 mg/kg ABZ/HPβCD, a 15% reduction in the weight of the animals was observed that was recovered after 72 hours (Figure 3.9)

20 Vehi cl e 50 mg/kg 18 40 mg/kg 25 mg/kg 16 10 mg/kg 5 mg/kg weight (g) weight

14

12

0 4 8 12 16 20 24 28 Days after treatment

Figure 3.9. Effect of various doses of ABZ/HPβCD on mice body weight. Mice treated with ABZ/HPβCD for 28 consecutive days.

Chapter 3Chapter

90

3.4. DISCUSSION

The aim of this chapter was to compare the pharmacokinetic profile of ABZ/HPβCD with a conventional suspension formulation of ABZ in HPMC. In addition, the in vitro anti-proliferative effect of the two formulations as well as their in vivo antitumor activity was evaluated.

Pharmacokinetic study revealed that the AUCs of ABZ, ABZSO, and ABZSO2 in plasma of animals that received ABZ/HPβCD were approximately 2.3, 7.3, and 3.8 fold greater than in mice that were given a corresponding dose of

ABZ/HPMC. Furthermore, peak plasma concentrations (Cmax) of ABZ, ABZSO, and ABZSO2 in ABZ/HPβCD-treated mice were found to be 34, 18, and 11 times higher than in ABZ/HPMC-treated animals. These results are consistent with other studies reporting that the use of the combination of acid and CD profoundly enhanced the bioavailability of ABZ compared with the conventional formulation 388, 389, 538, 544, 545.

Comparison between the cytotoxic effect of the two formulations in HCT-116, DU145, and HUVECs demonstrated that ABZ/HPβCD was approximately 13-, 9-, and 12- times more potent in inhibiting the growth the cells than ABZ/HPMC. These differences were solely attributed to the solubility of ABZ in HPβCD, as no significant difference between the IC50 values of ABZ/EtOH and ABZ/HPβCD was observed.

Consistent with the in vitro results, treatment with 50 mg/kg ABZ/HPβCD led to a significant increase in the survival of mice compared to that of 150 mg/kg ABZ/HPMC. Tumour growth and metastasis have been demonstrated to be angiogenesis dependent 546. In addition, it is well known that VEGF has a predominant role in angiogenesis 358. Therefore, the effect of both formulations on microvessel density, as a prognostic marker of metastatic risk 547, and VEGF in tumour tissue and plasma was evaluated. Results showed that treatment with ABZ/HPβCD led to a significant reduction in microvessel density and the expression of VEGF in both tumour and plasma. Additionally, ABZ/HPβCD led to a significant reduction in proliferation rate of the tumour cells as compared with vehicle and

ABZ/HPMC treated groups. No significant difference was observed in the degree of 3Chapter

91

apoptosis induced by the two formulations. This suggests that the higher efficacy of ABZ/HPβCD over ABZ/HPMC is most likely due to its greater ability to suppress the proliferation of the tumour cells, rather than induction of apoptosis.

Enhanced efficacy of ABZ/HPβCD may, at least in part, results from increased solubility and a better absorption of the drug. Soluble drug molecule can extravasate from leaky tumor vasculature and accumulate in the tumor tissue due to the absence of an effective lymphatic drainage 548. Improved therapeutic effect of ABZ/HPβCD can be also attributed to the higher plasma levels of ABZSO in mice that received ABZ/HPβCD. Another possible explanation for greater efficacy of ABZ/HPβCD is the delay in the oxidation of ABZSO to ABZSO2, as ABZSO2 is pharmacologically inactive and ABZSO is responsible for the therapeutic effect of the drug.

In addition to the higher solubility and improved pharmacokinetic parameters of ABZ as a result of the complexation with HPβCD, the possible effect of HPβCD on pharmacokinetic behavior of ABZ and therefore, its therapeutic effect should also be considered. HPβCD is known to be an efficient carrier for drugs as they are able to pass through the vascular endothelium 549. Furthermore, CDs have been shown to affect the interaction between the drug molecules and plasma proteins 550. However, this effect is dependent on the stability constant between the drug and CD, as well as the relative affinity of the drug to the plasma proteins. ABZ has been shown to have a moderate plasma protein binding of 70% 525 and its stability constant with HPβCD is within the optimum margin of 200-5000 M- 1 551. This stability constant and the moderate affinity of ABZ towards plasma proteins may reduce the binding of ABZ to plasma proteins. Since only the unbound fraction of the drugs have pharmacological effect, even a slight reduction in protein binding of ABZ may have a significant effect.

For pharmacokinetic study, both ABZ/HPβCD and ABZ/HPMC were administered at a dosage of 50 mg/kg. This dose was chosen based on the toxicity profile of ABZ/HPβCD, as doses above 50 mg/kg are toxic to the animals. Indeed, mice receiving an intraperitoneal dose of 60 mg/kg ABZ/HPβCD were euthanised within two hours post administration. Signs of poisoning, apparent 20 minutes after administration were sedation and Chapter 3Chapter convulsion. This toxic effect was due to the vehicle, as drug-free vehicle caused 92

the same effect in the animals. Since intraperitoneal administration of up to 10 mg/kg HPβCD is neither lethal nor toxic to mice 541, the observed toxic effect was attributed to exceeding the LD50 of acetic acid and/or sodium hydroxide. ABZ/HPMC dose for the anti-tumour experiment was selected according to our previous in vivo experiments in which it was demonstrated that 150 mg/kg ABZ had a significant anti-tumour effect with no adverse impact on animals’ wellbeing 398, 543.

In summary, the results show that the solubility of ABZ was highly improved by complexation with acetic acid/HPβCD. Indeed, a 10,000-fold increase in aqueous solubility of the drug was achieved. Further, ABZ/HPβCD exhibited significantly greater antitumour efficacy and higher plasma AUC and Cmax in comparison with ABZ/HPMC. These findings lead to the conclusion that the formulation may be suitable for parenteral administration.

Chapter 3Chapter

93

Chapter 4

CombinationCombination ooff AAlbendazolelbendazole wwiitthh ChemotherapeuticChemotherapeutic AAgentsgents

4.1. INTRODUCTION

The use of multiple cytotoxic drugs with non-overlapping toxicities plays a prominent role in the control and treatment of various types of malignancies.

Several approaches have been pursued in an attempt to design combination therapies. The most common strategy is to combine agents that possess different mechanisms of action 470. In this way, the probable emergence of drug resistance would diminish 552. Another strategy is to combine a new drug with as many established agents as possible 553. These approaches, although empirical, have crafted many successful combinations that have significantly improved patient outcomes.

In this chapter, combinations of ABZ with three antiparasitic (praziquantel, diethylcarbamazine, and ivermectin), and four cytotoxic drugs (paclitaxel, vinblastine, colchicine, and 2-methoxyestradiol) have been evaluated. The antiparasitic agents were chosen based on their synergistic interaction with ABZ in human parasites.

Praziquantel (PZQ, Biltricide) is an anthelmintic drug which is used against various cestode and trematode parasites, in particular schistosomes 554. PZQ increases the permeability of the parasite cell towards calcium ions with a secondary effect on its antigenicity and metabolism 555. In patients with hydatid cyst, a combination of ABZ and PZQ showed a significant improvement in symptoms in more than half of the cases 517. Diethylcarbamazine (DEC) is used as a single agent and in combination with ABZ to treat filariasis. DEC exerts its therapeutic efficacy through the inhibition of arachidonic acid metabolism in filarial microfilaria. This makes the microfilaria more susceptible to immune attack 518. In addition, DEC targets the cyclooxygenase pathway and inducible nitric oxide synthase (iNOS) 556. Ivermectib (IVE) is a broad-spectrum antiparasitic agent which is used to treat onchocerciasis, lymphatic filariasis, and strongiloidiasis in humans 557. In 1998, in “The Global

Programme to Eliminate Lymphatic Filariasis”, IVE in combination with ABZ was proven to be highly efficacious 519. IVE has been suggested to be an agonist for neurotransmitter function. It interacts with glutamate-gated chloride ion channels in 4 Chapter 95

nerve and muscle cells, and prevents their closure, which ultimately leads to hyperpolarization of the neuronal membrane, decrease in neuronal transmission, paralysis, and death of the parasite 558, 559. Besides its antiparasitic action, IVE has been shown to reverse resistance to doxorubicin in MDR mouse leukemic P388 cells 560. In a recent study, IVE has been shown to induce cell death in acute myeloid leukemia cell lines and delay tumour growth in three mouse models of leukemia 561.

As stated in chapter 1, PTX, VBL and CLC are microtubule-targeting agents (MTAs). MTAs include diverse compounds with different mechanisms of action. Nevertheless, all of them ultimately induce mitotic arrest 562. Since MTAs bind to different sites on tubulin, the combination of two of these agents has the potential to improve the efficacy and reduce the side effects 3.

4.2. RESULTS

4.2.1. Interaction of ABZ with Anti-parasitic Agents

4.2.1.1. Single Agent Effect

Before evaluating the combined drug effect, growth inhibitory effect of single agents was assessed using the SRB assay after 72 hours treatment. As shown in figure 4.1, PZQ and DEC at concentrations of 0.1 to 10 μM had no cytotoxic effect on HCT- 116 and DU145 cells. At 100 μM, however, a marginal effect was observed in both cell lines. In contrast, following IVE treatment, a concentration-dependent growth inhibition was observed in both cell lines, with a typical sigmoidal growth inhibition curve. The IC50 values for HCT-116 and DU145 were 3.2 ± 0.4 μM and 7.6 ± 0.3 μM, respectively. Due to the low cytotoxicity, IC50 value for PZQ and DEC could not be determined.

Chapter 4 Chapter

96

4.2.1.2. Effect of Combination Treatment

4.2.1.2.1. Combination of ABZ with DEC and PZQ

Because DEC and PZQ were not cytotoxic, no mutual effect and therefore, no synergism would be achievable. Nevertheless, the combination of these two compounds with ABZ was evaluated to assess if they potentiate the effect of ABZ. Figure 4.2 shows the dose-response curve for HCT-116 (top panel) and DU145 (bottom panel). Each curve represents a particular dose of PZQ (left panel) or DEC (right panel) in combination with various doses of ABZ. Combination therapy resulted in no additional cytotoxic effect compared to ABZ as a single agent. As it is demonstrated in the dose-response curve, where no shift to the left side of ABZ as a single agent was observed when DEC and PZQ were added.

4.2.1.2.2. Combination of ABZ and IVE

As opposed to DEC and PZQ, combination of ABZ with IVE resulted in a greater cytotoxicity compared with ABZ as a single agent. As depicted in figure 4.3, in both HCT-116 (top panel) and DU145 (bottom panel), all IVE concentrations exhibited a left-sided shift of the dose-response curve of ABZ.

Cytotoxic interaction was evaluated by median effect analysis method that quantitatively describes the interaction between the tested agents. As shown in figure 4.3 (right panel), combination of low dose IVE with high doses of ABZ resulted in an antagonism interaction in both cell lines whereas combination of ABZ at doses below 5 μM with IVE at concentrations above 1 μM resulted in an additive interaction.

Chapter 4 Chapter

97

100 100 100

75 75 75

50 50 50 % Control % Control % Co n tro l 25 25 25 IVE PZQ 0 0 DEC 0 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 10-8 10-7 10-6 10-5 10-4 10-3

Dose (M) Dose (M) Dose (M)

100 100 100

75 75 75

50 50 50 % Control % Co n tro l % Co n tro l 25 25 25 IVE DEC 0 PZQ 0 0 10 -8 10-7 10 -6 10-5 10-4 10-3 10-8 10-7 10-6 10-5 10-4 10-3 10-8 10-7 10-6 10-5 10-4 10-3

Dose (M) Dose (M) Dose (M)

Figure 4.1. Dose response curves for 72 hours PZQ, DEC and IVE treatment on HCT-116 (top panel) and DU-145 (bottom panel). Cells were treated with the drugs at concentrations ranging from 0.1 μM to 100 μM. After 72 hours incubation, cells were subjected to the SRB assay. Each point is shown as mean ± SEM (n=3). 98 Chapter 4

A.

100 100 ABZ ABZ 0.1 PM PZQ 0.1 PM DEC 75 75 1 PM PZQ 1 PM DEC 5 PM PZQ 50 50 10 PM DEC 10 PM PZQ 50 PM DEC % Control 100 PM PZQ % Control 25 25 100 PM DEC

0 0

10-9 10-8 10-7 10-6 10-5 10-4 10-9 10-8 10-7 10-6 10-5 10-4 Dose (M) Dose (M)

B. 100 100 ABZ ABZ 0.1 PM PZQ 0.1 PM DEC 75 75 1 PM PZQ 1 PM DEC 50 5 PM PZQ 50 10 PM DEC 10 PM PZQ 50 PM DEC % Control 100 PM PZQ % Control 100 PM DEC 25 25

0 0

10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10-9 10-8 10-7 10-6 10-5 10-4 Dose (M) Dose (M)

Figure 4.2. Effect of the combination of ABZ plus PZQ and ABZ plus DEC on HCT-116 (A) and DU145 (B) cell lines. Cytotoxicity was evaluated after treating the cells with vehicle and the drugs for 72 hours. Left panel shows dose response curve for PZQ and right panel shows dose response curve for DEC. Each point is shown as mean ± SEM (n=3).

99 Chapter 4

A. 4 0.1 PM IVE 100 ABZ 1 PM IVE 3 10 PM IVE 0.1 PM IVE 75 50 PM IVE 1 PM IVE 100 PM IVE 10 PM IVE 2 50 50 PM IVE

% Control Antagonism 100 PM IVE 1 25 Synergism Combination Index (CI) 0 0 10-9 10-8 10-7 10-6 10-5 10-4 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 ABZ (M) Dose (M)

4 B. 0.1 PM IVE 100 ABZ 1 PM IVE 3 0.1 PM IVE 10 PM IVE 75 1 PM IVE 50 PM IVE 10 PM IVE 100 PM IVE 2 50 50 PM IVE

% Control 100 PM IVE Antagonism 1 25 Synergism Combination Index (CI)

0 0 10-9 10-8 10-7 10-6 10-5 10-4 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 ABZ (M) Dose (M)

Figure 4.3. Effect of the combination of ABZ and IVE on HCT-116 (A) and DU-145 (B) cell lines. Cytotoxicity was evaluated after treating the cells with vehicle, ABZ, IVE and their combination for 72 hours. Left panel shows dose response curve and right panel shows median effect analysis. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3). 100

Chapter 4

4.2.2. Interaction between ABZ and MTAs

4.2.2.1. Single Agents Effect

Growth inhibitory effect of PTX at concentrations ranging from 0.5 to 10 nM, VBL at concentrations ranging from 0.5 to 10 nM, CLC concentrations ranging from 1 to 50 nM, and 2ME concentrations ranging from 0.01 to 100 μM, as single agents were assessed using the SRB assay after 72 hour treatment.

As shown in figure 4.4, a concentration-dependent growth inhibition was observed in both HCT-116 and DU145 cell lines. The IC50 value of each drug is shown in table 4.1.

HCT-116 DU145

Paclitaxel (nM) 2.3 ± 0.9 2.6 ± 1.1 Vinblastine (nM) 3.8 ± 1.2 5.5 ± 1.1 Colchicine (nM) 20 ± 12.1 27 ± 14.8 2-Methoxyestradiol (μM) 0.92 ± 0.21 1.2 ± 0.38

Table 4.1. IC50 values for 72-hour drug treatment on HCT-116 and DU145. Results are expressed as mean ± SEM (n=3).

Chapter 4 Chapter

101

100 PTX 100 VBL

75 75

50 50

% Control % Control 25 25

0 0

-1 0 -9 -8 -7 10 -1 0 10 -9 10 -8 10 -7 10 10 10 10 Dose (M) Dose (M)

100 PTX 100 VBL 75 75 50 50

% Control

25 % Control 25 0 0 10 -1 0 10 -9 10 -8 10 -7 10 -1 0 10 -9 10 -8 10 -7 Dose (M) Dose (M)

Figure 4.4. Dose response curves for 72-hour PTX and VBL treatment on HCT-116 (top panel) and DU145 (bottom panel) cell lines. Cells were treated with the drugs at concentrations ranging from 0.5 nM to 10 nM. After 72 hours incubation, cells were subjected to the SRB assay. Each point is shown as mean ± SEM (n=3). 102 Chapter 4

100 CLC 100 2ME

75 75 50 50 % Control % Control 25 25

0 0 -1 0 -9 -8 -7 -6 10 10 10 10 10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4

Dose (M) Dose (M)

100 CLC 100 2ME

75 75

50 50 % Control % Control 25 25

0 0

10 -1 0 10 -9 10 -8 10 -7 10 -6 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4

Dose (M) Dose (M)

Figure 4.5. Dose response curves for 72-hour CLC and 2ME treatment on HCT-116 (top panel) and DU145 (bottom panel). Cells were treated with CLC at concentrations ranging from 1 nM to 50 nM, and 2ME at concentration of 0.01 μM to 100 μM. After 72 hours incubation, cells were subjected to the SRB assay. Each point is shown as mean ± SEM (n=3). 103 Chapter 4

4.2.2.2. Effect of Combination Therapy

4.2.2.2.1. Combination of ABZ and PTX

As shown in figure 4.6, combination of ABZ and PTX produced a shift to the left side of ABZ dose-response curve. However, median effect analysis revealed that the shift is due to the cytotoxicity of PTX alone rather than its combination with ABZ. As shown in figure 4.6, (right panel), in both cell lines, the combination index for all tested concentrations were above 1, indicating antagonism. Indeed, as the concentration of PTX increased, CI changed from nearly additive to antagonistic effect.

4.2.2.2.2. Combination of ABZ and VBL

Similar to PTX, VBL in combination with ABZ produced a left-sided shift of ABZ dose-response curve that was due to the effect of VIN by itself (figure 4.7, left panel). Median effect analysis confirmed that the interaction between ABZ and VIN was antagonistic, as CI values were above 1 throughout the range of tested ABZ concentrations tested (figure 4.7, right panel).

Chapter 4Chapter

104

A. 4 100 ABZ 0.5 nM PTX 0.5 nM PTX 1 nM PTX 3 75 1 nM PTX 2.5 nM PTX 2.5 nM PTX 5 nM PTX 5 nM PTX 7.5 nM PTX 50 7.5 nM PTX 2 10 nM PTX 10 nM PTX % Control 25 Antagonism 1 Synergism Combination Index (CI) Index Combination 0 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10-9 10-8 10-7 10-6 10-5 10-4 Dose (M) ABZ (M)

B. 4 100 ABZ 0.5 nM PTX 0.5 nM PTX 1 nM PTX 75 1 nM PTX 3 2.5 nM PTX 2.5 nM PTX 5 nM PTX 5 nM PTX 7.5 nM PTX 50 7.5 nM PTX 2 10 nM PTX

% Control 10 nM PTX

25 Antagonism 1 Synergism 0 (CI) Index Combination 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 0 10-9 10-8 10-7 10-6 10-5 10-4 Dose (M) ABZ (M)

Figure 4.6. Effect of 72-hour simultaneous ABZ and PTX treatment for (A) HCT-116 and (B) DU145. Cytotoxicity was evaluated using the SRB assay. Left panel shows dose response curve and right panel shows median effect analysis. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3). 105 Chapter 4

A. 4 100 ABZ 0.5 nM VBL 0.5 nM VBL 1 nM VBL 3 75 1 nM VBL 2.5 nM VBL 2.5 nM VBL 5 nM VBL 5 nM VBL 7.5 nM VBL 50 7.5 nM VBL 2 10 nM VBL 10 nM VBL % Control Antagonism 25 1 Synergism Combination Index (CI) Index Combination 0 0 -9 -8 -7 -6 -5 -4 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 10 10 10 10 10 ABZ (M) Dose (M) B. 4 100 ABZ 0.5 nM VBL 0.5 nM VBL 1 nM VBL 1 nM VBL 3 75 2.5 nM VBL 2.5 nM VBL 5 nM VBL 5 nM VBL 7.5 nM VBL 50 7.5 nM VBL 2 10 nM VBL 10 nM VBL % Control 25 Antagonism 1 Synergism Combination Index (CI) Index Combination 0 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10-9 10-8 10-7 10-6 10-5 10-4 ABZ (M) Dose (M)

Figure 4.7. Effect of 72-hour simultaneous ABZ and VBL treatment for (A) HCT-116 and (B) DU145. Cytotoxicity was evaluated using the SRB assay Left panel shows dose response curve and right panel shows median effect analysis. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3). 106 Chapter 4

4.2.2.2.3. Combination of ABZ and CLC

Dose-response curves for HCT-116 and DU145 cell lines are shown in figure 4.8.A and 4.8.B (left panel), respectively. Each curve represents a particular dose of CLC in combination with various doses of ABZ.

In HCT-116 cell line, at concentrations below 1 μM ABZ, there was a shift to the left side of the combination curves compared to ABZ alone, indicating enhanced growth inhibitory effect of the combination therapy. However, at doses above 1 μM ABZ and 25 nM CLC, combination curves crossed over to the right side of the ABZ single agent curve, indicating that CLC was reducing the inhibitory effect of ABZ on cell growth. In DU145 cell line, left-sided shift of the combination curve was observed throughout the range of tested CLC concentrations.

Cytotoxic interaction of ABZ and CLC on HCT-116 and DU145 is shown in figure 4.8.A.and 4.8.B (right panel). In both cell lines, combination of ABZ at concentrations below 5 μM ABZ with CLC resulted in synergism. However, as the concentration of ABZ increased, the interaction changed from synergism to antagonism. Combination of CLC with 5 μM ABZ resulted in additivity and its combination with 10 μM ABZ, led to antagonism.

Chapter 4Chapter

107

A. 4 100 ABZ 1 nM CLC 1 nM CLC 3 2.5 nM CLC 75 2.5 nM CLC 5 nM CLC 5 nM CLC 7.5 nM CLC 7.5 nM CLC 10 nM CLC 50 10 nM CLC 2 25 nM CLC 25 nM CLC % Control 40 nM CLC 25 40 nM CLC Antagonism 1 50 nM CLC 50 nM CLC Synergism Combination Index (CI) Index Combination 0 0 10-9 10-8 10-7 10-6 10-5 10 -4 10-9 10 -8 10-7 10-6 10-5 10-4 ABZ (M) Dose (M) B. 4 100 ABZ 1 nM CLC 1 nM CLC 2.5 nM CLC 2.5 nM CLC 3 75 5 nM CLC 5 nM CLC 7.5 nM CLC 7.5 nM CLC 10 nM CLC 50 10 nM CLC 2 25 nM CLC 25 nM CLC % Control 40 nM CLC 25 40 nM CLC Antagonism 1 50 nM CLC 50 nM CLC Synergism Combination Index (CI) Index Combination 0 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10-9 10 -8 10-7 10-6 10-5 10-4 Dose (M) ABZ (M)

Figure 4.8. Effect of the combination of ABZ and CLC on HCT-116 (A) and DU145 (B) cell lines. Cytotoxicity was evaluated using the SRB assay after treating the cells with vehicle, ABZ, CLC and the combination of ABZ and CLC for 72 hours. Left panel shows dose response curve and right panel shows median effect analysis. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3). 108 Chapter 4

4.2.2.2.4. Combination of ABZ and 2ME

In HCT-116 cells, a low concentration of 2ME (0.25 μM) in combination with ABZ produced the same degree of growth inhibition as ABZ alone, as represented by their overlapping growth inhibition curves. However, increasing concentrations of 2ME in combination with ABZ led to a shift to the left side of ABZ dose-response curve, indicating higher cytotoxicity of combination treatment. Dose-response curves of high ABZ concentrations in combination with 1 μM and 2.5 μM 2ME crossed over the dose-response curve of ABZ alone, suggesting a reduction in cytotoxicity of the combination compared with ABZ alone (figure 4.9.A, left panel). In DU145, the shift to the left side of ABZ dose-response curve was observed throughout the range of tested 2ME concentrations and all combination curves crossed over the dose- response curve of ABZ at ABZ doses above 1 μM. This indicates that increasing concentrations of ABZ when combined with 2ME has a negative impact on the action of ABZ (figure 4.9.B, left panel). Median effect analysis of the interaction of ABZ and 2ME in HCT-116 cells revealed that the combination of 0.01 μM ABZ with 2ME resulted in antagonism, regardless of 2ME concentrations. In contrast, combination of 2ME with higher doses of ABZ ranging from 0.1 μM to 1 μM led to a synergistic interaction. However, as the concentration of 2ME increased, the interaction changed from synergism to additive. Combination of 2ME with high concentrations of ABZ (5 μM and 10 μM), resulted in antagonism (figure 4.9.A, right panel). In DU145 cells, 2ME at 50 μM was antagonistic with ABZ, regardless of ABZ concentration. In contrast to HCT-116, in DU145 cells, a low dose of ABZ (0.01 μM) was synergistic with 2ME, as were the concentrations below 5 μM ABZ. However, similar to HCT-116, at doses above 5 μM ABZ, an antagonistic interaction was observed when 2ME was added (figure 4.9.B, right panel). Chapter 4Chapter

109

A. 4 100 0.25 PM 2ME ABZ 3 0.5 PM 2ME 75 0.25 PM 2ME 0.75 PM 2ME 0.5 PM 2ME 1 PM 2ME 0.75 PM 2ME 50 2 2.5 PM 2ME 1 PM 2ME 5 PM 2ME

% Control 2.5 PM 2ME 10 PM 2ME 25 5 PM 2ME Antagonism 1 50 PM 2ME 10 PM 2ME Synergism Combination Index (CI) 0 50 PM 2ME 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10-9 10-8 10-7 10-6 10-5 10-4 ABZ (M) Dose (M)

B. 4 100 ABZ 0.25 PM 2ME 75 0.25 PM 2ME 3 0.5 PM 2ME 0.5 PM 2ME 0.75 PM 2ME 0.75 PM 2ME 1 PM 2ME 50 1 PM 2ME 2 2.5 PM 2ME 2.5 PM 2ME 5 PM 2ME % Control 25 5 PM 2ME Antagonism 10 PM 2ME 10 PM 2ME 1 50 PM 2ME Synergism 0 50 PM 2ME (CI) Index Combination

-9 -8 -7 -6 -5 -4 0 10 10 10 10 10 10 10-9 10 -8 10-7 10-6 10-5 10-4 Dose (M) ABZ (M)

Figure 4.9. Effect of the combination of ABZ and 2ME on HCT-116 (A) and DU145 (B) cell lines. Cytotoxicity was evaluated using the SRB assay after treating the cells with vehicle, ABZ, 2ME and the combination of ABZ and 2ME for 72 hours. Left panel shows dose response curve and right panel shows median effect analysis. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3).

110 Chapter 4

4.3. DISCUSSION

One of the main approaches in cancer therapy is to utilise a combination of chemotherapeutic agents with the objective of improving efficacy while reducing or maintaining the overall toxicity to an acceptable level. As a single agent, ABZ has been shown to be a promising anticancer agent both in vitro and in vivo. Nevertheless, its combination with other cytotoxic agents may improve its application. Hence, in this chapter the combination of ABZ with different chemotherapeutic drugs was investigated.

DEC, PZQ and IVE have been reported to have synergistic antiparasitic activity with ABZ. Following treatment of HCT-116 and DU145 cells with DEC and PZQ as single agents, only a slight cytotoxic effect was observed at highest doses of the drugs (100 μM). Furthermore, DEC and PZQ did not potentiate the effect of ABZ when used in combination with ABZ. Conversely, IVE induced cell death in both cell lines at low micromolar concentrations and its combination with ABZ led to a synergistic effect. In a recent study, it has been shown that IVE has an anti- proliferative effect on a leukemia cancer cell line 561. The study has also reported that the combination of IVE with ABZ had CI values of 1.59, 1.09, and 0.89 on ED25, ED50, and ED75, respectively 561. Based on these CI values, the authors suggested that the interaction was antagonistic. However, according to Chou and Talalay method, CI value of 0.89 and 1.09 are considered synergistic and nearly additive, respectively 470.

Given the proven success of MTAs such as vinca alkaloids and taxanes in cancer therapy and the fact that MTAs can synergise with one another, the combination of ABZ with other MTAs was evaluated. As previously stated, three established binding sites on tubulin are taxane-, vinca-, and colchicine-binding sites. Therefore, PTX, VBL and CLC were chosen as representative drugs that interact with these binding sites. In both cell lines, the combination of ABZ with PTX resulted in antagonism throughout PTX doses used. Likewise, VBL had an antagonistic interaction with ABZ regardless of the concentration tested. Surprisingly, a dose-

dependent synergistic interaction between ABZ and CLC was observed. Several 4Chapter

111

studies suggested that benzimidazoles bind to the colchicine-binding site on β- tubulin 563, 564. In contrast, a recent study reported that benomyl, an antifungal agent from BZ class, binds to a novel site on tubulin and that benomyl had synergistic interaction with CLC 565. More recently, flubendazole, another compound from BZ group, was reported to interact with a site on tubulin “similar to colchicine-binding site” 566. This study also showed that flubendazole had a synergistic interaction with CLC in leukemia cell lines and with vinblastine and vincristine in both in vitro and in a leukemia xenograft model.

Despite its efficacy, CLC is not being used in cancer therapy due to its toxicity 3. Therefore, 2ME, which is a CLC-domain binder, was tested in combination with ABZ. It was hypothesised that because 2ME shares the same binding site on β- tubulin and its structure and the mechanism of action are similar to CLC, it may act synergistically with ABZ. As expected, combination of ABZ and 2ME in HCT-116 and DU145 resulted in synergism in a dose-dependent manner. Synergistic interaction of 2ME with several chemotherapeutic agents such as paclitaxel 567, vinorelbine 568, laulimalide 569, and tamoxifen 570 have been previously demonstrated. More recently, 2ME has been evaluated in clinical trials for the treatment solid tumours both as a single agent and in combination with docetaxel 467, 571.

This chapter demonstrated that ABZ acts synergistically with CLC and 2ME. In subsequent chapter, the mechanism underlying this synergism as well as the effect of combination therapy in a HCT-116 xenograft model will be investigated.

Chapter 4Chapter

112

Chapter 5

TheThe EffectEffect ofof AlbendazoleAlbendazole andand 2-Methoxyestradiol2-Methoxyestradiol aass a singlesingle aagentgent aandnd iinn CCombinationombination IInn VVitroitro aandnd IInn VVivoivo

5.1. INTRODUCTION

Combination therapy has a prominent role in cancer treatment. The main aims of using drug combination are to achieve synergistic therapeutic effect, to reduce the dose and toxicity, and to minimise or delay drug resistance 468.

In the previous chapter, it was shown that ABZ and CLC exhibited a synergistic anti- proliferative effect on HCT-116 and DU145 cell lines. Since CLC is not being used in cancer therapy due to its toxicity 3, the interaction between ABZ and 2ME, a related and a structurally similar compound to colchicine was evaluated. Similar to CLC, 2ME was shown to be synergistic with ABZ in inhibiting the proliferation of HCT-116 and DU145 cells. 2ME has been shown to be active against a variety of cancer cells, both in vitro and in vivo. Additionally, it does not exhibit myelosupression and other haematological toxicities associated with MTAs 572. This property makes 2ME an ideal candidate for the combination with other MTAs, since overlapping toxicities being the major limiting factor in combination therapy would be greatly diminished.

2ME has been evaluated in combination with various microtubule-targeting agents. A synergistic induction of apoptosis has been reported when 2ME is combined with paclitaxel in human endometrial cancer cell lines 567, with vinorelbine in breast cancer cell line 568, with laulimalide in lung carcinoma cells and breast cancer cells 569, and with tamoxifen in estrogen receptor-positive and estrogen receptor-negative breast cancer cell lines 570.

In this chapter, the effect of the sequential and simultaneous combination therapy on HCT-116 cell lines is evaluated. Furthermore, the underlying mechanism(s) of synergistic interaction between ABZ and 2ME is investigated. Finally, the efficacy of ABZ and 2ME as single agents and in combination, on the survival of mice- bearing HCT-116 xenograft is evaluated.

Chapter 5Chapter

114

5.2. RESULTS

5.2.1. Interaction between ABZ and 2ME: Simultaneous versus Sequential Treatment

Cytotoxic interaction between ABZ and 2ME administered simultaneously or sequentially were assessed over a period of 48 and 72 hours. The dose-response curves for 48 and 72 hours simultaneous treatment of HCT-116, are shown in figure 5.1.A and 5.2.A (left panel), respectively. Each curve represents a particular dose of 2ME in combination with various dose of ABZ.

In simultaneous treatment, at doses below 1 μM ABZ, there was an increase in growth inhibition when 2ME was added. Combined treatment produced a shift to the left side of ABZ dose response curve, indicating increased cytotoxicity of combination therapy. In contrast, combination of 2ME with 1 μM and 10 μM ABZ caused no additional cytotoxic effect on the cells. The interaction between ABZ and 2ME was quantified using median effect analysis. As shown in figure 5.1.A (right panel), combination of ABZ at doses ranging from 0.1 μM to 1 μM with 2ME resulted in synergism, regardless of 2ME doses. At 10 μM ABZ, however, antagonism was observed throughout the range of tested 2ME doses.

The effect of sequential treatment was determined by treating the cells with ABZ alone for 24 hours, followed by 2ME for another 24 hours (figure 5.1.B) or 48 hours (figure 5.2.B). Treating the cells with low concentration of 2ME (0.25 μM) resulted in a similar cytotoxicity to ABZ being used alone, as represented by their overlapping growth inhibition curves. Increasing concentrations of 2ME in combination with ABZ led to a shift to the left side of the ABZ dose response curve, indicating higher cytotoxicity of combination treatment. However, similar to simultaneous treatment, high ABZ concentrations in combination with 2ME crossed over the dose response curve of ABZ, representing reduction in cytotoxicity of the combination. The interaction between ABZ and 2ME is shown in figure 5.1.B (right panel). ABZ at 10 μM was antagonistic with all tested concentrations of 2ME. There was also an antagonism between low concentrations of ABZ (0.01 μM, 0.1 Chapter 5Chapter

115

μM and 0.25 μM) with 0.25 μM 2ME that became synergistic at 0.5 μM and 1 μM ABZ.

Figure 5.1.C and 5.2.C show the effect of pre-treatment of the cells with 2ME for 24 hours followed by ABZ addition for a further 24 or 48 hours. The curves of low concentrations of 2ME (0.25 μM and 0.5 μM) represented a shift to the right, indicating reduced cytotoxic effect of this combination compared to single agent. However, at 0.75 μM, 1 μM and 10 μM 2ME, a slight left-sided shift in combination curves were observed. Once again, 10 μM ABZ curve crossed over the dose response curve of ABZ.

A similar effect was observed for 72 hours treatment in all tested schedule treatments with synergism and antagonism being more pronounced compared with 48 hours treatment. The interaction between ABZ and 2ME over a period of 72 hours was already evaluated in the previous chapter. This experiment was repeated in order to provide a constant experimental condition between sequential and concurrent treatment.

5Chapter 116

A. 4 100 ABZ 0.25 PM 2ME 0.25 PM 2ME 3 0.5 PM 2ME 75 0.5 PM 2ME 0.75 PM 2ME 0.75 PM 2ME 1 PM 2ME 50 2 10 PM 2ME 1 PM 2ME

% Control 10 PM 2ME 25 Antagonism 1 Synergism 0 Index (CI) Combination 0 10-9 10-8 10-7 10-6 10 -5 10-4 -9 -8 -7 -6 -5 -4 10 10 10 10 10 10 Dose (M) ABZ (M)

B. 100 4 0.25 PM 2ME ABZ 0.5 PM 2ME 75 3 0.25 PM 2ME 0.75 PM 2ME 0.5 PM 2ME 1 PM 2ME 50 0.75 PM 2ME 2 10 PM 2ME 1 PM2ME % Control 25 10 PM2ME Antagonism 1 Synergism

0 Index (CI) Combination

10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 0 10-9 10 -8 10 -7 10 -6 10 -5 10 -4 Dose (M) ABZ (M)

C. 6 100 10 PM 2ME 5 ABZ 1 PM 2ME 0.25 PM 2ME 75 4 0.75 PM 2ME 0.5 PM 2ME 0.5 PM 2ME 50 0.75 PM 2ME 3 0.25 PM 2ME 1 PM 2ME % Control 25 10 PM 2ME 2 Antagonism 1 0 Index (CI) Combination Synergism

-9 -8 -7 -6 -5 -4 0 10 10 10 10 10 10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 Dose (M) ABZ (M)

Figure 5.1. Dose-response curves (left) and cytotoxic interaction between ABZ and 2ME (right). HCT-116 cells were exposed to ABZ and 2ME for 48 hours, simultaneously (A), or treated with ABZ for 24 hours followed by additional 24 hours treatment with 2ME (B), or treated with 2ME for 24 hours followed by additional 24 hours treatment with ABZ (C). CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM. Chapter 5Chapter

117

A. 4 100 ABZ 0.25 PM 2ME 0.5 PM 2ME 0.25 PM 2ME 3 75 0.75 PM 2ME 0.5 PM 2ME 1 PM 2ME 0.75 PM 2ME 2 10 PM 2ME 50 1 PM 2ME

% Control 10 PM 2ME Antagonism 25 1 Synergism Combination Index (CI) Combination 0 0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 ABZ (M) Dose (M)

B. 5 100 ABZ 0.25 PM 2ME 0.25 PM 2ME 4 0.5 PM 2ME 75 0.5 PM 2ME 0.75 PM 2ME

0.75 PM2ME 3 1 PM 2ME 50 1 PM 2ME 10 PM 2ME

% Control 10 PM 2ME 2 25 Antagonism 1 Synergism 0 Index (CI) Combination

-9 -8 -7 -6 -5 -4 0 10 10 10 10 10 10 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 Dose (M) ABZ (M)

C. 5 100 ABZ 0.25 PM 2ME 0.25 PM 2ME 4 0.5 PM 2ME 75 0.5 PM2ME 0.75 PM 2ME 0.75 PM 2ME 3 1 PM 2ME 50 1 PM 2ME 10 PM 2ME

% Control 10 PM 2ME 2 25 Antagonism 1

0 Index (CI) Combination Synergism

10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 0 10-9 10 -8 10 -7 10 -6 10-5 10 -4 Dose (M) ABZ (M)

Figure 5.2. Dose response curves (left) and cytotoxic interaction between ABZ and 2ME (right). HCT-116 cells were exposed to ABZ and 2ME for 72 hours, simultaneously (A), or treated with ABZ for 24 hours followed by additional 48 hours treatment with 2ME (B), or treated with 2ME for 24 hours followed by additional 48 hours treatment with ABZ (C). CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM. Chapter 5Chapter

118

5.2.2. Interaction between CLC, CA4, ABZ and 2ME

It is generally believed that drugs with similar mechanisms of action cannot interact synergistically. In the previous chapter, a synergism between ABZ and CLC, and ABZ and 2ME were observed. Additionally, whereas several studies have shown that ABZ binds to the colchicine-binding site on β-tubulin, one report suggested that benomyl, an agent belongs to benzimidazole group, binds to a novel site on β-tubulin 565. Therefore, to gain further insight into the action of colchicine-domain binders, 2ME, CA4 and CLC were evaluated for their interactions with CLC and with one another. In addition, the effect of the combination of ABZ and CA4 was also evaluated. It was postulated that if the combination of CA4 and 2ME with CLC, and with each other exhibit antagonism, it would be reasonable to conclude that either ABZ binds to a distinct site on β-tubulin or ABZ is an exception in terms of representing a synergistic interaction with CLC-domain binders.

Before evaluating the effect of CA4 in combination with other agents, its IC50 value as a single agent on HCT-116 and DU-145 was assessed. As shown in figure 5.3 following CA4 treatment, a concentration-dependent growth inhibition was observed in both cell lines. The IC50 values for HCT-116 and DU145 were 0.29 ± 0.11 nM and 0.39 ± 0.14 nM, respectively.

5Chapter

119

A. 100 CA4 75

50

% Control 25

0 10 -1 1 10 -1 0 10 -9 10 -8 Dose (M)

B. 100 CA4 75

50

% Control 25

0 10 -1 1 10 -1 0 10 -9 10 -8 Dose (M)

Figure 5.3. Dose-response curves for 72-hour CA4 treatment on HCT-116 (A) and DU145 (B). Cells were treated with CA4 at concentrations ranging from 0.05 nM to 1 nM. After 72 hours incubation, cells were subjected to the SRB assay. Each point is shown as mean ± SEM.

5Chapter 120

5.2.2.1. Interaction between ABZ and CA4

Cytotoxic interaction between ABZ and CA4 was assessed over a period of 72 hours treatment. Dose-response curves for HCT-116 and DU-145 cell lines are shown in figure 5.4.A and 5.4.B (left panel), respectively. Each curve represents a particular dose of CA4 in combination with various doses of ABZ.

In both cell lines, at doses below 1 μM ABZ, there was a left-sided shift of the combination curves compared to ABZ alone, indicating enhanced growth inhibitory effect of the combination. However, at doses above 1 μM ABZ, combination curves crossed over to the right side of the ABZ dose-response curve, indicating that CA4 was reducing the inhibitory effect of ABZ on cell viability. CA4 at doses below 0.5 nM caused approximately 90% growth inhibition with almost no additional effect when ABZ was added.

Interaction between ABZ and CA4 in HCT-116 cell line is shown in figure 5.4.A. (right panel). Regardless CA4 concentrations, combination of ABZ concentrations ranging from 0.1 μM to 0.75 μM with CA4 doses ranging from 0.05 nM to 0.5 nM resulted in synergism. At 10 μM ABZ, however, antagonism was observed throughout the range of tested CA4 concentrations.

In DU145, combination of ABZ concentrations below 1 μM with CA4 at concentrations up to 0.5 nM resulted in synergism. However, there was an increase in CI values as the concentration of CA4 was increased from 0.5 nM to 1 nM, indicating that interaction was changing from synergism to antagonism (figure 5.4.B, right panel). Chapter 5Chapter

121

A. 4 100 ABZ 0.05 nM CA4 0.05 nM CA4 0.1 nM CA4 0.1 nM CA4 75 3 0.2 nM CA4 0.2 nM CA4 0.3 nM CA4 0.3 nM CA4 50 0.4 nM CA4 2 0.4 nM CA4 0.5 nM CA4

% Control 0.5 nM CA4 0.75 nM CA4 25 Antagonism 0.75 nM CA4 1 nM CA4 1 Synergism 1 nM CA4 0 (CI) Index Combination

-9 -8 -7 -6 -5 -4 0 10 10 10 10 10 10 10-9 10-8 10-7 10-6 10-5 10-4 Dose (M) ABZ (M)

B. 4 100 ABZ 0.05 nM CA4 0.05 nM CA4 0.1 nM CA4 0.1 nM CA4 3 75 0.2 nM CA4 0.2 nM CA4 0.3 nM CA4 0.3 nM CA4 0.4 nM CA4 50 0.4 nM CA4 2 0.5 nM CA4 0.5 nM CA4 % Control 0.75 nM CA4 25 0.75 nM CA4 Antagonism 1 nM CA4 1 1 nM CA4 Synergism 0 (CI) Index Combination

-9 -8 -7 -6 -5 -4 0 10 10 10 10 10 10 10-9 10-8 10-7 10-6 10 -5 10-4 Dose (M) ABZ (M)

Figure 5.4. Effect of the combination of ABZ and CA4 on HCT-116 (A) and DU145 (B) cell lines. Cytotoxicity was evaluated after treating the cells with ABZ, CA4 and their combination for 72 hours. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3).

122 Chapter 5

5.2.2.2. Interaction between CLC and CA4

Interaction between CA4 and CLC was evaluated following 72 hours simultaneous treatment. The dose-response curves for HCT-116 and DU145 cell lines are shown in the left panel of figure 5.5.A and 5.5.B, respectively. Each curve represents a particular dose of CA4 in combination with various doses of CLC.

In both cell lines, there was a left-sided shift of the combination curves compared with CLC alone, implying an increase in anti-proliferative effect of the combination compared with CLC as a single agent.

The interaction between CLC and CA4 in HCT-116 and DU145 cell lines are shown in right panel of figure 5.5.A and 5.5.B, respectively. In both cell lines, addition of CA4 at doses below 0.5 nM to doses up to 50 nM CLC was synergistic. 1 nM CLC was antagonistic with CA4 regardless of CA4 concentrations. In addition, CA4 at concentrations above 0.5 nM was antagonistic with all CLC concentrations. Overall, at fractions affected higher than 0.9 (>90% cell kill), the interaction between the two agents was antagonistic. Chapter 5Chapter

123

A. 4 100 CLC 0.05 nM CA4 0.05 nM CA4

0.1 nM CA4 0.1 nM CA4 75 3 0.2 nM CA4 0.2 nM CA4 0.3 nM CA4 0.3 nM CA4 50 0.4 nM CA4 2 0.4 nM CA4 0.5 nM CA4

% Control 0.5 nM CA4 0.75 nM CA4 25 Antagonism 0.75 nM CA4 1 nM CA4 1 Synergism 1 nM CA4

(CI) Index Combination 0 0 10 -1 0 10 -9 10 -8 10 -7 10 -6 10-10 10-9 10-8 10-7 10-6 Dose (M) CLC (M) B. 4 100 CLC 0.05 nM CA4 0.05 nM CA4 0.1 nM CA4 75 3 0.1 nM CA4 0.2 nM CA4 0.2 nM CA4 0.3 nM CA4 0.3 nM CA4 50 0.4 nM CA4 2 0.4 nM CA4 0.5 nM CA4

% Control 0.5 nM CA4 0.75 nM CA4 25 Antagonism 0.75 nM CA4 1 nM CA4 1 Synergism 1 nM CA4 (CI) Index Combination 0

-1 0 -9 -8 -7 -6 0 10 10 10 10 10 10-10 10-9 10-8 10-7 10-6 Dose (M) CLC (M)

Figure 5.5. Effect of the combination of CLC and CA4 on HCT-116 (A) and DU145 (B) cell lines. Cytotoxicity was evaluated after treating the cells with CLC, CA4 and their combination for 72 hours. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3).

124 Chapter 5

5.2.2.3. Interaction between CLC and 2ME

The dose-response curves for 72 hours simultaneous treatment of HCT-116 and DU145 cells are shown in the left panel of figure 5.6.A and 5.6.B, respectively. Each curve represents a particular dose of CLC in combination with various doses of CLC.

Overall, in both cell lines, combination of CLC with 2ME resulted in enhanced growth inhibition compared to CLC alone. At concentrations below 2.5 nM CLC, there was a shift to the left side of the curves compared to the growth inhibitory curve of CLC as a single agent. However, at higher concentrations of CLC, a right- sided shift in combination curve was observed.

The cytotoxic interaction between CLC and 2ME on HCT-116 and DU145 are shown in the figure 5.6.A and 5.6.B (right panel), respectively. In both cell lines, combination of 2ME ranging from 0.25 μM to 5 μM with doses below 0.1 μM CLC resulted in synergism, whereas addition of 10 and 50 μM 2ME to CLC resulted in an antagonistic interaction.

Chapter 5Chapter

125

A. 4 100 CLC 0.25 PM 2ME 0.25 PM 2ME 3 0.5 PM 2ME 75 0.5 PM 2ME 0.75 PM 2ME 0.75 PM 2ME 1 PM 2ME 50 1 PM 2ME 2 2.5 PM 2ME 2.5 PM 2ME 5 PM 2ME % Control 5 PM 2ME 25 Antagonism 10 PM 2ME 10 PM 2ME 1 Synergism 50 PM 2ME 50 PM 2ME (CI) Index Combination 0 0 10 -1 0 10 -9 10 -8 10 -7 10 -6 10-10 10-9 10-8 10 -7 10-6 CLC (M) Dose (M) B. 4 100 CLC 0.25 PM 2ME 0.25 PM 2ME 0.5 PM 2ME 75 0.5 PM 2ME 3 0.75 PM 2ME 0.75 PM 2ME 1 PM 2ME 50 1 PM 2ME 2 2.5 PM 2ME 2.5 PM 2ME % Control 5 PM 2ME 5 PM 2ME 25 Antagonism 10 PM 2ME 10 PM 2ME 1 50 PM 2ME Synergism 50 PM 2ME 0 Combination Index (CI)

-1 0 -9 -8 -7 -6 0 10 10 10 10 10 10-10 10-9 10-8 10-7 10-6 Dose (M) CLC (M)

Figure 5.6. Effect of the combination of CLC and 2ME on HCT-116 (A) and DU145 (B) cell lines. Cytotoxicity was evaluated after treating the cells with CLC, 2ME and their combination for 72 hours. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3).

126 Chapter 5

5.2.2.4. Interaction between 2ME and CA4

Interaction between 2ME and CA4 was evaluated following 72 hours simultaneous treatment. The dose-response curves for HCT-116 and DU145 cell lines are shown in the left panel of figure 5.7.A and 5.7.B, respectively. Each curve represents a particular dose of CA4 in combination with various doses of 2ME.

At doses below 5 μM 2ME in HCT-116 and below 2.5 μM 2ME in DU145, there was a left-sided shift of the combination curves, indicating increased cytotoxic effect in combination treatment. Higher concentrations of 2ME resulted in a reduction in anti-proliferative effect as shown by a right-sided shift to the 2ME dose-response curve (Figure 5.7, left panel).

Cytotoxic interaction between CA4 and 2ME on HCT-116 and DU145 are shown in the right panel of figure 5.7.A and 5.7.B, respectively. CA4 concentrations below 0.75 nM and 2ME below 10 μM in HCT-116 represented a synergistic interaction, whereas at higher doses, antagonism was observed. In DU145 cells, at doses below 50 μM 2ME a synergistic interaction was observed between the two agents. However, treatment with doses higher than 50 μM 2ME resulted in antagonism.

Chapter 5Chapter

127

A. 4 100 2ME 0.05 nM CA4 0.05 nM CA4 3 0.1 nM CA4 75 0.1 nM CA4 0.2 nM CA4 0.2 nM CA4 0.3 nM CA4 50 0.3 nM CA4 2 0.4 nM CA4 0.4 nM CA4 0.5 nM CA4 % Control 0.5 nM CA4 25 Antagonism 0.75 nM CA4 0.75 nM CA4 1 Synergism 1 nM CA4 1 nM CA4 (CI) Index Combination 0 0 -7 -6 -5 -4 10-7 10-6 10-5 10-4 10-3 10 10 10 10 Dose (M) 2ME (M)

B. 4 0.05 nM CA4 100 2ME 0.1 nM CA4 0.05 nM CA4 3 75 0.1 nM CA4 0.2 nM CA4 0.2 nM CA4 0.3 nM CA4 50 0.3 nM CA4 2 0.4 nM CA4 0.4 nM CA4 0.5 nM CA4

% Control 0.5 nM CA4 Antagonism 0.75 nM CA4 25 1 0.75 nM CA4 Synergism 1 nM CA4 1 nM CA4 (CI) Index Combination 0 0 -7 -6 -5 -4 -3 10 -7 10 -6 10 -5 10 -4 10 10 10 10 10 2ME (M) Dose (M)

Figure 5.7. Effect of 2ME and CA4 on HCT-116 (A) and DU-145 (B) cell lines. Cytotoxicity was evaluated after treating the cells with CA4, 2ME and their combination for 72 hours. CI<1 indicates synergism, CI >1 indicates antagonism and CI=1 shows additive effect. Each point is shown as mean ± SEM (n=3).

128 Chapter 5

5.2.3. Interaction between ABZ and 2ME

In order to gain further insights into the mechanism of synergistic interaction between ABZ and 2ME, the effect of the combination therapy on tubulin polymerisation, acetylated tubulin, angiogenesis-related proteins, and apoptosis- related proteins were investigated. In all experiments, cells were treated simultaneously with ABZ and 2ME. With regard to time point, preliminary experiments showed that the effect of both ABZ and 2ME could be observed after 24 hours treatment. The optimal concentrations of ABZ and 2ME in combination were determined in a 24-hours growth inhibition assay and low concentrations, which had minimal effect on cell proliferation, were chosen to evaluate the drug effect on apoptosis-related proteins and tubulin depolymerisation (table 5.1). In this way, the sensitivity for the detection of the combined effect was maximised. The IC50 values of 24 hours treatment were 10 ± 9 μM for ABZ and 28 ± 17 μM for 2ME. To assess the drug effect on angiogenesis-related protein, high concentrations of ABZ and 2ME were used.

2ME (0.75 μM) Combination ABZ ABZ Effect Effect Effect Dose (μM) (% cell kill) (% cell kill) (% cell kill)

0.1 11.31 ± 3.19 20.18 ± 4.32 39.77 ± 4.09 0.25 16.27 ± 2.97 50.71 ± 5.30

0.5 26.75 ± 5.63 65.33 ± 6.03

Table 5.1. Percentage of cells that were affected by the combination of ABZ and 2ME following 24 hours treatment

Chapter 5Chapter

129

5.2.3.1. Effect of Combination of ABZ and 2ME on Microtubule--related Proteins

5.2.3.1.1. α-tubulin

It has been previously shown that both ABZ and 2ME depolymerise tubulin in a dose-dependent manner 573, 574. The aim of this experiment was to determine if the synergy between ABZ and 2ME resulted from their synergistic effect on tubulin depolymerisation. This effect was analysed using a quantitative tubulin polymerisation assay in HCT-116 cells. The assay is based on the fact that during the extraction of tubulin, polymerised tubulins remain in the pellet form after centrifugation, as they are not soluble in microtubule-stabilising buffer 574. Therefore, tubulin-depolymerising agents increase the level of soluble tubulin in the supernatant whereas tubulin-stabilising agents enhance insoluble tubulin in the pellet.

As shown in figure 5.8, combination of ABZ and 2ME had no effect on microtubule polymerisation compared with control (p>0.05). Similarly, combination therapy did not increased de-polymerisation of tubulin compared with control and single agents.

Chapter 5Chapter

130

A.

ABZ (μM) - 0.1 0.25 - 0.1 0.25

2ME (μM) - - - 0.75 0.75 0.75

P S P S P S P S P S P S

B.

50 Control 0.1 PM ABZ 40 0.25 PM ABZ 30 0.75 PM 2ME

20 0.1 PM ABZ+ 2ME 0.25 PM ABZ+2ME 10 % Polymerised Tubulin Polymerised % 0

Figure 5.8. Effect of ABZ and 2ME on tubulin polymerisation in HCT-116 cells. A. Representative blot from four experiments. Cells were treated with the indicated concentrations of ABZ, 2ME, and their combination for 24 hours. Cells were then lysed with a microtubule-stabilising buffer, and polymerised (P) and the soluble (S) tubulin fractions were separated by centrifugation. Each fraction was resolved on 12% gels and probed with an antibody against α-tubulin. B. The percentage of polymerised tubulin (% P) was determined by dividing the densitometric value of polymerised tubulin by the total tubulin content.

Chapter 5 Chapter

131

5.2.3.1.2. Acetylated α-tubulin

Membranes from tubulin polymerisation assay were re-probed with antibody against acetylated α-tubulin. Tubulin acetylation is an established marker of microtubule stability. 2ME has been shown to reduce tubulin acetylation in MDA-MB-231 breast cancer and 1A9 ovarian cancer cell lines 447, 575. Additionally, in rat orthotopic brain tumour model, 2ME-induced de-acetylation has been reported to be correlated with the suppression of tumour growth 447.

Representative membranes from four experiments are shown in figure 5.9. Similar to tubulin polymerisation assay, neither ABZ nor 2ME had effect on tubulin acetylation compared with control (p>0.05). In addition, no reduction in tubulin acetylation was observed in cells that were treated with the combination of ABZ and 2ME compared with single agents or vehicle-treated cells.

Chapter 5Chapter

132

A.

ABZ (μM) - 0.1 0.25 - 0.1 0.25

2ME (μM) - - - 0.75 0.75 0.75

P S P S P S P S P S P S

B.

60 Control 0.1 PM ABZ 40 0.25 PM ABZ 0.75 PM 2ME 0.1 PM ABZ+2ME 20 0.25 PM ABZ+2ME

% Acetylated% Tubulin 0

Figure 5.9. Effect of ABZ and 2ME on tubulin acetylation in HCT-116 cells. A. representative blot from four experiments. Membranes from tubulin polymerization assay were re-probed with antibodies against acetylated α-tubulin. B. The percentage of acetylated tubulin was determined by dividing the densitometric value of polymerised tubulin by the total tubulin content.

Chapter 5 Chapter

133

5.2.3.2. Effect of Combination of ABZ and 2ME on Apoptosis--related Proteins

ABZ induces apoptosis through intrinsic pathway of apoptosis as evidenced by the release of cytochrome c from mitochondria in a variety of human cancer cell lines 402. 2ME activates both the extrinsic pathway 321 and the intrinsic pathway of apoptosis 576, 577.

To investigate whether the observed cytotoxic effect of the combination of ABZ and 2ME occurred via induction of apoptosis, activities of caspase 8 and caspase 9, as the initiators of extrinsic and intrinsic pathways of apoptosis were assessed. In addition, the activity of the downstream effector, caspase 3 was also evaluated.

As depicted in figure 5.10.A and 5.10.B, Caspase-8 and -3 were activated after 16 hours in cells that were treated with ABZ, 2ME and ABZ plus 2ME, as evidenced by the release of pNA from the caspases substrates. The concentrations of pNA were statistically significantly higher in cells treated with the combination of ABZ and 2ME compared to the cells that were treated with single agent (p<0.05). In contrast, neither single agents nor their combination altered the activity of caspase 9, suggesting that the apoptosis induced by the drugs was not mediated through intrinsic pathway of apoptosis (figure 5.10.C).

To further investigate the signaling events involved in the apoptosis induced by combination therapy, the effect of the combination treatment on DR5 protein levels was determined using western blot analysis. As shown in figure 5.11, ABZ and 2ME significantly upregulated the expression of DR5 compared with vehicle-treated cells (p<0.05). In addition, combination of the two agents further increased the levels of DR5 protein compared with single agents (p<0.05). These results suggest that the activation of caspase-8 is, at least in part, dependent on death receptor signaling.

Because HCT-116 cells harbor wild-type p53, it was hypothesised that upregulation of DR5 could be a consequence of p53-induced growth arrest. To test this hypothesis, the levels of p53 protein were determined by western blotting. As shown in figure 5.12, no significant difference in p53 levels was observed among control

and treated cells, suggesting that the drug-induced cell kill, as well as upregulation 5Chapter of DR5 receptor is independent from p53. 134

A. 0.6 4 hrs 8 hrs 16 hrs 0.4 24 hrs

0.2 Caspase Activity

0.0 M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M 2ME M 2ME M 2ME M 2ME Control Control Control Control P P P P P P P P P P P P

0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25 M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME 0.75 0.75 0.75 0.75 P P P P P P P P 0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25 B. 0.8 4 hrs 8 hrs 0.6 16 hrs 24 hrs 0.4

0.2 Caspase Activity

0.0 M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M 2ME M 2ME M 2ME M 2ME Control Control Control Control P P P P P P P P P P P P

0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25 M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME 0.75 0.75 0.75 0.75 P P P P P P P P 0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25

C. 0.4 4 hrs 8 hrs 0.3 16 hrs 24 hrs

0.2

0.1 Caspase Activity 0.0

M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M ABZ M 2ME M 2ME M 2ME M 2ME Control Control Control Control P P P P P P P P P P P P

0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25 M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME M ABZ+2ME 0.75 0.75 0.75 0.75 P P P P P P P P

0.1 0.1 0.1 0.1 0.25 0.25 0.25 0.25

Figure 5.10. Effect of ABZ, 2ME, and their combintion on caspase activation. HCT-116 cells were incubated with vehicle (2% ethanol), ABZ (0.1 and 0.25 μM), 2ME (0.75 μM), and their combination for 4, 8, 16, 24 hours. Caspase activity was determined using caspase substrates labeled with p-nitroaniline (pNA) using colorimetic assay that detects pNA released by A. activated caspase -8, B. activated caspase-3, and C. activated caspase-9 in lysates of the cells. The data represent the mean values for duplicate measurements from Chapter 5Chapter three experiments and error bars represent SEM. 135

A.

ABZ (μM) - 0.1 0.25 0.5 - 0.1 0.25 0.5

2ME (0.75 μM) - - - - + + + +

B.

3 Control

0.1 PM ABZ 0.25 PM ABZ 2 0.5 PM ABZ 0.75 PM 2ME

1 0.1 PM ABZ+2ME 0.25 PM ABZ+2ME

Relative Intensity 0.5 PM ABZ+2ME 0

Figure 5.11. Effect of ABZ, 2ME and their combination on DR5 protein expression. A. Immunoblot analysis of DR5 expression. HCT-116 cells were treated with vehicle, ABZ, 2ME, and ABZ plus 2ME at indicated concentration for 24 hours. Cells were then lysed and subjected to immunoblot analysis for DR5 protein. The blot is representative of three separate experiments. B. The bar graph represents the mean DR5 expression of three individual experiments, with error bars showing SEM.

5 Chapter

136

A.

ABZ (μM) - 0.1 0.25 0.5 - 0.1 0.25 0.5

2ME (0.75 μM) - - - - + + + +

B.

β-actin

C.

1.5 Control

0.1 PM ABZ 1.0 0.25 PM ABZ 0.5 PM ABZ 0.75 PM 2ME 0.5 0.1 PM ABZ+2ME Relative Intensity 0.25 PM ABZ+2ME 0.0

Figure 5.12. Effect of ABZ, 2ME and their combination on P53 protein expression. A. Immunoblot analysis of P53 expression. Membranes from DR5 detection (figure 5.11) were re-probed with antibodies against P53. The blot is representative of three separate experiments. β-actin was used as a control for equal protein loading. B. The bar graph represents the mean P53 expression of three individual experiments, with error bars showing SEM.

Chapter 5 Chapter

137

5.2.3.3. Effect of Combination of ABZ and 2ME on Angiogenesis-related Proteins

Both 2ME and ABZ have been shown to inhibit angiogenesis in various in vivo models. This anti-angiogenic effect is at least in part, due to the down-regulation of HIF-1α protein 353, 404, 406. In vitro inhibition of HIF-1α and VEGF required higher concentrations of the drugs compared with the concentrations that inhibit cell proliferation. Therefore, according to previous studies, high concentrations of ABZ and 2ME for these experiments were used.

5.3.3.3.1. Hypoxia Inducible Factor 1α (HIF-1α)

Given that microtubule-targeted drugs down-regulate HIF-1α protein 356, it was hypothesised that ABZ and 2ME may synergistically suppress the expression of HIF-1α. To test this hypothesis, HCT-116 cells were treated with 1 μM ABZ, 50 μM 2ME and the combination of the two agents. Cells were then left in normoxia or exposed to hypoxia for six hours.

As shown in figure 5.13, combination of ABZ and 2ME significantly reduced the expression of HIF-1α compared with single agents and control (p<0.05). In normoxia condition, no HIF-1α was detected.

5Chapter

138

CTL ABZ 2ME ABZ+2ME

HIF-1α

β-actin

1.0

0.8

0.6

0.4

Intensity Relative 0.2

0.0 Control ABZ 2ME ABZ+2ME

Figure 5.13. Effect of ABZ, 2ME, and their combination on HIF-1α expression in hypoxia. HCT-116 cells were treated with 1 μM ABZ, 50 μM 2ME, and their combination. Following six hours incubation in hypoxia, the cells were lysed and subjected to immunoblot analysis for HIF-1α. The blot is representative of three separate experiments. β- actin was used as a control for equal protein loading. The bar graph represents the mean HIF-1α expression of three individual experiments, with error bars showing SEM.

Chapter 5 Chapter

139

5.2.3.3.2. Vascular Endothelia Growth Factor (VEGF)

To determine the effect of the combination of ABZ and 2ME on transcriptional activity of HIF-1α, VEGF levels were measured in conditioned medium from HCT- 116 cells that were treated with ABZ and 2ME as single agents and in combination. To ensure that the effect of the drugs on VEGF levels was not due to the cytotoxicity, cytotoxicity assay was performed in parallel with measurement of VEGF levels in the cell supernatants.

Treating the cells with ABZ and 2ME significantly decreased the level of VEGF in hypoxia condition (p<0.05). In contrast, no significant reduction in VEGF levels was observed in cells that were incubated in normoxia condition, as the percentage of VEGF reduction was directly proportional to the percentage of cell kill (p>0.05) (figure 5.14). Similar to single agent treatment, VEGF expression was not significantly affected by combined therapy under normoxia condition, whereas in hypoxia condition, combination therapy resulted in a reduction in VEGF levels. However, this synergistic interaction in suppression of VEGF was only evident at the highest 2ME concentration (p=0.016) (figure 5.15.C). As shown in figure 5.15.A and 5.15.B, treatment with 5 μM and 10 μM 2ME in combination with ABZ resulted in a marginal and non-significant reduction of VEGF (p>0.05).

Interestingly, hypoxic HCT-116 cells were less sensitive to the anti-proliferative effects of ABZ and 2ME as single agents compared to the normoxic cells (figure 5.14). This effect was observed in all three tested concentrations of ABZ and 2ME. Similar to the single agents, hypoxic cells were also more resistant to the combination therapy compared with the normoxic cells (figure 5.15).

Chapter 5Chapter

140

A. B. 80 80 Cytotoxicity Cytotoxicity VEGF Reduction 60 VEGF Reduction 60

40 40 % Control

20 % Control 20

0 15 10 0 15 10 Dose (PM) Dose (PM)

C. D.

60 60 Cytotoxicity Cytotoxicity VEGF Reduction VEGF Reduction 40 40

% Control 20 % Control 20

0 0 510 50 510 50

Dose (PM) Dose (PM)

Figure 5.14. Effect of single-agent treatment on VEGF and cytotoxicity. HCT-116 were plated at a density of 14000 cells/well and were treated with various concentrations of ABZ (A,B) and 2ME (C,D) in serum-free medium and incubated for 16 hours. The plates were then incubated for an additional 6 hours in the incubator in normoxia (left panel) or placed in hypoxia chamber (right panel). The supernatants were collected subjected to VEGF ELISA assay and SRB assay was performed on cells.

Chapter 5Chapter

141

80 5 PM 2ME 80 10 PM 2ME 80 50 PM 2ME

60 60 60

40 40 40 % Control % Control % Control 20 20 20

0 0 0 15 10 15 10 15 10 ABZ (PM) ABZ (PM) ABZ (PM)

5 PM 2ME 80 80 10 PM 2ME 100 50 PM 2ME

60 60 80

60 40 40

% Control 40 % Control 20 20 % Control 20 0 0 15 10 15 10 0 15 10 ABZ (PM) ABZ (PM) ABZ (PM)

Cytotoxicity- ABZ VEGF Reduction-ABZ Cytotoxicity-ABZ+2ME VEGF Reduction-ABZ+2ME

Figure 5.15. Effect of combination therapy on VEGF and cytotoxicity. Combination of ABZ with 2ME in normoxia (top panel) and hypoxia (bottom panel) condition. HCT-116 were plated at a density of 14000 cells/well treated with various concentrations of ABZ, 2ME and their combination in serum free medium and incubated for 16 hours. The plates were then incubated for an additional 6 hours in the incubator in normoxia or placed in hypoxia chamber . The supernatants were collected subjected to VEGF ELISA assay and SRB assay was performed on cells.

142 Chapter 5

5.2.4. Comparison of the Effect of 2ME-ethanol and 2ME/CMC/HPßCD In Vitro

It has been demonstrated that water-soluble polymers such as HPMC and CMC influence the interaction between drug and cyclodextrins by increasing the solubilising effect of cyclodextrins 578. Therefore, a solution of 2ME containing 0.5% CMC and 25% HPβCD was prepared and its cytotoxicity was evaluated in HCT-116 and DU145 cells. In addition, the cytotoxicity of 2ME/CMC/HPβCD was compared with 2ME dissolved in ethanol (2ME/EtOH). Cells were incubated with various concentrations of 2ME/CMC/HPβCD and 2ME/EtOH ranging from 0.01 to 100 μM. After 72 hours incubation, cytotoxicity was assessed by SRB assay. To ascertain whether drug-free vehicle had an effect on cell viability, cytotoxicity of CMC/HPβCD was also evaluated.

As shown in figure 5.16.A and 5.16.B, Drug-free vehicle had no cytotoxic effect on the cells. 2ME/CMC/HPβCD formulation showed a dose-dependent toxicity on HCT-116 and DU-145 cell lines. The IC50 values of 2ME/CMC/HPβCD and 2ME/EtOH for HCT-116 were 1.1 ± 0.3 μM and 0.93 ± 0.2 μM (p>0.05), respectively. In DU-145, the IC50 values were 2.1 ± 0.6 μM 2ME/CMC/HPβCD and 1.9 ± 0.9 μM for 2ME/EtOH (p>0.05). These results suggest that the cytotoxic effect of 2ME was not hampered by the formulation.

Chapter 5 Chapter

143

A. 100 2ME/EtOH 2ME/CMC/HPECD 75 Vehicle (CMC/HPECD) 50 % Control 25

0 10 -9 10 -8 10 -7 10 -6 10 -5 10 -4 10 -3 Dose (M)

B. 100 2ME/EtOH

2ME/CMC/HPECD 75 Vehicle (CMC/HPECD)

50

% Control 25

0 -9 -8 -7 -6 -5 -4 -3 10 10 10 10 10 10 10 Dose (M)

Figure 5.16. Dose response curves of 2ME/CMC/HPβCD and 2ME/EtOH in HCT-116 (A) and DU145 (B) cells. Cells were exposed to various concentrations of 2ME/CMC/HPβCD and 2ME/EtOH ranging from 0.01 to 100 μM, incubated for 72 hours and subjected to SRB assay.

5 Chapter 144

5.2.5. Toxicity Evaluation of Simultaneous Administration of ABZ and 2ME Combination In Vivo

Prior to in vivo combination experiment, a pilot study was carried out to evaluate the possible toxicity of simultaneous administration of ABZ and 2ME. Animals were treated with 50 mg/kg ABZ in combination with 25 mg/kg 2ME treatment, as well as the combination of ABZ vehicle and 2ME vehicle. Following administration, mice manifested evidence of acute toxicity such as sedation, convulsion, and haematuria. These manifestations were also observed in vehicle-treated mice, indicating that the toxicity resulted from the combination of the vehicles rather than the drugs, themselves.

5.2.6. Comparison of the Efficacy of Various Dose of ABZ and 2ME on Tumour Growth In Vivo

An initial in vivo dose-response experiment was carried out in order to evaluate the efficacy of various doses of ABZ and 2ME in combination. Based on the data from toxicity experiment (section 5.3.5) sequential schedule was chosen for in vivo experiment.

Mice were inoculated subcutaneously with HCT-116 cells in the hind leg. When tumour size reached 100 mm3, animals were randomised in five treatment groups containing 4 mice in each group. Mice were treated with ABZ followed by 2ME after 24 hours. After three weeks, mice were euthanized and tumours were excised. As shown in figure 5.17, ABZ at concentrations of 25 mg/kg and 50 mg/kg in combination with 25 mg/kg 2ME had a statistically significant inhibitory effect on tumour growth compared with control group (p=0.0017 and p=0.0006, respectively). In contrast, combination of 10 mg/kg 2ME with either doses of ABZ had no inhibitory effect on tumour growth. Indeed, an antagonistic interaction was observed when 10 mg/kg 2ME was added to the 50 mg/kg ABZ. In contrast to simultaneous treatment, sequential administration of ABZ and 2ME had no adverse effect on animals’ well-being. Chapter 5 Chapter

145

A.

1000 Control 3) 800 50 mg/kg ABZ+10 mg/kg 2ME

600 25 mg/kg ABZ+10 mg/kg 2ME 50 mg/kg ABZ+25 mg/kg 2ME 400 25 mg/kg ABZ+25 mg/kg 2ME

200 Tumor Volume (mm 0

0 3 6 9 12 15 18 21 Day

B. 1.0 Control 0.8 50 mg/kg ABZ+10 mg/kg 2ME 25 mg/kg ABZ+10 mg/kg 2ME 0.6 50 mg/kg ABZ+25 mg/kg 2ME 25 mg/kg ABZ+25 mg/kg 2ME 0.4

Tumor wei0.2 ght (g)

0.0 Treatment groups

Figure. 5.17. Effect of the various concentrations of ABZ and 2ME on HCT-116 xenograft tumour growth. 2x106 HCT-116 cells were injected subcutaneously to the hind leg of animals. When tumour volume reached approximately 100 mm3, animals were randomly assigned to five groups (four mice per group) and given the vehicle or the combination of ABZ and 2ME. Drugs were administered sequentially with 24 hours interval for three weeks. Tumour size was measured every three days. Results are expressed as mean tumour volume (A) and mean tumour weight (B) and error bars represented SEM.

5 Chapter

146

5.2.7. Effect of the Combination of ABZ and 2ME on the Survival of Tumour Bearing Mice

On the basis of the dose-response results (figure 5.17), in a subsequent experiment, mice were treated with 25 mg/kg and 50 mg/kg ABZ in combination with 25 mg/kg 2ME. Mice were inoculated with 2x106 HCT-116 cells. When the tumour size reached approximately 100 mm3, animals were randomized into 6 groups of 8-9 animals and were treated with the vehicle, 50 mg/kg ABZ, 25 mg/kg ABZ and 25 mg/kg 2ME and the combination of ABZ plus 2ME. To assess the effect of individual drugs, animals were treated with ABZ on day 1 and the vehicle of 2ME on day 2, or ABZ vehicle on day 1 and 2ME on day 2. Combination therapy group received ABZ on day 1 followed by 2ME 24 h later (day 2). Control animals received vehicle alone. Mice were euthanized when the tumour size reached 1000 mm3.

Treatment with 50 mg/kg ABZ resulted in a significant delay in tumour growth over control (p=0.0011). In contrast, 25 mg/kg 2ME as a single agent had no significant effect on the survival of the mice compared to the vehicle-treated group (p>0.05). Additionally, no survival benefit was observed in mice that were treated with the combination of 50 mg/kg ABZ and 25 mg/kg 2ME. In fact, the combination exhibited an antagonistic response, as the median survival of the animals that were treated with the combination of ABZ and 2ME was less than that in mice that were given ABZ as a single agent (29.5 days versus 41 days) (figure 5.18). This result was consistent with in vitro drug interaction analysis where combination of 2ME with high doses of ABZ resulted in an antagonistic effect on HCT-116 and DU145 cell proliferation (figure 5.1.B).

In contrast to the combination of high dose of ABZ with 2ME, 25 mg/kg ABZ combined with 25 mg/kg 2ME provided a survival advantage over single agent treatment (fig 5.19). Median survival of mice that were treated with 25 mg/kg ABZ and 25 mg/kg 2ME were 31 and 29.5 days, respectively, whereas in animals that received the combination of the two agents, median survival was 40.5 days. Log-rank P-values

for the comparison between treated and untreated mice are represented in Table 5.2 and 5 Chapter 5.3. 147

100 Control 80 50 mg/kg ABZ

60 25 mg/kg 2ME Combination 40

Percent survival Percent 20

0 10 20 30 40 50 Days post-treatment

Figure 5.18. In vivo response of HCT-116 xenografts to high dose of ABZ in combination with 2ME. Mice were inoculated with 2x106 HCT-116 cells. When the tumour size reached approximately 100 mm3, animals were treated with the vehicle control, 50 mg/kg ABZ, 25 mg/kg 2ME and a combination of the two agents. To assess the effect of individual drugs, animals were treated with ABZ on day 1 and the vehicle of 2ME on day 2, or ABZ vehicle on day 1 and 2ME on day 2. Combination therapy group received ABZ on day 1 followed by 2ME 24 h later (day 2). Control animals received vehicle alone. Mice were euthanized when the tumour size reached 1000 mm3.

Median Survival (Day) Log-Rank Control 23 - ABZ 41.5 0.0011 2ME 29.5 0.1032 Combination 29.5 0.1117

Table 5.2. Median survival analysis of animals that were treated with the vehicle, 50 mg/kg ABZ, 25 mg/kg 2ME and the combination of the two agents. The median survival was calculated using Kaplan-Meier statistics. The log-rank P-value is the comparison between each treatment group with untreated mice.

5 Chapter

148

100 Control

80 25 mg/kg ABZ

60 25 mg/kg 2ME Combination 40

Percent survival Percent 20

0 10 20 30 40 50 Days post-treatment

Figure 5.19. In vivo response of HCT-116 xenografts to low dose of ABZ in combination with 2ME.. Mice were inoculated with 2x106 HCT-116 cells. When the tumour size reached approximately 100 mm3, animals were treated with the vehicle, 25 mg/kg ABZ, 25 mg/kg 2ME and a combination of the two agents. To assess the effect of individual drugs, animals were treated with ABZ on day 1 and the vehicle of 2ME on day 2, or ABZ vehicle on day 1 and 2ME on day 2. Combination therapy group received ABZ on day 1 followed by 2ME 24 h later (day 2). Control animals received vehicle alone. Mice were euthanized when the tumour size reached 1000 mm3.

Median Survival (Days) Log-Rank P-value Control 23 - ABZ 31 0.0444 2ME 29.5 0.1032 Combination 40.5 0.0019

Table 5.3. Median survival analysis of animals treated the vehicle, 25 mg/kg ABZ, 25 mg/kg 2ME and the combination of the two agents. The median survival was calculated using Kaplan- Meier statistics. The log-rank P-value is the comparison between each treatment group with untreated mice.

5 Chapter

149

5.2.8. Effect of the Combination of ABZ and 2ME on Xenograft Tumour Cell Proliferation

To assess whether the observed anti-tumour effects of the drugs as a single agent and in combination are associated with the suppression of tumour cell proliferation, immunohistochemistry analysis of Ki67 was performed on tumour sections from all treatment groups (Figure 5.20). Ki67 is a nuclear antigen that is exclusively expressed by cells in the Gl-, G2-, S-, and M-phases of the cell cycle but not by cells in the G0- phase 579.

While both 50 mg/kg and 25 mg/kg ABZ as single agents significantly reduced the proliferation rate of tumour cells (p<0.05), 2ME had no effect on ki-67 rate. Combination therapy with 50 mg/kg ABZ and 2ME resulted in a significant decrease in tumour proliferation rate in comparison with mice that were treated with vehicle (p<0.05). However, 50 mg/kg ABZ as a single agent was more effective in the suppression of proliferation rate than the combination therapy (p<0.05). Conversely, combination of 25 mg/kg ABZ with 2ME led to a statistically significant reduction in proliferation rate, compared with vehicle-treated mice and the mice that received ABZ and 2ME as single agents (p<0.05). These results suggest that increase in survival rate of animals that were treated with the combination of 25 mg/kg ABZ and 2ME was, at least in part, due to the suppression of the tumor cells proliferation.

5 Chapter 150

A.

Vehicle 25 mg/kg 2ME 50 mg/kg ABZ

2ME + 50 mg/kg ABZ 25 mg/kg ABZ 2ME + 50 mg/kg ABZ

B. 100 Vehicle 80 25 mg/kg 2ME 50 mg/kg ABZ 60 50 mg/kg ABZ+2ME

25 mg/kg ABZ 40 25 mg/kg ABZ+2ME

% Ki67 Positive % Ki67 20

0

Figure 5.20. Effect of ABZ, 2ME and the combination of ABZ and 2ME on inhibition of tumour cell proliferation in HCT-116 xenograft mice. Paraffin-embedded sections derived from subcutaneous tumours were immunostained for the cell proliferation marker, Ki67. Ki67- positive cells were counted and reported as the percentage of the total number of cells in proliferative phases in each field. A. Representative images of Ki67-stained (brown) sections B. Average number of Ki67-positive cells. Error bars indicate SEM. Chapter 5 Chapter

151

5.2.9. Effect of the Combination of ABZ and 2ME on Tumour Angiogenesis

As discussed in chapter 1, both ABZ and 2ME have been reported to suppress tumour angiogenesis. Therefore, to examine whether inhibition of tumour growth following combination treatment was associated with a reduction in the vascularisation level of the tumours, tumour tissues were subjected to immunohistochemistry assay with an antibody against CD31, a marker of endothelial cells of blood vessels.

As shown in figure 5.21, animals that were treated with single agents had a significant decrease in the tumoral CD31 compared with mice that received no treatment (p<0.05). Similarly, tumour sections from mice that received the combination of 50 mg/kg ABZ with 2ME displayed a significant reduction in CD31 antigen in comparison with vehicle-treated group (p<0.01). However, 50 mg/kg ABZ as a single agent was more effective in reducing CD31 levels compared with the combination therapy (p<0.01). Additionally, no differences in the degree of vascularisation were observed in combination-treated group in comparison with 2ME-treated group (P>0.05). In contrast, in animals that received the combination of 25 mg/kg ABZ and 2ME, suppression of angiogenesis was more pronounced than in mice that were treated with the single agents (p<0.001). These results imply that the survival benefit of animals that were given the combination of 25 mg/kg ABZ and 2ME was in part, due to the suppression of angiogenesis.

Chapter 5 Chapter

152

A.

Vehicle 25 mg/kg 2ME 50 mg/kg ABZ

2ME + 50 mg/kg ABZ 25 mg/kg ABZ 2ME + 25 mg/kg ABZ

B.

10 Vehicle 25 mg/kg 2ME 8 50 mg/kg ABZ 6 50 mg/kg ABZ+2ME 25 mg/kg ABZ pixels)

3 4 25 mg/kg ABZ+2ME

(10 2

0 Area CD31-positive Mean

Figure 5.21. Effect of ABZ, 2ME and their combination on angiogenesis in HCT-116 xenograft tumours. Frozen sections derived from subcutaneous tumours were stained with anti- CD31 antibody. CD31-positive vessel area was measured per high-power field (103 pixels) A. Representative images of CD31-stained (brown) sections, B. Average number of CD31-positive area. Error bars indicate SEM. Chapter 5 Chapter

153

5.2.10. Effect of the Combination of ABZ and 2ME on Apoptosis in Tumour Cells

TUNEL assay was performed to determine whether ABZ, 2ME and their combination induce apoptotic cell death in tumour cells (Figure 5.22). TUNEL-positive cells in tumors that were treated with ABZ alone at both 25 and 50 mg/kg concentrations were significantly higher than vehicle-treated tumors (p<0.01). In contrast, the number of TUNEL-positive cells in animals that were treated with 2ME was not significantly different from those that received no treatment (p>0.05). Combination therapy with 50 mg/kg ABZ and 2ME was significantly less effective in inducing apoptosis in tumor cells, compared with the effect of 50 mg/kg ABZ as a single agent (p<0.01). In contrast, in animals that received the combination of 25 mg/kg ABZ and 2ME, TUNEL-positive cells were markedly higher than those that were treated with single agents (p<0.01). These results suggest that besides suppression of the proliferation rate and angiogenesis, induction of apoptosis was contributed to the survival benefit of the animals that were treated with the combination of 25 mg/kg ABZ and 2ME.

Chapter 5 Chapter

154

A.

Vehicle 25 mg/kg 2ME 50 mg/kg ABZ

2ME + 50 mg/kg ABZ 25 mg/kg ABZ 2ME + 50 mg/kg ABZ

B.

10 Vehi cl e 25 mg/kg 2ME 8 50 mg/kg ABZ 6 50 mg/kg ABZ+2ME 25 mg/kg ABZ 4 25 mg/kg ABZ+2ME

2 % of Apoptotic Cells Apoptotic of %

0

Figure 5.22. Effect of ABZ, 2ME and the combination on apoptosis in HCT-116 xenograft tumours. Paraffin-embedded sections derived from subcutaneous tumours were evaluated by TUNEL assay. A. Representative images of apoptotic cells (brown) B. Average number of apoptotic cells. Error bars indicate SEM.

5 Chapter

155

5.2.11. Effect of the Combination of ABZ and 2ME on Tumour and Plasma VEGF

To further explore the mechanism underlying the interaction between ABZ and 2ME, tumours and plasma samples of the animals from each treatment group were analysed for VEGF expression.

As shown in figure 5.23, combination of 50 mg/kg ABZ significantly reduced the expression of VEGF in tumour (p<0.01). In contrast, 25 mg/kg ABZ and 2ME as single agents had no significant effect on VEGF expression (p>0.05). Consistent with results from immunohistochemistry, combination of 50 mg/kg ABZ and 2ME was less effective than 50 mg/kg ABZ as a single agent. Conversely, 25 mg/kg ABZ combined with 2ME markedly suppressed VEGF expression.

As depicted in figure 5.24, both 25 mg/kg ABZ and 2ME failed to reduce the VEGF levels in plasma (p>0.05). In contrast, VEGF levels were significantly decreased in animals treated with 50 mg/kg ABZ (p<0.01). Combination therapy with 50 mg/kg ABZ and 2ME, had no effect on VEGF levels in comparison with vehicle (p>0.05). However, a significant reduction in VEGF levels was observed in mice treated with the combination of 25 mg/kg ABZ with 2ME compared with vehicle-treated group (p<0.01) and animals which were treated with ABZ and 2ME as single agents (p<0.01).

Chapter 5 Chapter

156

1 2 3 4 5 6

VEGF

β-actin

1.0 Vehicle 0.8 25 mg/kg ABZ 50 mg/kg ABZ 0.6 25 mg/kg 2ME 25 mg/kg ABZ+2ME 0.4 50 mg/kg ABZ+2ME

Relative Intensity 0.2

0.0

Figure 5.23. Effect of ABZ, 2ME and their combination on VEGF levels in subcutaneous HCT-116 xenograft tumour. The blot is representative of four experiments. From each treatment group, four tumours were analysed for VEGF levels. β-actin was used as a control for equal protein loading. [1] Control, [2] 25 mg/kg ABZ, [3] 50 mg/kg ABZ, [4] 25 mg/kg 2ME, [5] 25 mg/kg ABZ+2ME, [6] 50 mg/kg ABZ + 2ME. The graph shows densitometric analysis of VEGF immunoblot. Each column is shown as the mean ± SEM (n = 4).

Chapter 5 Chapter

157

25 Control

20 25 mg/kg ABZ 50 mg/kg ABZ 15 25 mg/kg 2ME 25 mg/kg ABZ+2ME 10

VEGF (pg/ml)VEGF 50 mg/kg ABZ+2ME 5

0

Figure 5.24. VEGF levels in plasma of mice-bearing HCT-116 xenograft treated with ABZ, 2ME and their combination. Following euthanasia, blood was collected by cardiac puncture and plasma samples were subjected to ELISA assay for VEGF levels. Each column represents mean VEGF levels ± SEM (n = 8-9).

Chapter 5 Chapter

158

5.3. DISCUSSION

In the present chapter, the mechanisms underlying synergy between ABZ and 2ME, two tubulin depolymerising agents were examined. Additionally, the effect of combination therapy on mice-bearing subcutaneous HCT-116 tumour was investigated.

The interaction between ABZ and 2ME was found to be dose- and schedule-dependent. While simultaneous treatment with ABZ and 2ME resulted in the most synergistic interactions compared with other schedules, pre-incubation with 2ME led to antagonism in most tested concentartions.

Synergism between MTAs has been reported previously in both pre-clinical models and in clinical trial. For example, PTX has been reported to act synergistically with vinblastine in KB3-1 cells 580, and with estramustine in metastatic hormone-refractory prostate cancer 581. Estramustin plus vinblastine in hormone-refractory prostate cancer 582, 583, and vinorelbine plus docetaxel in metastatic breast cancer 584 are superior to either drug alone, as are vinorelbine and PTX in P388 murine leukemia cells 585, and docetaxel and CI-980 586, a CLC analogue, in KB3-1 cells. Yet, it is uncommon for two agents to interact synergistically while they bind to the same binding site. More often, this kind of combination leads to additivity or antagonism, as the drugs cannot bind to the same site simultaneously. The only exception reported is a recent study by Martello et. al. which demonstrated that taxol and discodermolide represented a synergistic drug combination in four human cancer cell lines 258, and in a subsequent study on the mechanism of synergism, it was shown that taxol and discodermolide synergistically suppress the microtubule dynamics 260.

Several studies have suggested that BZs bind to the colchicine-binding site of mammalian tubulin 563, 564, 587, 588. In contrast, a recent study reported that benomyl, an antifungal agent and a member of BZ compounds, did not inhibit the binding of CLC to its binding site 589, and in a subsequent study, the combination of benomyl and CLC was shown to be synergistic 565. These data led to a conclusion that benomyl binds to a novel site of tubulin. Previous chapter showed that ABZ had a synergistic interaction with Chapter 5 Chapter CLC and 2ME. Therefore, it was hypothesised that antagonism between colchicine- 159

binding agents would further confirm that ABZ does not bind to the colchicine-binding site. To test this hypothesis, the interactions between 2ME plus CLC and CA4 plus CLC were assessed. In addition, the combination of these three colchicine-binding agents with each other was evaluated. Surprisingly, it was found that all four colchicine- domain binders represented a concentration-dependent synergistic cytotoxic effect on the proliferation of HCT-116 and DU145 cell lines.

As discussed in chapter 1, at clinically relevant doses, both microtubule-polymerising and -depolymerising compounds inhibit microtubule dynamics. It has been shown that the IC50 values for inhibiting the cell proliferation by 2ME is 10- to 100-fold lower than the concentration required for tubulin de-polymerisation 418. As for CA4, an IC50 of 7 nM for growth inhibition in L1210 murine and tubulin polymerisation inhibition at 2.5 μM have been reported 590. In 1A9 ovarian cancer cells the concentration required for tubulin depolymerisation by ABZ, is 10-fold higher than the IC50 values for inhibiting cell growth 573. These results suggest that there is only a modest correlation between the concentrations that lead to the cell death, and the concentrations that change microtubule polymer mass.

There are several possible explanations for the observed synergism between CLC- binding agents. Similar to other MTAs, at low concentrations, CLC suppresses the dynamic instability of microtubules with no effect on the polymer mass 99. As a result, while the stoichiometry of its binding to soluble tubulin is approximately 1 mol per mol tubulin 591, much less CLC with very little binding is required to inhibit the dynamic instability of microtubules 592. Considering that each micrometer of microtubule contains 1690 tubulin dimers 593, a great number of binding sites are available for drug molecules. Therefore, it is conceivable that additional drugs could be bound to microtubules.

Another possible explanation is that the drugs may exhibit different affinity towards different isotypes of tubulin. In mammals, eight isotypes of α-tubulin and seven isotypes of β-tubulin have been recognised 8. CLC has been proven to have the highest affinity 594

for αβIV and the lowest affinity for αβIII . Unfortunately, there is no published data 5 Chapter regarding the binding affinities of ABZ, 2ME, and CA4 to various tubulin isotypes. 160

Nevertheless, if the drugs discriminate among the various isotypes or among various forms of microtubules (spindle, cytoskeletal, etc.), and the preference of each drug in terms of binding to a specific isotype of tubulin differs from the other drug, then it is plausible to conclude that the synergy may result from different affinity of the drugs towards different forms or isotypes of microtubules.

Another possibility is that the synergy could be entirely unrelated to tubulin. However, because all tested CLC-domain binders synergise with each other, it is more likely that the compounds share a common mechanism of synergy that is related to the microtubule.

With regard to the mechanism of synergism between ABZ and 2ME, the effects of the combination therapy on tubulin polymerisation, acetylated tubulin, and apoptosis-related proteins were investigated. To increase the sensitivity of the assays, low concentrations of ABZ and 2ME with a marginal effect as a single agent were used. However, to evaluate the combination effect on angiogenesis-related proteins, high doses of each drug were studied. As expected, comparison between the percentage of polymerised tubulin in cells that were treated with single agents and with the combination of the drugs revealed that the doses at which synergistic anti-proliferative effect were observed, did not depolymerise tubulin. Similarly, combination therapy had no impact on de-acetylation of tubulin compared to single agents. These results further confirm that the effect of the combination of ABZ and 2ME on cell proliferation is unlikely to be mediated through the inhibition of tubulin polymerisation.

Activation of the caspase cascade has been proven to be the central mechanism promoting apoptosis in response to chemotherapy. Therefore, to elucidate whether the cell death induced by ABZ and 2ME is mediated through apoptosis, the activity of caspase-3, caspase-8 and caspase-9 were assessed. As stated earlier, two major apoptosis pathways, the extrinsic pathway and the intrinsic pathway converge on caspases activation 305. The extrinsic pathway is activated through death receptors (DRs). The interaction of DRs with tumour necrosis factor-related apoptosis–inducing

ligand (TRAIL) induces the activation caspase-8, which in turn, triggers the cascade of 5 Chapter activated procapases that follows 305, 595, 596. The intrinsic pathway is controlled by Bcl-2 161

family of proteins and involves in the release of apoptosome containing caspase 9 595. Finally, activation of initiator caspase-8 and -9 result in the cleavage of caspase-3 597, 598. Results from caspase activity assays revealed that while both single agent treatment and combination therapy failed to activate caspase-9, they activated caspase-8 and caspase-3 in a time-dependent manner. Additionally, combination therapy was significantly more effective in activating caspase-8 and caspase-3 in comparison with single drug treatment. The expression of DR5 was also induced in the cells that were treated with either single agents or the combination of the agents, with combination treatment representing more pronounced effect compared to the single drugs. These results suggest that the extrinsic death receptor apoptotic pathway is involved in drug-induced apoptosis in HCT-116 cells. The mechanism of upregulation of DR5 is not clear but most likely is independent from p53, as no increase in the protein levels of P53 was observed. DR5 has been shown to be activated in both ligand-dependent and ligand-independent manner. While p53 has been implicated in ligand-independent upregulation of DR5 599, it is not required for the apoptotic response to TRAIL {Kim, 2000 #1088}. Possible role of TRAIL in DR5 overexpression and the upstream signal that lies between drug exposure and activation of caspases are unknown.

Historically, MTAs-induced apoptosis is mediated primarily by the mitochondria/caspase 9 activation pathway. Yet, it has been reported that the extrinsic pathway may also be engaged by MTAs. For instance, PTX has been shown to up- regulate DR5 protein and sensitise prostate cancer cell lines to the cytotoxic effects of TRAIL 600. However, these effects appear to be cell line-dependent as they were not observed in NSCLC and breast cancer cell lines 601, 602. In agreement with current results, 2ME has also been reported to overexpress DR5 in a variety of human cancer cell lines including breast, cervical, prostate and glioma cells 321.

In the next set of experiments, it was sought to determine whether ABZ and 2ME synergistically suppressed the expression of HIF-1α and VEGF, the two proteins that have pivotal role in angiogenesis. ABZ and 2ME have been demonstrated to downregulate the expression of HIF-1α, both in vitro and in vivo 353, 404. Therefore it was hypothesised that the combination of ABZ and 2ME may further suppress HIF-1α and 5 Chapter 162

its target protein, VEGF. While incubation of HCT-116 cells at 1% oxygen led to a marked accumulation of HIF-1α, no HIF-1α was detected in normoxia condition. The latter could be explained by the fact that in the presence of oxygen, hydroxylated HIF- 1α degraded rapidly by the proteasome pathway. Following treatment with ABZ and 2ME, a significant reduction in HIF-1α expression was observed in HCT-116 cells. This effect was more pronounced in the cells that were exposed to the combination of the two agents. Similar to HIF-1α, VEGF levels were also decreased in hypoxia condition, with the combination of 50 μM 2ME with ABZ, being more effective than the single agent. Conversely, no effect on VEGF levels were detected in normoxia, as the percentage of VEGF reduction was directly proportional to the percentage of cell kill. The latter finding is in contradiction with previous report by Mabjeesh et. al. where they found that in addition to hypoxia condition, 2ME also suppressed VEGF and HIF-1α in normoxia 353. This discrepancy may be due to the difference in experimental conditions as well as in the cell lines used. The study by Mabjeesh et. al. also revealed a link between disruption of microtubules and dysregulation of HIF-1α. They showed that depolymerisation of microtubules by 2ME was necessary for suppression of HIF-1α to occur. This finding explains why much higher concentrations of 2ME are required to downregulate HIF-1α compared with the concentrations that inhibit the cell proliferation. Likewise, inhibition of tube formation, cell migration, VEGFR2 suppression, and HIF-1α down-regulation require much higher concentrations of ABZ compared with the concentrations that inhibited cell proliferation 404, 603. It is therefore plausible to conclude that the suppression of angiogenesis is independent from inhibition of the cell proliferation and that there is a moderate correlation between the two events.

One could argue that the concentrations that inhibit HIF and VEGF, greatly exceed the clinically relevant doses and that the in vitro activities that require ABZ and 2ME doses as high as 10-50 μM may not be clinically significant. Although this concept seems reasonable, several reports suggested otherwise. ABZ at 150 mg/kg has been shown to suppress VEGF, HIF-1α and angiogenesis in pre-clinical models 404, 406. In a mouse orthotopic breast cancer model, treatment with 150 mg/kg 2ME not only inhibited Chapter 5 Chapter angiogenesis, but also depolymerised interphase and mitotic microtubules 353. Also in 163

mouse model of endometriosis, it has been shown that 2ME significantly suppressed the expression of HIF-1α 604. These results suggest that ABZ and 2ME may have additional or different effects that are dependent on their activity on tumour itself, as opposed to a monolayer tumour cells and that the complexity of the tumour-host interaction may not be reflected in in vitro assays.

Finally, although beyond the scope of this thesis, hypoxic HCT-116 cells were markedly less responsive to the drugs compared to normoxic cells. This could be due to the alteration in pro- and anti-apoptotic genes. HIF-1α directly regulates Bcl-xL, an anti- apoptotic protein of the Bcl-2 family. Overexpression of Bcl-xl has been reported to protect cancer cells from apoptosis 605. In addition, activation of HIF-1α leads to the down-regulation of pro-apoptotic proteins, Bid and Bad, which contribute to the drug resistance 606.

Evaluating the combination therapy in mice-bearing HCT-116 tumours indicated that 50 mg/kg ABZ as a single agent was more effective in prolonging the survival of animals compared with combination therapy. These results were consistent with the in vitro assay where the combination of high concentrations of ABZ with 2ME represented an antagonistic antiproliferative effect on HCT-116 cells. In addition, Immunohistochemistry analysis of tumour sections revealed that ABZ alone was more effective in reducing microvessel density, suppressing the proliferation, and inducing apoptosis, than the combination of the two agents. In contrast, combination of low dose of ABZ (25 mg/kg) with 2ME resulted in a significant increase in the survival of mice compared with treatment with single agents. In addition, in mice treated with the combination, a statistically significantly less tumour cell proliferation was observed compared with mice treated with either drug alone or vehicle. Moreover, analysis of tumour samples from these mice revealed that the combination therapy significantly decreased microvessel density and markedly increased the number of apoptotic tumour cells. Finally, tumour and plasma of mice that were treated with the combination, exhibited a significantly lower VEGF levels in comparison with animals that received single agents or the vehicle. Chapter 5 Chapter

164

Overall, this chapter demonstrated that the combination of low dose of ABZ and 2ME represent a synergistic anti-tumor effect. High effectiveness of the combination therapy stemmed from inhibition of the proliferation of tumor cells, suppression of angiogenesis, and induction of apoptosis. Chapter 5 Chapter

165

Chapter 6 SummarySummary andand FutureFuture DirectionsDirections

Tubulin is one of the most highly conserved proteins in evolution 5, 607 , and one of the most validated targets in cancer therapy. Microtubule-targeting agents (MTAs) have been successfully used in cancer therapy and many new agents from this class are under investigation and development. Albendazole (ABZ) is a MTA that binds to colchicine-binding site on β-tubulin and is effective against a wide range of human cancer cells. ABZ was originally described as an antiparasitic drug, but several studies have confirmed its antiproliferative and anti-angiogenic properties that show promise in cancer therapy.

This project was designed to improve the efficacy of ABZ as an anti-cancer agent. To this end, two strategies were employed: The first strategy was to formulate ABZ with hydroxypropyl-β-cyclodextrin (HPβCD) and acetic acid with the aim of improving its aqueous solubility and as a result, its anti-tumour efficacy (chapter 3), and the second approach was to develop a novel therapeutic strategy using combination therapy (chapter 4 and chapter 5).

6.1. ABZ FORMULATION

ABZ is a lipophilic compound with very low water solubility (0.2 μg/ml), allowing its preparation only as a suspension 524. Complexation of ABZ with HPβCD in combination with ionisation with acetic acid, resulted in a 10,000-fold increase in its aqueous solubility. Furthermore, in comparison with a conventional suspension of ABZ in hydroxypropyl methylcellulose (ABZ/HPMC), ABZ/HPβCD was 10 times more cytotoxic against HCT-116, DU145 and HUVEC cells. Pharmacokinetic study of the formulation in nude mice showed that the area under the curve (AUC) and

Cmax of ABZ-sulfoxide (ABZSO), the active metabolite of ABZ, in plasma of animals administered ABZ/HPβCD were approximately 7 and 18 fold higher compared to the mice that received the corresponding dose of ABZ/HPMC. This improvement in pharmacokinetic profiles was also translated into the anti-tumour efficacy of the formulation, as ABZ/HPβCD significantly delayed the tumour growth and prolonged the survival of HCT-116 tumour-bearing mice compared with ABZ/HPMC. These findings demonstrated that complexation of ABZ with HPβCD Chapter 6Chapter improves its pharmacokinetic profile and anti-tumour efficacy. 167

Cyclodextrins (CDs) are classified as hydrophobic, hydrophilic and ionisable derivatives 539. While hydrophilic CDs such as HPβCD are used in fast release formulations 608, hydrophobic derivatives are utilised for prolonged release of water- soluble drugs 609-611. The use of the combination of hydrophobic and hydrophilic CDs may also be a promising approach. For example, it has been shown that the combination of HPβCD, with triacetyl- β- cyclodextrin (TAβCD), in an appropriate ratio resulted in a significant slow drug release with prolonged maintenance of plasma level 612. In such a system, the hydrophilic portion (HPβCD) acts as a fast releasing fraction, whereas the hydrophobic portion (TAβCD) acts as a sustained releasing fraction. On the other hand, ionisable CDs such as Sulfobutylether- β- cyclodextrin (SEM-β-CD) and carboxymethyl ethyl-β-cyclodextrin (CME-β-CD) improve inclusion property of compounds and alter the dissolution rate of drugs 539, 613. Hence, it would be valuable to have studies examining the effect of ionisable cyclodextrins, or the combination of hydrophilic and hydrophobic CDs in the solubilisation of ABZ.

6.2.COMBINATION THERAPY

The second aim of this thesis was to evaluate the interaction between ABZ and other chemotherapeutic agents. In chapter 4, combination of ABZ and three antiparasitic agents, ivermectin, diethylcarbamazine and praziquantel, was evaluated, as these drugs synergised with ABZ in the treatment of nemathodes 517-519. In addition, the interaction between ABZ and three tubulin-binding agents that differ in their binding sites on β-tubulin was examined. Results revealed that the combination of ABZ and CLC was synergistic and of interest, both agents bind to the same binding site on β- tubulin. Since CLC is not being used in cancer therapy due to its toxicity 3, the interaction between ABZ and 2-methoxyestradiol (2ME), a related and a structurally similar compound to colchicine was assessed. Similar to CLC, 2ME also synergised with ABZ in inhibiting HCT-116 and DU145 cell proliferation.

In chapter 5, the mechanism of synergism between ABZ and 2ME was explored and the effect of combination therapy on HCT-116 xenograft tumour in nude mice was evaluated. In vitro results showed that ABZ and 2ME have a schedule-dependent synergistic interaction on HCT-116 cells. This interaction was, at least in part, 6Chapter

168

mediated through the apoptotic mode of cell death. Interestingly, combination therapy triggered the extrinsic pathway of apoptosis, as evidenced by upregulation of DR5 and activation of caspase 8. It was also found that the interaction between ABZ and 2ME was unrelated to depolymerisation of tubulin. In vivo, combination of 25 mg/kg ABZ with 25 mg/kg 2ME significantly prolonged the survival of mice compared with treatment with single agents. This effect was accompanied by a decrease in proliferation rate of tumour cells and microvessel density. Additionally, tumour and plasma of mice that were treated with the combination of ABZ and 2ME, exhibited a significantly lower VEGF levels in comparison with animals that received single agents or the vehicle. In contrast, 50 mg/kg ABZ combined with 25 mg/kg 2ME had an antagonistic effect on the survival of animals compared to ABZ alone. These results were consistent with the in vitro cytotoxicity assay in which, the combination of high concentrations of ABZ with 2ME represented an antagonistic antiproliferative effect on HCT-116 cells.

In vivo assessment of synergy was carried out on subcutaneous xenograft models. Although this ectopical model is useful in illustrating the in vivo activity of the combination therapy and to monitor the possible toxicity of the combined treatment, it does not reflect clinical response, as in clinical settings, most compounds and combinations are evaluated in patients with advanced diseases which are resistant to the standard chemotherapy. Therefore, the use of orthotopic or experimental metastasis models to demonstrate activity of the combination of ABZ and 2ME would be ideal. 2ME has been shown to be highly efficacious when it is administered at doses ranging 75-150 mg/kg. Additionally, in vitro drug interaction analysis suggested that simultaneous treatment resulted in the most synergistic interaction. Therefore, it would be valuable to study the effect of higher concentrations of 2ME in combination with ABZ, preferentially in a simultaneous treatment regimen.

Another finding of this thesis, presented in chapter 5, was that CLC-domain binders, ABZ, CLC, 2ME and CA4 represented a synergistic antiproliferative interaction on HCT-116 and DU145 cell lines. Indeed, at doses close to their IC50, ABZ, 2ME and CA4 not only were synergistic with CLC, but also synergised with one another. This

finding challenges the hypothesis that synergy cannot occur between two agents that 6Chapter

169

bind to the same binding site. Therefore, further investigations are needed to uncover the nature of this interaction. For example, it would be valuable to investigate whether the synergy occurs through the suppression of microtubule dynamics or additional targets are involved.

Tubulin-binding agents continue to be one of the most promising compounds in cancer therapy and novel colchicine-domain binders offer promise especially as anti- angiogenic agents. The finding that the combination of two colchicine-domain binders can act synergistically, suggests that such drug combinations, which would not normally be considered due to the similar mechanism of action and identical binding sites may nevertheless provide therapeutic benefit in cancer therapy. Chapter 6Chapter

170

References

1. Mollinedo F, Gajate C. Microtubules, microtubule-interfering agents and apoptosis. Apoptosis 2003;8(5):413-50.

2. Etienne-Manneville S. From signaling pathways to microtubule dynamics: the key players. Curr Opin Cell Biol;22(1):104-11.

3. Jordan MA, Wilson L. Microtubules as a target for anticancer drugs. Nat Rev Cancer 2004;4(4):253-65.

4. Wilson L, Panda D, Jordan MA. Modulation of microtubule dynamics by drugs: a paradigm for the actions of cellular regulators. Cell Struct Funct 1999;24(5):329-35.

5. Downing KH, Nogales E. Tubulin structure: insights into microtubule properties and functions. Current opinion in structural biology 1998;8(6):785-91.

6. Montenegro Gouveila S, Akhmanova A. Cell and molecular biology of microtubule plus end and tracking proteins: end binding proteins and their partners. 2010.

7. Wade RH. Microtubules: an overview. Methods in molecular medicine 2007;137:1-16.

8. Luduena RF. Multiple forms of tubulin: different gene products and covalent modifications. Int Rev Cytol 1998;178:207-75.

9. Hammond JW, Cai D, Verhey KJ. Tubulin modifications and their cellular functions. Curr Opin Cell Biol 2008;20(1):71-6.

10. Song YH, Mandelkow E. The anatomy of flagellar microtubules: polarity, seam, junctions, and lattice. J Cell Biol 1995;128(1-2):81-94.

11. Allen C, Borisy GG. Structural polarity and directional growth of microtubules of Chlamydomonas flagella. J Mol Biol 1974;90(2):381-402.

12. Bornens M. Organelle positioning and cell polarity. Nat Rev Mol Cell Biol 2008;9(11):874-86.

13. Kavallaris M. Microtubules and resistance to tubulin-binding agents. Nat Rev Cancer 2010;10(3):194-204.

14. Verdier-Pinard P, Pasquier E, Xiao H, et al. Tubulin proteomics: towards breaking the code. Analytical biochemistry 2009;384(2):197-206.

15. Verrills NM, Kavallaris M. Improving the targeting of tubulin-binding agents: lessons from drug resistance studies. Curr Pharm Des 2005;11(13):1719-33.

16. Gan PP, McCarroll JA, Po'uha ST, Kamath K, Jordan MA, Kavallaris M. Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of betaIII-tubulin. Mol Cancer Ther 2010;9(5):1339-48.

172

17. Vilmar AC, Santoni-Rugiu E, Sorensen JB. Class III beta-tubulin in advanced NSCLC of adenocarcinoma subtype predicts superior outcome in a randomized trial. Clin Cancer Res 2011;17(15):5205-14.

18. Ferrandina G, Zannoni GF, Martinelli E, et al. Class III beta-tubulin overexpression is a marker of poor clinical outcome in advanced ovarian cancer patients. Clin Cancer Res 2006;12(9):2774-9.

19. Pasquier E, Kavallaris M. Microtubules: a dynamic target in cancer therapy. IUBMB Life 2008;60(3):165-70.

20. Kanakkanthara A, Wilmes A, O'Brate A, et al. Peloruside- and laulimalide- resistant human ovarian carcinoma cells have betaI-tubulin mutations and altered expression of betaII- and betaIII-tubulin isotypes. Mol Cancer Ther 2011;10(8):1419-29.

21. Nogales E. Structural insight into microtubule function. Annu Rev Biophys Biomol Struct 2001;30:397-420.

22. Barra HS, Arce CA, Argarana CE. Posttranslational tyrosination/detyrosination of tubulin. Mol Neurobiol 1988;2(2):133-53.

23. Lafanechere L, Courtay-Cahen C, Kawakami T, et al. Suppression of tubulin tyrosine ligase during tumor growth. J Cell Sci 1998;111 ( Pt 2):171-81.

24. Mialhe A, Lafanechere L, Treilleux I, et al. Tubulin detyrosination is a frequent occurrence in breast cancers of poor prognosis. Cancer Res 2001;61(13):5024-7.

25. Badin-Larcon AC, Boscheron C, Soleilhac JM, et al. Suppression of nuclear oscillations in Saccharomyces cerevisiae expressing Glu tubulin. Proc Natl Acad Sci U S A 2004;101(15):5577-82.

26. Peris L, Thery M, Faure J, et al. Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends. J Cell Biol 2006;174(6):839-49.

27. Piperno G, LeDizet M, Chang XJ. Microtubules containing acetylated alpha- tubulin in mammalian cells in culture. J Cell Biol 1987;104(2):289-302.

28. Hubbert C, Guardiola A, Shao R, et al. HDAC6 is a microtubule-associated deacetylase. Nature 2002;417(6887):455-8.

29. Gaertig J, Cruz MA, Bowen J, Gu L, Pennock DG, Gorovsky MA. Acetylation of lysine 40 in alpha-tubulin is not essential in Tetrahymena thermophila. J Cell Biol 1995;129(5):1301-10.

30. Zhang Y, Li N, Caron C, et al. HDAC-6 interacts with and deacetylates tubulin and microtubules in vivo. EMBO J 2003;22(5):1168-79.

173

31. Matsuyama A, Shimazu T, Sumida Y, et al. In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J 2002;21(24):6820-31.

32. Tran AD, Marmo TP, Salam AA, et al. HDAC6 deacetylation of tubulin modulates dynamics of cellular adhesions. J Cell Sci 2007;120(Pt 8):1469-79.

33. Fourest-Lieuvin A, Peris L, Gache V, et al. Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1. Molecular biology of the cell 2006;17(3):1041-50.

34. Verhey KJ, Gaertig J. The tubulin code. Cell Cycle 2007;6(17):2152-60.

35. Redeker V, Levilliers N, Schmitter JM, et al. Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules. Science 1994;266(5191):1688-91.

36. Edde B, Rossier J, Le Caer JP, Desbruyeres E, Gros F, Denoulet P. Posttranslational glutamylation of alpha-tubulin. Science 1990;247(4938):83-5.

37. Thazhath R, Liu C, Gaertig J. Polyglycylation domain of beta-tubulin maintains axonemal architecture and affects cytokinesis in Tetrahymena. Nat Cell Biol 2002;4(3):256-9.

38. Westermann S, Weber K. Post-translational modifications regulate microtubule function. Nat Rev Mol Cell Biol 2003;4(12):938-47.

39. Boucher D, Larcher JC, Gros F, Denoulet P. Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein Tau and tubulin. Biochemistry 1994;33(41):12471-7.

40. Bonnet C, Boucher D, Lazereg S, et al. Differential binding regulation of microtubule-associated proteins MAP1A, MAP1B, and MAP2 by tubulin polyglutamylation. J Biol Chem 2001;276(16):12839-48.

41. Desai A, Mitchison TJ. Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 1997;13:83-117.

42. Howard J, Hyman AA. Growth, fluctuation and switching at microtubule plus ends. Nat Rev Mol Cell Biol 2009;10(8):569-74.

43. Singh P, Rathinasamy K, Mohan R, Panda D. Microtubule assembly dynamics: an attractive target for anticancer drugs. IUBMB Life 2008;60(6):368-75.

44. van der Vaart B, Akhmanova A, Straube A. Regulation of microtubule dynamic instability. Biochem Soc Trans 2009;37(Pt 5):1007-13.

45. Rodionov VI, Borisy GG. Microtubule treadmilling in vivo. Science 1997;275(5297):215-8.

46. Margolis RL, Wilson L. Microtubule treadmilling: what goes around comes around. Bioessays 1998;20(10):830-6. 174

47. Shaw SL, Kamyar R, Ehrhardt DW. Sustained microtubule treadmilling in Arabidopsis cortical arrays. Science 2003;300(5626):1715-8.

48. Drechsel DN, Kirschner MW. The minimum GTP cap required to stabilize microtubules. Curr Biol 1994;4(12):1053-61.

49. Calligaris D, Verdier-Pinard P, Devred F, Villard C, Braguer D, Lafitte D. Microtubule targeting agents: from biophysics to proteomics. Cell Mol Life Sci 2010;67(7):1089-104.

50. Andersen SS. Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18. Trends Cell Biol 2000;10(7):261-7.

51. Bhat KM, Setaluri V. Microtubule-associated proteins as targets in cancer chemotherapy. Clin Cancer Res 2007;13(10):2849-54.

52. Buee L, Bussiere T, Buee-Scherrer V, Delacourte A, Hof PR. Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res Brain Res Rev 2000;33(1):95-130.

53. Rouzier R, Rajan R, Wagner P, et al. Microtubule-associated protein tau: a marker of paclitaxel sensitivity in breast cancer. Proc Natl Acad Sci U S A 2005;102(23):8315-20.

54. Pentheroudakis G, Kalogeras KT, Wirtz RM, et al. Gene expression of estrogen receptor, progesterone receptor and microtubule-associated protein Tau in high-risk early breast cancer: a quest for molecular predictors of treatment benefit in the context of a Hellenic Cooperative Oncology Group trial. Breast Cancer Res Treat 2009;116(1):131-43.

55. Pusztai L, Jeong JH, Gong Y, et al. Evaluation of microtubule-associated protein-Tau expression as a prognostic and predictive marker in the NSABP-B 28 randomized clinical trial. J Clin Oncol 2009;27(26):4287-92.

56. Baquero MT, Lostritto K, Gustavson M, et al. Evaluation of prognostic and predictive value of microtubule associated protein tau in two independent cohorts. Breast cancer research : BCR 2011;13(5):R85.

57. Smoter M, Bodnar L, Duchnowska R, Stec R, Grala B, Szczylik C. The role of Tau protein in resistance to paclitaxel. Cancer Chemother Pharmacol 2011;68(3):553-7.

58. Andre F, Hatzis C, Anderson K, et al. Microtubule-associated protein-tau is a bifunctional predictor of endocrine sensitivity and chemotherapy resistance in estrogen receptor-positive breast cancer. Clin Cancer Res 2007;13(7):2061-7.

59. Wosnitzer MS, Domingo-Domenech J, Castillo-Martin M, et al. Predictive Value of Microtubule Associated Proteins Tau and Stathmin in Patients With Nonmuscle Invasive Bladder Cancer Receiving Adjuvant Intravesical Taxane Therapy. J Urol 2011.

175

60. Sanchez C, Diaz-Nido J, Avila J. Phosphorylation of microtubule-associated protein 2 (MAP2) and its relevance for the regulation of the neuronal cytoskeleton function. Prog Neurobiol 2000;61(2):133-68.

61. Shafit-Zagardo B, Kalcheva N. Making sense of the multiple MAP-2 transcripts and their role in the neuron. Mol Neurobiol 1998;16(2):149-62.

62. Liu Y, Mangini J, Saad R, et al. Diagnostic value of microtubule-associated protein-2 in Merkel cell carcinoma. Appl Immunohistochem Mol Morphol 2003;11(4):326-9.

63. Soltani MH, Pichardo R, Song Z, et al. Microtubule-associated protein 2, a marker of neuronal differentiation, induces mitotic defects, inhibits growth of melanoma cells, and predicts metastatic potential of cutaneous melanoma. Am J Pathol 2005;166(6):1841-50.

64. Song Z, He CD, Sun C, et al. Increased expression of MAP2 inhibits melanoma cell proliferation, invasion and tumor growth in vitro and in vivo. Exp Dermatol 2010;19(11):958-64.

65. Veitia R, David S, Barbier P, et al. Proteolysis of microtubule associated protein 2 and sensitivity of pancreatic tumours to docetaxel. Br J Cancer 2000;83(4):544-9.

66. Bauer JA, Chakravarthy AB, Rosenbluth JM, et al. Identification of markers of taxane sensitivity using proteomic and genomic analyses of breast tumors from patients receiving neoadjuvant paclitaxel and radiation. Clin Cancer Res 2010;16(2):681-90.

67. Ookata K, Hisanaga S, Bulinski JC, et al. Cyclin B interaction with microtubule-associated protein 4 (MAP4) targets p34cdc2 kinase to microtubules and is a potential regulator of M-phase microtubule dynamics. J Cell Biol 1995;128(5):849-62.

68. Nguyen HL, Gruber D, Bulinski JC. Microtubule-associated protein 4 (MAP4) regulates assembly, protomer-polymer partitioning and synthesis of tubulin in cultured cells. J Cell Sci 1999;112 ( Pt 12):1813-24.

69. Shiina N, Tsukita S. Mutations at phosphorylation sites of Xenopus microtubule-associated protein 4 affect its microtubule-binding ability and chromosome movement during mitosis. Mol Biol Cell 1999;10(3):597-608.

70. Chang W, Gruber D, Chari S, et al. Phosphorylation of MAP4 affects microtubule properties and cell cycle progression. J Cell Sci 2001;114(Pt 15):2879- 87.

71. Poruchynsky MS, Giannakakou P, Ward Y, et al. Accompanying protein alterations in malignant cells with a microtubule-polymerizing drug-resistance phenotype and a primary resistance mechanism. Biochem Pharmacol 2001;62(11):1469-80.

176

72. Kavallaris M, Tait AS, Walsh BJ, et al. Multiple microtubule alterations are associated with Vinca alkaloid resistance in human leukemia cells. Cancer Res 2001;61(15):5803-9.

73. Howell B, Larsson N, Gullberg M, Cassimeris L. Dissociation of the tubulin- sequestering and microtubule catastrophe-promoting activities of oncoprotein 18/stathmin. Mol Biol Cell 1999;10(1):105-18.

74. Mistry SJ, Atweh GF. Role of stathmin in the regulation of the mitotic spindle: potential applications in cancer therapy. Mt Sinai J Med 2002;69(5):299- 304.

75. Brattsand G, Roos G, Marklund U, et al. Quantitative analysis of the expression and regulation of an activation-regulated phosphoprotein (oncoprotein 18) in normal and neoplastic cells. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 1993;7(4):569-79.

76. Brattsand G. Correlation of oncoprotein 18/stathmin expression in human breast cancer with established prognostic factors. Br J Cancer 2000;83(3):311-8.

77. Wang F, Wang LX, Li SL, et al. Downregulation of stathmin is involved in malignant phenotype reversion and cell apoptosis in esophageal squamous cell carcinoma. J Surg Oncol 2011;103(7):704-15.

78. Mitra M, Kandalam M, Sundaram CS, et al. Reversal of stathmin-mediated microtubule destabilization sensitizes retinoblastoma cells to a low dose of antimicrotubule agents: a novel synergistic therapeutic intervention. Invest Ophthalmol Vis Sci 2011;52(8):5441-8.

79. Alli E, Bash-Babula J, Yang JM, Hait WN. Effect of stathmin on the sensitivity to antimicrotubule drugs in human breast cancer. Cancer Res 2002;62(23):6864-9.

80. Yoshie M, Miyajima E, Kyo S, Tamura K. Stathmin, a microtubule regulatory protein, is associated with hypoxia-inducible factor-1alpha levels in human endometrial and endothelial cells. Endocrinology 2009;150(5):2413-8.

81. Giodini A, Kallio MJ, Wall NR, et al. Regulation of microtubule stability and mitotic progression by survivin. Cancer Res 2002;62(9):2462-7.

82. Altieri DC. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 2003;22(53):8581-9.

83. Ambrosini G, Adida C, Altieri DC. A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nature medicine 1997;3(8):917-21.

84. Zhou J, O'Brate A, Zelnak A, Giannakakou P. Survivin deregulation in beta- tubulin mutant ovarian cancer cells underlies their compromised mitotic response to taxol. Cancer Res 2004;64(23):8708-14.

85. Kapoor TM, Mitchison TJ. Eg5 is static in bipolar spindles relative to tubulin: evidence for a static spindle matrix. J Cell Biol 2001;154(6):1125-33. 177

86. Ding S, Xing N, Lu J, et al. Overexpression of Eg5 predicts unfavorable prognosis in non-muscle invasive bladder urothelial carcinoma. Int J Urol 2011;18(6):432-8.

87. Turner J, Anderson R, Guo J, Beraud C, Fletterick R, Sakowicz R. Crystal structure of the mitotic spindle kinesin Eg5 reveals a novel conformation of the neck-linker. J Biol Chem 2001;276(27):25496-502.

88. Calligaris D, Verdier-Pinard P, Devred F, Villard C, Braguer D, Lafitte D. Microtubule targeting agents: from biophysics to proteomics. Cell Mol Life Sci;67(7):1089-104.

89. Perez EA. Microtubule inhibitors: Differentiating tubulin-inhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol Cancer Ther 2009;8(8):2086-95.

90. Zhou J, Giannakakou P. Targeting microtubules for cancer chemotherapy. Curr Med Chem Anticancer Agents 2005;5(1):65-71.

91. Weisenberg RC, Borisy GG, Taylor EW. The colchicine-binding protein of mammalian brain and its relation to microtubules. Biochemistry 1968;7(12):4466- 79.

92. Borisy GG, Taylor EW. The mechanism of action of colchicine. Colchicine binding to sea urchin eggs and the mitotic apparatus. The Journal of cell biology 1967;34(2):535-48.

93. Borisy GG, Taylor EW. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. The Journal of cell biology 1967;34(2):525-33.

94. Wallace SL. Colchicine. Seminars in arthritis and rheumatism 1974;3(4):369- 81.

95. Brossi A, Yeh HJ, Chrzanowska M, et al. Colchicine and its analogues: recent findings. Medicinal research reviews 1988;8(1):77-94.

96. Hillmann G, Ruthmann A. Effect of mitotic inhibitors on the ultrastructure of root meristem cells. Planta 1982;155(2):124-132.

97. Bhattacharyya B, Panda D, Gupta S, Banerjee M. Anti-mitotic activity of colchicine and the structural basis for its interaction with tubulin. Medicinal research reviews 2008;28(1):155-83.

98. Hastie SB. Interactions of colchicine with tubulin. Pharmacology & therapeutics 1991;51(3):377-401.

99. Panda D, Daijo JE, Jordan MA, Wilson L. Kinetic stabilization of microtubule dynamics at steady state in vitro by substoichiometric concentrations of tubulin-colchicine complex. Biochemistry 1995;34(31):9921-9.

178

100. Taylor EW. The Mechanism of Colchicine Inhibition of Mitosis. I. Kinetics of Inhibition and the Binding of H3-Colchicine. The Journal of cell biology 1965;25:SUPPL:145-60.

101. Kanthou C, Tozer GM. Microtubule depolymerizing vascular disrupting agents: novel therapeutic agents for oncology and other pathologies. Int J Exp Pathol 2009;90(3):284-94.

102. Tozer GM, Kanthou C, Baguley BC. Disrupting tumour blood vessels. Nature reviews. Cancer 2005;5(6):423-35.

103. Pettit GR, Cragg GM, Singh SB. Antineoplastic agents, 122. Constituents of Combretum caffrum. Journal of natural products 1987;50(3):386-91.

104. Beauregard DA, Thelwall PE, Chaplin DJ, Hill SA, Adams GE, Brindle KM. Magnetic resonance imaging and spectroscopy of combretastatin A4 prodrug- induced disruption of tumour perfusion and energetic status. British journal of cancer 1998;77(11):1761-7.

105. Dark GG, Hill SA, Prise VE, Tozer GM, Pettit GR, Chaplin DJ. Combretastatin A-4, an agent that displays potent and selective toxicity toward tumor vasculature. Cancer research 1997;57(10):1829-34.

106. Chaplin DJ, Pettit GR, Hill SA. Anti-vascular approaches to solid tumour therapy: evaluation of combretastatin A4 phosphate. Anticancer research 1999;19(1A):189-95.

107. Tozer GM, Prise VE, Wilson J, et al. Combretastatin A-4 phosphate as a tumor vascular-targeting agent: early effects in tumors and normal tissues. Cancer research 1999;59(7):1626-34.

108. Holwell SE, Cooper PA, Grosios K, et al. Combretastatin A-1 phosphate a novel tubulin-binding agent with in vivo anti vascular effects in experimental tumours. Anticancer Res 2002;22(2A):707-11.

109. Kanthou C, Tozer GM. The tumor vascular targeting agent combretastatin A- 4-phosphate induces reorganization of the actin cytoskeleton and early membrane blebbing in human endothelial cells. Blood 2002;99(6):2060-9.

110. Tozer GM, Kanthou C, Parkins CS, Hill SA. The biology of the combretastatins as tumour vascular targeting agents. International journal of experimental pathology 2002;83(1):21-38.

111. Hori K, Saito S. Microvascular mechanisms by which the combretastatin A-4 derivative AC7700 (AVE8062) induces tumour blood flow stasis. Br J Cancer 2003;89(7):1334-44.

112. Nihei Y, Suzuki M, Okano A, et al. Evaluation of antivascular and antimitotic effects of tubulin binding agents in solid tumor therapy. Japanese journal of cancer research : Gann 1999;90(12):1387-95.

179

113. Cai SX, Drewe J, Kemnitzer W. Discovery of 4-aryl-4H-chromenes as potent apoptosis inducers using a cell- and caspase-based Anti-cancer Screening Apoptosis Program (ASAP): SAR studies and the identification of novel vascular disrupting agents. Anticancer Agents Med Chem 2009;9(4):437-56.

114. Cai SX. Small molecule vascular disrupting agents: potential new drugs for cancer treatment. Recent Pat Anticancer Drug Discov 2007;2(1):79-101.

115. Yoshimatsu K, Yamaguchi A, Yoshino H, Koyanagi N, Kitoh K. Mechanism of action of E7010, an orally active sulfonamide antitumor agent: inhibition of mitosis by binding to the colchicine site of tubulin. Cancer Res 1997;57(15):3208- 13.

116. Banerjee M, Poddar A, Mitra G, Surolia A, Owa T, Bhattacharyya B. Sulfonamide drugs binding to the colchicine site of tubulin: thermodynamic analysis of the drug-tubulin interactions by isothermal titration calorimetry. J Med Chem 2005;48(2):547-55.

117. Luo Y, Hradil VP, Frost DJ, et al. ABT-751, a novel tubulin-binding agent, decreases tumor perfusion and disrupts tumor vasculature. Anti-cancer drugs 2009;20(6):483-92.

118. Kasibhatla S, Baichwal V, Cai SX, et al. MPC-6827: a small-molecule inhibitor of microtubule formation that is not a substrate for multidrug resistance pumps. Cancer Res 2007;67(12):5865-71.

119. Cox DS, Scott KR, Gao H, Raje S, Eddington ND. Influence of multidrug resistance (MDR) proteins at the blood-brain barrier on the transport and brain distribution of enaminone anticonvulsants. J Pharm Sci 2001;90(10):1540-52.

120. Nicholson B, Lloyd GK, Miller BR, et al. NPI-2358 is a tubulin- depolymerizing agent: in-vitro evidence for activity as a tumor vascular-disrupting agent. Anti-cancer drugs 2006;17(1):25-31.

121. Johnson IS, Wright HF, Svoboda GH, Vlantis J. Antitumor principles derived from Vinca rosea Linn. I. Vincaleukoblastine and leurosine. Cancer Res 1960;20:1016-22.

122. Cutts JH, Beer CT, Noble RL. Biological properties of Vincaleukoblastine, an alkaloid in Vinca rosea Linn, with reference to its antitumor action. Cancer Res 1960;20:1023-31.

123. Bai RL, Pettit GR, Hamel E. Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and vinca alkaloid sites. The Journal of biological chemistry 1990;265(28):17141-9.

124. Wilson L, Jordan MA, Morse A, Margolis RL. Interaction of vinblastine with steady-state microtubules in vitro. Journal of molecular biology 1982;159(1):125-49.

125. Jordan MA, Wilson L. Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations. Biochemistry 1990;29(11):2730-9.

180

126. Jordan MA, Margolis RL, Himes RH, Wilson L. Identification of a distinct class of vinblastine binding sites on microtubules. Journal of molecular biology 1986;187(1):61-73.

127. Singer WD, Jordan MA, Wilson L, Himes RH. Binding of vinblastine to stabilized microtubules. Molecular pharmacology 1989;36(3):366-70.

128. Jordan MA. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Current medicinal chemistry. Anti-cancer agents 2002;2(1):1-17.

129. Erickson HP. Negatively stained vinblastine aggregates. Annals of the New York Academy of Sciences 1975;253:51-2.

130. Himes RH. Interactions of the catharanthus (Vinca) alkaloids with tubulin and microtubules. Pharmacology & therapeutics 1991;51(2):257-67.

131. Geldof AA, Minneboo A, Heimans JJ. Vinca-alkaloid neurotoxicity measured using an in vitro model. Journal of neuro-oncology 1998;37(2):109-13.

132. Zhou XJ, Placidi M, Rahmani R. Uptake and metabolism of vinca alkaloids by freshly isolated human hepatocytes in suspension. Anticancer research 1994;14(3A):1017-22.

133. Budman DR. Vinorelbine (Navelbine): a third-generation vinca alkaloid. Cancer investigation 1997;15(5):475-90.

134. Photiou A, Sheikh MN, Bafaloukos D, Retsas S. Antiproliferative activity of vinorelbine (Navelbine) against six human melanoma cell lines. Journal of cancer research and clinical oncology 1992;118(4):249-54.

135. Uckun FM, Morar S, Qazi S. Vinorelbine-based salvage chemotherapy for therapy-refractory aggressive leukaemias. British journal of haematology 2006;135(4):500-8.

136. Bunn PA, Jr., Kelly K. New chemotherapeutic agents prolong survival and improve quality of life in non-small cell lung cancer: a review of the literature and future directions. Clinical cancer research : an official journal of the American Association for Cancer Research 1998;4(5):1087-100.

137. Hill BT. Vinflunine, a second generation novel Vinca Alkaloid with a distinctive pharmacological profile, now in clinical development and prospects for future mitotic blockers. Current pharmaceutical design 2001;7(13):1199-212.

138. Ngan VK, Bellman K, Panda D, Hill BT, Jordan MA, Wilson L. Novel actions of the antitumor drugs vinflunine and vinorelbine on microtubules. Cancer research 2000;60(18):5045-51.

139. Jordan MA, Horwitz SB, Lobert S, Correia JJ. Exploring the mechanisms of action of the novel microtubule inhibitor vinflunine. Seminars in oncology

2008;35(3 Suppl 3):S6-S12.

181

140. Kruczynski A, Barret JM, Etievant C, Colpaert F, Fahy J, Hill BT. Antimitotic and tubulin-interacting properties of vinflunine, a novel fluorinated Vinca alkaloid. Biochemical pharmacology 1998;55(5):635-48.

141. Kruczynski A, Hill BT. Vinflunine, the latest Vinca alkaloid in clinical development. A review of its preclinical anticancer properties. Critical reviews in oncology/hematology 2001;40(2):159-73.

142. Okouneva T, Hill BT, Wilson L, Jordan MA. The effects of vinflunine, vinorelbine, and vinblastine on centromere dynamics. Mol Cancer Ther 2003;2(5):427-36.

143. Lobert S, Ingram JW, Hill BT, Correia JJ. A comparison of thermodynamic parameters for vinorelbine- and vinflunine-induced tubulin self-association by sedimentation velocity. Mol Pharmacol 1998;53(5):908-15.

144. Kruczynski A, Poli M, Dossi R, et al. Anti-angiogenic, vascular-disrupting and anti-metastatic activities of vinflunine, the latest vinca alkaloid in clinical development. Eur J Cancer 2006;42(16):2821-32.

145. Holwell SE, Hill BT, Bibby MC. Anti-vascular effects of vinflunine in the MAC 15A transplantable adenocarcinoma model. Br J Cancer 2001;84(2):290-5.

146. Duflos A, Kruczynski A, Barret JM. Novel aspects of natural and modified vinca alkaloids. Current medicinal chemistry. Anti-cancer agents 2002;2(1):55-70.

147. Kingston DG. Tubulin-interactive natural products as anticancer agents. Journal of natural products 2009;72(3):507-15.

148. Gralla RJ, Gatzemeier U, Gebbia V, Huber R, O'Brien M, Puozzo C. Oral vinorelbine in the treatment of non-small cell lung cancer: rationale and implications for patient management. Drugs 2007;67(10):1403-10.

149. Aapro MS, Conte P, Esteban Gonzalez E, Trillet-Lenoir V. Oral vinorelbine: role in the management of metastatic breast cancer. Drugs 2007;67(5):657-67.

150. Mano M. Vinorelbine in the management of breast cancer: New perspectives, revived role in the era of targeted therapy. Cancer Treat Rev 2006;32(2):106-18.

151. von Mehren M, Balcerzak SP, Kraft AS, et al. Phase II Trial of Dolastatin- 10, a Novel Anti-Tubulin Agent, in Metastatic Soft Tissue Sarcomas. Sarcoma 2004;8(4):107-11.

152. Banerjee S, Wang Z, Mohammad M, Sarkar FH, Mohammad RM. Efficacy of selected natural products as therapeutic agents against cancer. Journal of natural products 2008;71(3):492-6.

153. Singh R, Sharma M, Joshi P, Rawat DS. Clinical status of anti-cancer agents derived from marine sources. Anticancer Agents Med Chem 2008;8(6):603-17.

182

154. Miyazaki K, Kobayashi M, Natsume T, et al. Synthesis and antitumor activity of novel dolastatin 10 analogs. Chem Pharm Bull (Tokyo) 1995;43(10):1706-18.

155. Watanabe J, Endo Y, Shimada N, Natsume T, Sasaki T, Kobayashi M. Antiangiogenic activity of TZT-1027 (soblidotin) on chick chorioallantoic membrane and human umbilical vein endothelial cells. In Vivo 2007;21(2):297-304.

156. Natsume T, Watanabe J, Ogawa K, Yasumura K, Kobayashi M. Tumor- specific antivascular effect of TZT-1027 (Soblidotin) elucidated by magnetic resonance imaging and confocal laser scanning microscopy. Cancer Sci 2007;98(4):598-604.

157. Cigler T, Vahdat LT. Eribulin mesylate for the treatment of breast cancer. Expert Opin Pharmacother 2010;11(9):1587-93.

158. Jordan MA, Kamath K, Manna T, et al. The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth. Mol Cancer Ther 2005;4(7):1086-95.

159. Kuznetsov G, TenDyke K, Yu M, Littlefield B. Antiproliferative effects of halichondrin B analog eribulin mesylate (E7389) against paclitaxel-resistant human cancer cells in vitro. In; 2007; 2007. p. 275.

160. Towle MJ, Salvato KA, Budrow J, et al. In vitro and in vivo anticancer activities of synthetic macrocyclic ketone analogues of halichondrin B. Cancer Res 2001;61(3):1013-21.

161. Fornier MN. Approved agents for metastatic breast cancer. Seminars in oncology 2011;38 Suppl 2:S3-10.

162. Bacher G, Nickel B, Emig P, et al. D-24851, a novel synthetic microtubule inhibitor, exerts curative antitumoral activity in vivo, shows efficacy toward multidrug-resistant tumor cells, and lacks neurotoxicity. Cancer Res 2001;61(1):392- 9.

163. Wienecke A, Bacher G. Indibulin, a novel microtubule inhibitor, discriminates between mature neuronal and nonneuronal tubulin. Cancer Res 2009;69(1):171-7.

164. He X, Li S, Huang H, et al. A pharmacokinetic and safety study of single dose intravenous combretastatin A4 phosphate in Chinese patients with refractory solid tumours. Br J Clin Pharmacol 2011;71(6):860-70.

165. Koh DM, Blackledge M, Collins DJ, et al. Reproducibility and changes in the apparent diffusion coefficients of solid tumours treated with combretastatin A4 phosphate and bevacizumab in a two-centre phase I clinical trial. Eur Radiol 2009;19(11):2728-38.

166. Meyer T, Gaya AM, Dancey G, et al. A phase I trial of radioimmunotherapy with 131I-A5B7 anti-CEA antibody in combination with combretastatin-A4-

183

phosphate in advanced gastrointestinal carcinomas. Clin Cancer Res 2009;15(13):4484-92.

167. Rustin GJ, Shreeves G, Nathan PD, et al. A Phase Ib trial of CA4P (combretastatin A-4 phosphate), carboplatin, and paclitaxel in patients with advanced cancer. Br J Cancer 2010;102(9):1355-60.

168. Bilenker JH, Flaherty KT, Rosen M, et al. Phase I trial of combretastatin a-4 phosphate with carboplatin. Clin Cancer Res 2005;11(4):1527-33.

169. Dahut WL, Lakhani NJ, Gulley JL, et al. Phase I clinical trial of oral 2- methoxyestradiol, an antiangiogenic and apoptotic agent, in patients with solid tumors. Cancer biology & therapy 2006;5(1):22-7.

170. James J, Murry DJ, Treston AM, et al. Phase I safety, pharmacokinetic and pharmacodynamic studies of 2-methoxyestradiol alone or in combination with docetaxel in patients with locally recurrent or metastatic breast cancer. Investigational new drugs 2007;25(1):41-8.

171. Kulke MH, Chan JA, Meyerhardt JA, et al. A prospective phase II study of 2- methoxyestradiol administered in combination with bevacizumab in patients with metastatic carcinoid tumors. Cancer Chemother Pharmacol 2011;68(2):293-300.

172. Matei D, Schilder J, Sutton G, et al. Activity of 2 methoxyestradiol (Panzem NCD) in advanced, platinum-resistant ovarian cancer and primary peritoneal carcinomatosis: a Hoosier Oncology Group trial. Gynecologic oncology 2009;115(1):90-6.

173. Rajkumar SV, Richardson PG, Lacy MQ, et al. Novel therapy with 2- methoxyestradiol for the treatment of relapsed and plateau phase multiple myeloma. Clinical cancer research : an official journal of the American Association for Cancer Research 2007;13(20):6162-7.

174. Sweeney C, Liu G, Yiannoutsos C, et al. A phase II multicenter, randomized, double-blind, safety trial assessing the pharmacokinetics, pharmacodynamics, and efficacy of oral 2-methoxyestradiol capsules in hormone-refractory prostate cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2005;11(18):6625-33.

175. Tevaarwerk AJ, Holen KD, Alberti DB, et al. Phase I trial of 2- methoxyestradiol NanoCrystal dispersion in advanced solid malignancies. Clin Cancer Res 2009;15(4):1460-5.

176. Zhou Q, Gustafson D, Nallapareddy S, et al. A phase I dose-escalation, safety and pharmacokinetic study of the 2-methoxyestradiol analog ENMD-1198 administered orally to patients with advanced cancer. Investigational new drugs 2011;29(2):340-6.

177. Fox E, Maris JM, Cohn SL, et al. Pharmacokinetics of orally administered ABT-751 in children with neuroblastoma and other solid tumors. Cancer Chemother

Pharmacol 2010;66(4):737-43.

184

178. Fox E, Maris JM, Widemann BC, et al. A phase I study of ABT-751, an orally bioavailable tubulin inhibitor, administered daily for 21 days every 28 days in pediatric patients with solid tumors. Clin Cancer Res 2008;14(4):1111-5.

179. Mauer AM, Cohen EE, Ma PC, et al. A phase II study of ABT-751 in patients with advanced non-small cell lung cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2008;3(6):631-6.

180. Meany HJ, Sackett DL, Maris JM, et al. Clinical outcome in children with recurrent neuroblastoma treated with ABT-751 and effect of ABT-751 on proliferation of neuroblastoma cell lines and on tubulin polymerization in vitro. Pediatr Blood Cancer 2010;54(1):47-54.

181. Michels J, Ellard SL, Le L, et al. A phase IB study of ABT-751 in combination with docetaxel in patients with advanced castration-resistant prostate cancer. Ann Oncol 2010;21(2):305-11.

182. Rudin CM, Mauer A, Smakal M, et al. Phase I/II study of pemetrexed with or without ABT-751 in advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2011;29(8):1075-82.

183. Tsimberidou AM, Akerley W, Schabel MC, et al. Phase I clinical trial of MPC-6827 (Azixa), a microtubule destabilizing agent, in patients with advanced cancer. Mol Cancer Ther 2010;9(12):3410-9.

184. Mita MM, Spear MA, Yee LK, et al. Phase 1 first-in-human trial of the vascular disrupting agent plinabulin(NPI-2358) in patients with solid tumors or lymphomas. Clin Cancer Res 2010;16(23):5892-9.

185. Jameson MB, Sharp DM, Sissingh JI, et al. Transient retinal effects of 5,6- dimethylxanthenone-4-acetic acid (DMXAA, ASA404), an antitumor vascular- disrupting agent in phase I clinical trials. Invest Ophthalmol Vis Sci 2009;50(6):2553-9.

186. Jameson MB, Thompson PI, Baguley BC, et al. Clinical aspects of a phase I trial of 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent. Br J Cancer 2003;88(12):1844-50.

187. Rustin GJ, Bradley C, Galbraith S, et al. 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a novel antivascular agent: phase I clinical and pharmacokinetic study. Br J Cancer 2003;88(8):1160-7.

188. McKeage MJ, Reck M, Jameson MB, et al. Phase II study of ASA404 (vadimezan, 5,6-dimethylxanthenone-4-acetic acid/DMXAA) 1800mg/m(2) combined with carboplatin and paclitaxel in previously untreated advanced non- small cell lung cancer. Lung cancer 2009;65(2):192-7.

189. Arakelyan N, Jais JP, Delwail V, et al. Reduced versus full doses of irradiation after 3 cycles of combined doxorubicin, bleomycin, vinblastine, and dacarbazine in early stage Hodgkin lymphomas: results of a randomized trial. Cancer 2010;116(17):4054-62. 185

190. Dumontet C, Jordan MA. Microtubule-binding agents: a dynamic field of cancer therapeutics. Nat Rev Drug Discov 2010;9(10):790-803.

191. Griffiths G, Hall R, Sylvester R, Raghavan D, Parmar MK. International phase III trial assessing neoadjuvant cisplatin, methotrexate, and vinblastine chemotherapy for muscle-invasive bladder cancer: long-term results of the BA06 30894 trial. J Clin Oncol 2011;29(16):2171-7.

192. Armitage JO. Overview of rational and individualized therapeutic strategies for non-Hodgkin's lymphomas. Clin Lymphoma 2002;3 Suppl 1:S5-11.

193. Borchmann P, Herbrecht R, Wilhelm M, et al. Phase I/II study of pixantrone in combination with cyclophosphamide, vincristine, and prednisone in patients with relapsed aggressive non-Hodgkin lymphoma. Leukemia & lymphoma 2011;52(4):620-8.

194. Faderl S, Thomas DA, O'Brien S, et al. Augmented hyper-CVAD based on dose-intensified vincristine, dexamethasone, and asparaginase in adult acute lymphoblastic leukemia salvage therapy. Clin Lymphoma Myeloma Leuk 2011;11(1):54-9.

195. Mascarenhas L, Lyden ER, Breitfeld PP, et al. Randomized phase II window trial of two schedules of irinotecan with vincristine in patients with first relapse or progression of rhabdomyosarcoma: a report from the Children's Oncology Group. J Clin Oncol 2010;28(30):4658-63.

196. Anton A, Barnadas A, Florian J, et al. Biweekly vinorelbine and tegafur/uracil in patients with metastatic breast cancer previously treated with anthracyclines and taxanes: GEICAM 2000-02 phase II study. Clin Transl Oncol 2011;13(4):281-6.

197. Massa E, Dessi M, Gaspardini G, Saba F, Cherchi V, Mantovani G. Phase II study of vinorelbine/cetuximab in patients with recurrent/metastatic squamous cell carcinoma of the head and neck progressing after at least two chemotherapy regimens. Oral Oncol 2010;46(11):818-21.

198. Karampeazis A, Vamvakas L, Agelidou A, et al. Docetaxel vs. vinorelbine in elderly patients with advanced non--small-cell lung cancer: a hellenic oncology research group randomized phase III study. Clin Lung Cancer 2011;12(3):155-60.

199. Schwarzenberger P, Fariss A, Linares L, Nedzi L, Salazar OM. Dose escalation of once weekly oral vinorelbine concurrent with weekly split dose hypofractionated chest radiation for palliation of advanced non-small cell lung cancer: a phase I/II study. Am J Med Sci 2011;341(6):454-9.

200. Segawa Y, Kiura K, Takigawa N, et al. Phase III trial comparing docetaxel and cisplatin combination chemotherapy with mitomycin, vindesine, and cisplatin combination chemotherapy with concurrent thoracic radiotherapy in locally advanced non-small-cell lung cancer: OLCSG 0007. J Clin Oncol 2010;28(20):3299- 306.

186

201. Santo A, Genestreti G, Terzi A, et al. Gemcitabine (GEM) and vindesine (VDS) in advanced non-small cell lung cancer (NSCLC): a phase II study in elderly or poor performance status patients. Lung cancer 2006;53(3):355-60.

202. van Luijk IF, Coens C, van der Burg ME, et al. Phase II study of bleomycin, vindesine, mitomycin C and cisplatin (BEMP) in recurrent or disseminated squamous cell carcinoma of the uterine cervix. Ann Oncol 2007;18(2):275-81.

203. Bellmunt J, Theodore C, Demkov T, et al. Phase III trial of vinflunine plus best supportive care compared with best supportive care alone after a platinum- containing regimen in patients with advanced transitional cell carcinoma of the urothelial tract. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2009;27(27):4454-61.

204. Hainsworth JD, Meluch AA, Lane CM, et al. Single agent vinflunine in the salvage treatment of patients with castration-resistant prostate cancer: a phase II trial of the Sarah Cannon research consortium. Cancer Invest 2010;28(3):275-9.

205. Krzakowski M, Ramlau R, Jassem J, et al. Phase III trial comparing vinflunine with docetaxel in second-line advanced non-small-cell lung cancer previously treated with platinum-containing chemotherapy. J Clin Oncol 2010;28(13):2167-73.

206. Spigel DR, Hainsworth JD, Lane CM, Clark B, Burris HA, Greco FA. Phase II trial of vinflunine in relapsed small cell lung cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2010;5(6):874-8.

207. Yardley DA, McCleod M, Schreiber F, et al. A phase II trial of vinflunine as monotherapy or in combination with trastuzumab as first-line treatment of metastatic breast cancer. Cancer Invest 2010;28(9):925-31.

208. Fornier MN, Morris PG, Abbruzzi A, et al. A phase I study of dasatinib and weekly paclitaxel for metastatic breast cancer. Ann Oncol 2011.

209. Goel S, Mita AC, Mita M, et al. A phase I study of eribulin mesylate (E7389), a mechanistically novel inhibitor of microtubule dynamics, in patients with advanced solid malignancies. Clin Cancer Res 2009;15(12):4207-12.

210. Tan AR, Rubin EH, Walton DC, et al. Phase I study of eribulin mesylate administered once every 21 days in patients with advanced solid tumors. Clin Cancer Res 2009;15(12):4213-9.

211. Kuppens IE, Witteveen PO, Schot M, et al. Phase I dose-finding and pharmacokinetic trial of orally administered indibulin (D-24851) to patients with solid tumors. Invest New Drugs 2007;25(3):227-35.

212. Oostendorp RL, Witteveen PO, Schwartz B, et al. Dose-finding and pharmacokinetic study of orally administered indibulin (D-24851) to patients with advanced solid tumors. Invest New Drugs 2010;28(2):163-70.

187

213. Horti J, Juhasz E, Monostori Z, Maeda K, Eckhardt S, Bodrogi I. Phase I study of TZT-1027, a novel synthetic dolastatin 10 derivative, for the treatment of patients with non-small cell lung cancer. Cancer chemotherapy and pharmacology 2008;62(1):173-80.

214. Patel S, Keohan ML, Saif MW, et al. Phase II study of intravenous TZT-1027 in patients with advanced or metastatic soft-tissue sarcomas with prior exposure to anthracycline-based chemotherapy. Cancer 2006;107(12):2881-7.

215. Yamamoto N, Andoh M, Kawahara M, Fukuoka M, Niitani H. Phase I study of TZT-1027, a novel synthetic dolastatin 10 derivative and inhibitor of tubulin polymerization, given weekly to advanced solid tumor patients for 3 weeks. Cancer Sci 2009;100(2):316-21.

216. Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 1971;93(9):2325-7.

217. Derry WB, Wilson L, Jordan MA. Low potency of taxol at microtubule minus ends: implications for its antimitotic and therapeutic mechanism. Cancer Res 1998;58(6):1177-84.

218. Nogales E, Whittaker M, Milligan RA, Downing KH. High-resolution model of the microtubule. Cell 1999;96(1):79-88.

219. Terwogt JM, Nuijen B, Huinink WW, Beijnen JH. Alternative formulations of paclitaxel. Cancer Treat Rev 1997;23(2):87-95.

220. Gelderblom H, Verweij J, Nooter K, Sparreboom A. Cremophor EL: the drawbacks and advantages of vehicle selection for drug formulation. Eur J Cancer 2001;37(13):1590-8.

221. Sparreboom A, van Zuylen L, Brouwer E, et al. Cremophor EL-mediated alteration of paclitaxel distribution in human blood: clinical pharmacokinetic implications. Cancer Res 1999;59(7):1454-7.

222. Ng SS, Figg WD, Sparreboom A. Taxane-mediated antiangiogenesis in vitro: influence of formulation vehicles and binding proteins. Cancer Res 2004;64(3):821- 4.

223. Parekh H, Wiesen K, Simpkins H. Acquisition of taxol resistance via P- glycoprotein- and non-P-glycoprotein-mediated mechanisms in human ovarian carcinoma cells. Biochem Pharmacol 1997;53(4):461-70.

224. Gligorov J, Lotz JP. Preclinical pharmacology of the taxanes: implications of the differences. Oncologist 2004;9 Suppl 2:3-8.

225. Dranitsaris G, Coleman R, Gradishar W. nab-Paclitaxel weekly or every 3 weeks compared to standard docetaxel as first-line therapy in patients with metastatic breast cancer: an economic analysis of a prospective randomized trial.

Breast Cancer Res Treat 2010;119(3):717-24.

188

226. Gradishar WJ, Krasnojon D, Cheporov S, et al. Significantly longer progression-free survival with nab-paclitaxel compared with docetaxel as first-line therapy for metastatic breast cancer. J Clin Oncol 2009;27(22):3611-9.

227. Teneriello MG, Tseng PC, Crozier M, et al. Phase II evaluation of nanoparticle albumin-bound paclitaxel in platinum-sensitive patients with recurrent ovarian, peritoneal, or fallopian tube cancer. J Clin Oncol 2009;27(9):1426-31.

228. Stinchcombe TE. Nanoparticle albumin-bound paclitaxel: a novel Cremphor- EL-free formulation of paclitaxel. Nanomedicine (Lond) 2007;2(4):415-23.

229. Shionoya M, Jimbo T, Kitagawa M, Soga T, Tohgo A. DJ-927, a novel oral taxane, overcomes P-glycoprotein-mediated multidrug resistance in vitro and in vivo. Cancer Sci 2003;94(5):459-66.

230. Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular, and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 1999;39:361-98.

231. Cisternino S, Bourasset F, Archimbaud Y, Semiond D, Sanderink G, Scherrmann JM. Nonlinear accumulation in the brain of the new taxoid TXD258 following saturation of P-glycoprotein at the blood-brain barrier in mice and rats. British journal of pharmacology 2003;138(7):1367-75.

232. Tsao CK, Seng S, Oh WK, Galsky MD. Clinical development of cabazitaxel for the treatment of castration-resistant prostate cancer. Clin Med Insights Oncol 2011;5:163-9.

233. Polizzi D, Pratesi G, Tortoreto M, et al. A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts. Cancer Res 1999;59(5):1036-40.

234. Sampath D, Discafani CM, Loganzo F, et al. MAC-321, a novel taxane with greater efficacy than paclitaxel and docetaxel in vitro and in vivo. Mol Cancer Ther 2003;2(9):873-84.

235. Metzger-Filho O, Moulin C, de Azambuja E, Ahmad A. Larotaxel: broadening the road with new taxanes. Expert opinion on investigational drugs 2009;18(8):1183-9.

236. Bradley MO, Swindell CS, Anthony FH, et al. Tumor targeting by conjugation of DHA to paclitaxel. J Control Release 2001;74(1-3):233-6.

237. Bollag DM, McQueney PA, Zhu J, et al. Epothilones, a new class of microtubule-stabilizing agents with a taxol-like mechanism of action. Cancer Res 1995;55(11):2325-33.

238. Altmann KH, Wartmann M, O'Reilly T. Epothilones and related structures--a new class of microtubule inhibitors with potent in vivo antitumor activity. Biochim Biophys Acta 2000;1470(3):M79-91.

189

239. Nettles JH, Li H, Cornett B, Krahn JM, Snyder JP, Downing KH. The binding mode of epothilone A on alpha,beta-tubulin by electron crystallography. Science 2004;305(5685):866-9.

240. Chou TC, Zhang XG, Harris CR, et al. Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel. Proc Natl Acad Sci U S A 1998;95(26):15798-802.

241. Kowalski RJ, Giannakakou P, Hamel E. Activities of the microtubule- stabilizing agents epothilones A and B with purified tubulin and in cells resistant to paclitaxel (Taxol(R)). J Biol Chem 1997;272(4):2534-41.

242. Hari M, Loganzo F, Annable T, et al. Paclitaxel-resistant cells have a mutation in the paclitaxel-binding region of beta-tubulin (Asp26Glu) and less stable microtubules. Mol Cancer Ther 2006;5(2):270-8.

243. Rothermel J, Wartmann M, Chen T, Hohneker J. EPO906 (epothilone B): a promising novel microtubule stabilizer. Seminars in oncology 2003;30(3 Suppl 6):51-5.

244. Chen SM, Meng LH, Ding J. New microtubule-inhibiting anticancer agents. Expert opinion on investigational drugs 2010;19(3):329-43.

245. Abrey L, Wen P, Govindan R, et al. Activity of patupilone for the treatment of recurrent or progressive brain metastases in patients (pts) with non-small cell lung cancer (NSCLC): an open-label, multicenter, phase II study. Journal of Clinical Oncology 2007;25(18_suppl):18058.

246. Conlin AK, Seidman AD. Point: combination versus single-agent chemotherapy: the argument for sequential single agents. J Natl Compr Canc Netw 2007;5(8):668-72.

247. Sparano JA, Vrdoljak E, Rixe O, et al. Randomized phase III trial of ixabepilone plus capecitabine versus capecitabine in patients with metastatic breast cancer previously treated with an anthracycline and a taxane. J Clin Oncol 2010;28(20):3256-63.

248. Klar U, Buchmann B, Schwede W, Skuballa W, Hoffmann J, Lichtner RB. Total synthesis and antitumor activity of ZK-EPO: the first fully synthetic epothilone in clinical development. Angew Chem Int Ed Engl 2006;45(47):7942-8.

249. Galmarini CM. Sagopilone, a microtubule stabilizer for the potential treatment of cancer. Curr Opin Investig Drugs 2009;10(12):1359-71.

250. Hoffmann J, Fichtner I, Lemm M, et al. Sagopilone crosses the blood-brain barrier in vivo to inhibit brain tumor growth and metastases. Neuro-oncology 2009;11(2):158-66.

251. Stupp R, Tosoni A, Bromberg JE, et al. Sagopilone (ZK-EPO, ZK 219477) for recurrent glioblastoma. A phase II multicenter trial by the European Organisation for Research and Treatment of Cancer (EORTC) Brain Tumor Group. Ann Oncol 2011;22(9):2144-9. 190

252. Lee JJ, Swain SM. The epothilones: translating from the laboratory to the clinic. Clin Cancer Res 2008;14(6):1618-24.

253. Fumoleau P, Coudert B, Isambert N, Ferrant E. Novel tubulin-targeting agents: anticancer activity and pharmacologic profile of epothilones and related analogues. Ann Oncol 2007;18 Suppl 5:v9-15.

254. Overmoyer B, Waintraub S, Kaufman P, et al. Phase II trial of KOS-862 (epothilone D) in anthracycline and taxane pretreated metastatic breast cancer. Journal of Clinical Oncology 2005;23(16_suppl):778.

255. Zhou Y, Zhong Z, Liu F, et al. KOS-1584: a rationally designed epothilone D analog with improved potency and pharmacokinetic (PK) properties. Proceedings of the American Association for Cancer Research 2005;2005(1):595.

256. Villalona-Calero M, Goel S, Schaaf L, et al. First-in-human phase I trial of a novel epothilone, KOS-1584. Journal of Clinical Oncology 2006;24(18_suppl):2003.

257. Kowalski RJ, Giannakakou P, Gunasekera SP, Longley RE, Day BW, Hamel E. The microtubule-stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (Taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel- resistant cells. Mol Pharmacol 1997;52(4):613-22.

258. Martello LA, McDaid HM, Regl DL, et al. Taxol and discodermolide represent a synergistic drug combination in human carcinoma cell lines. Clin Cancer Res 2000;6(5):1978-87.

259. Altmann KH. Microtubule-stabilizing agents: a growing class of important anticancer drugs. Curr Opin Chem Biol 2001;5(4):424-31.

260. Honore S, Kamath K, Braguer D, et al. Synergistic suppression of microtubule dynamics by discodermolide and paclitaxel in non-small cell lung carcinoma cells. Cancer Res 2004;64(14):4957-64.

261. Mita A, Lockhart AC, Chen TL, et al. A phase I pharmacokinetic (PK) trial of XAA296A (Discodermolide) administered every 3 wks to adult patients with advanced solid malignancies. In; 2004; 2004. p. 2025.

262. Long BH, Carboni JM, Wasserman AJ, et al. Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol). Cancer Res 1998;58(6):1111-5.

263. Attard G, Greystoke A, Kaye S, De Bono J. Update on tubulin-binding agents. Pathol Biol (Paris) 2006;54(2):72-84.

264. McDaid HM, Bhattacharya SK, Chen XT, et al. Structure-activity profiles of eleutherobin analogs and their cross-resistance in Taxol-resistant cell lines. Cancer Chemother Pharmacol 1999;44(2):131-7.

265. Hamel E, Sackett DL, Vourloumis D, Nicolaou KC. The coral-derived natural products eleutherobin and sarcodictyins A and B: effects on the assembly of 191

purified tubulin with and without microtubule-associated proteins and binding at the polymer taxoid site. Biochemistry 1999;38(17):5490-8.

266. Nicolaou KC, Pfefferkorn J, Xu J, et al. Total synthesis and chemical biology of the sarcodictyins. Chem Pharm Bull (Tokyo) 1999;47(9):1199-213.

267. Mulzer J, Ohler E. Microtubule-stabilizing marine metabolite laulimalide and its derivatives: synthetic approaches and antitumor activity. Chem Rev 2003;103(9):3753-86.

268. Gapud EJ, Bai R, Ghosh AK, Hamel E. Laulimalide and paclitaxel: a comparison of their effects on tubulin assembly and their synergistic action when present simultaneously. Mol Pharmacol 2004;66(1):113-21.

269. Mooberry SL, Tien G, Hernandez AH, Plubrukarn A, Davidson BS. Laulimalide and isolaulimalide, new paclitaxel-like microtubule-stabilizing agents. Cancer Res 1999;59(3):653-60.

270. Crown J, O'Leary M, Ooi WS. Docetaxel and paclitaxel in the treatment of breast cancer: a review of clinical experience. Oncologist 2004;9 Suppl 2:24-32.

271. Kumar S, Mahdi H, Bryant C, Shah JP, Garg G, Munkarah A. Clinical trials and progress with paclitaxel in ovarian cancer. Int J Womens Health 2010;2:411-27.

272. Stebbing J, Wildfire A, Portsmouth S, et al. Paclitaxel for anthracycline- resistant AIDS-related Kaposi's sarcoma: clinical and angiogenic correlations. Ann Oncol 2003;14(11):1660-6.

273. Yamamoto Y, Kawano I, Iwase H. Nab-paclitaxel for the treatment of breast cancer: efficacy, safety, and approval. Onco Targets Ther 2011;4:123-36.

274. Katsumata N. Docetaxel: an alternative taxane in ovarian cancer. Br J Cancer 2003;89 Suppl 3:S9-S15.

275. Lyseng-Williamson KA, Fenton C. Docetaxel: a review of its use in metastatic breast cancer. Drugs 2005;65(17):2513-2531.

276. Michael A, Syrigos K, Pandha H. Prostate cancer chemotherapy in the era of targeted therapy. Prostate Cancer Prostatic Dis 2009;12(1):13-6.

277. Baas P, Szczesna A, Albert I, et al. Phase I/II study of a 3 weekly oral taxane (DJ-927) in patients with recurrent, advanced non-small cell lung cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2008;3(7):745-50.

278. Saif MW, Sarantopoulos J, Patnaik A, Tolcher AW, Takimoto C, Beeram M. Tesetaxel, a new oral taxane, in combination with capecitabine: a phase I, dose- escalation study in patients with advanced solid tumors. Cancer Chemother Pharmacol 2011.

192

279. Pivot X, Koralewski P, Hidalgo JL, et al. A multicenter phase II study of XRP6258 administered as a 1-h i.v. infusion every 3 weeks in taxane-resistant metastatic breast cancer patients. Ann Oncol 2008;19(9):1547-52.

280. Villanueva C, Awada A, Campone M, et al. A multicentre dose-escalating study of cabazitaxel (XRP6258) in combination with capecitabine in patients with metastatic breast cancer progressing after anthracycline and taxane treatment: a phase I/II study. Eur J Cancer 2011;47(7):1037-45.

281. Yoo GH, Kafri Z, Ensley JF, et al. XRP6258-induced gene expression patterns in head and neck cancer carcinoma. Laryngoscope 2010;120(6):1114-9.

282. Dieras V, Limentani S, Romieu G, et al. Phase II multicenter study of larotaxel (XRP9881), a novel taxoid, in patients with metastatic breast cancer who previously received taxane-based therapy. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 2008;19(7):1255-60.

283. Robert F, Harper K, Ackerman J, Gupta S. A phase I study of larotaxel (XRP9881) administered in combination with carboplatin in chemotherapy-naive patients with stage IIIB or stage IV non-small cell lung cancer. Cancer Chemother Pharmacol 2010;65(2):227-34.

284. Yamamoto N, Boku N, Minami H. Phase I study of larotaxel administered as a 1-h intravenous infusion every 3 weeks to Japanese patients with advanced solid tumours. Cancer chemotherapy and pharmacology 2009;65(1):129-36.

285. Zatloukal P, Gervais R, Vansteenkiste J, et al. Randomized multicenter phase II study of larotaxel (XRP9881) in combination with cisplatin or gemcitabine as first-line chemotherapy in nonirradiable stage IIIB or stage IV non-small cell lung cancer. Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer 2008;3(8):894-901.

286. Chi KN, Beardsley E, Eigl BJ, et al. A phase 2 study of patupilone in patients with metastatic castration-resistant prostate cancer previously treated with docetaxel: Canadian Urologic Oncology Group study P07a. Ann Oncol 2011.

287. Fogh S, Machtay M, Werner-Wasik M, et al. Phase I trial using patupilone (epothilone B) and concurrent radiotherapy for central nervous system malignancies. Int J Radiat Oncol Biol Phys 2010;77(4):1009-16.

288. Hussain A, DiPaola RS, Baron AD, Higano CS, Tchekmedyian NS, Johri AR. Phase II trial of weekly patupilone in patients with castration-resistant prostate cancer. Ann Oncol 2009;20(3):492-7.

289. Rubin EH, Rothermel J, Tesfaye F, et al. Phase I dose-finding study of weekly single-agent patupilone in patients with advanced solid tumors. J Clin Oncol 2005;23(36):9120-9.

290. Ten Bokkel Huinink WW, Sufliarsky J, Smit WM, et al. Safety and efficacy of patupilone in patients with advanced ovarian, primary fallopian, or primary peritoneal cancer: a phase I, open-label, dose-escalation study. J Clin Oncol 2009;27(19):3097-103. 193

291. Huang H, Menefee M, Edgerly M, et al. A phase II clinical trial of ixabepilone (Ixempra; BMS-247550; NSC 710428), an epothilone B analog, in patients with metastatic renal cell carcinoma. Clin Cancer Res 2010;16(5):1634-41.

292. Peereboom DM, Supko JG, Carson KA, et al. A phase I/II trial and pharmacokinetic study of ixabepilone in adult patients with recurrent high-grade gliomas. Journal of neuro-oncology 2010;100(2):261-8.

293. DeConti RC, Algazi AP, Andrews S, et al. Phase II trial of sagopilone, a novel epothilone analog in metastatic melanoma. Br J Cancer 2010;103(10):1548-53.

294. Arnold D, Voigt W, Kiewe P, et al. Weekly administration of sagopilone (ZK-EPO), a fully synthetic epothilone, in patients with refractory solid tumours: results of a phase I trial. Br J Cancer 2009;101(8):1241-7.

295. Morrow PK, Divers S, Provencher L, et al. Phase II study evaluating the efficacy and safety of sagopilone (ZK-EPO) in patients with metastatic breast cancer that has progressed following chemotherapy. Breast Cancer Res Treat 2010;123(3):837-42.

296. Schmid P, Kiewe P, Possinger K, et al. Phase I study of the novel, fully synthetic epothilone sagopilone (ZK-EPO) in patients with solid tumors. Ann Oncol 2010;21(3):633-9.

297. Silvani A, Gaviani P, Fiumani A, et al. Systemic sagopilone (ZK-EPO) treatment of patients with recurrent malignant gliomas. Journal of neuro-oncology 2009;95(1):61-4.

298. Beer TM, Higano CS, Saleh M, et al. Phase II study of KOS-862 in patients with metastatic androgen independent prostate cancer previously treated with docetaxel. Invest New Drugs 2007;25(6):565-70.

299. Monk JP, Villalona-Calero M, Larkin J, et al. A phase 1 study of KOS-862 (Epothilone D) co-administered with carboplatin (Paraplatin(R)) in patients with advanced solid tumors. Invest New Drugs 2011.

300. Musacchio A, Salmon ED. The spindle-assembly checkpoint in space and time. Nature reviews. Molecular cell biology 2007;8(5):379-93.

301. Gascoigne KE, Taylor SS. How do anti-mitotic drugs kill cancer cells? Journal of cell science 2009;122(Pt 15):2579-85.

302. Gascoigne KE, Taylor SS. Cancer cells display profound intra- and interline variation following prolonged exposure to antimitotic drugs. Cancer Cell 2008;14(2):111-22.

303. Shi J, Orth JD, Mitchison T. Cell type variation in responses to antimitotic drugs that target microtubules and kinesin-5. Cancer research 2008;68(9):3269-76.

304. Storey S. Targeting apoptosis: selected anticancer strategies. Nature reviews.

Drug discovery 2008;7(12):971-2.

194

305. Okada H, Mak TW. Pathways of apoptotic and non-apoptotic death in tumour cells. Nature reviews. Cancer 2004;4(8):592-603.

306. Varbiro G, Veres B, Gallyas F, Jr., Sumegi B. Direct effect of Taxol on free radical formation and mitochondrial permeability transition. Free radical biology & medicine 2001;31(4):548-58.

307. Andre N, Carre M, Brasseur G, et al. Paclitaxel targets mitochondria upstream of caspase activation in intact human neuroblastoma cells. FEBS letters 2002;532(1-2):256-60.

308. Du L, Lyle CS, Chambers TC. Characterization of vinblastine-induced Bcl- xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction. Oncogene 2005;24(1):107-17.

309. Pathan N, Aime-Sempe C, Kitada S, Basu A, Haldar S, Reed JC. Microtubule-targeting drugs induce bcl-2 phosphorylation and association with Pin1. Neoplasia 2001;3(6):550-9.

310. Haldar S, Chintapalli J, Croce CM. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996;56(6):1253-5.

311. Scatena CD, Stewart ZA, Mays D, et al. Mitotic phosphorylation of Bcl-2 during normal cell cycle progression and Taxol-induced growth arrest. J Biol Chem 1998;273(46):30777-84.

312. Tudor G, Aguilera A, Halverson DO, Laing ND, Sausville EA. Susceptibility to drug-induced apoptosis correlates with differential modulation of Bad, Bcl-2 and Bcl-xL protein levels. Cell death and differentiation 2000;7(6):574-86.

313. Jones NA, Turner J, McIlwrath AJ, Brown R, Dive C. Cisplatin- and paclitaxel-induced apoptosis of ovarian carcinoma cells and the relationship between bax and bak up-regulation and the functional status of p53. Mol Pharmacol 1998;53(5):819-26.

314. Sunters A, Fernandez de Mattos S, Stahl M, et al. FoxO3a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines. J Biol Chem 2003;278(50):49795-805.

315. Pourroy B, Carre M, Honore S, et al. Low concentrations of vinflunine induce apoptosis in human SK-N-SH neuroblastoma cells through a postmitotic G1 arrest and a mitochondrial pathway. Mol Pharmacol 2004;66(3):580-91.

316. Giannakakou P, Robey R, Fojo T, Blagosklonny MV. Low concentrations of paclitaxel induce cell type-dependent p53, p21 and G1/G2 arrest instead of mitotic arrest: molecular determinants of paclitaxel-induced cytotoxicity. Oncogene 2001;20(29):3806-13.

317. Bressin C, Bourgarel-Rey V, Carre M, et al. Decrease in c-Myc activity enhances cancer cell sensitivity to vinblastine. Anti-cancer drugs 2006;17(2):181-7.

195

318. Marchenko ND, Zaika A, Moll UM. Death signal-induced localization of p53 protein to mitochondria. A potential role in apoptotic signaling. J Biol Chem 2000;275(21):16202-12.

319. Mihara M, Erster S, Zaika A, et al. p53 has a direct apoptogenic role at the mitochondria. Mol Cell 2003;11(3):577-90.

320. Rovini A, Savry A, Braguer D, Carre M. Microtubule-targeted agents: when mitochondria become essential to chemotherapy. Biochim Biophys Acta 2011;1807(6):679-88.

321. LaVallee TM, Zhan XH, Johnson MS, et al. 2-methoxyestradiol up-regulates death receptor 5 and induces apoptosis through activation of the extrinsic pathway. Cancer research 2003;63(2):468-75.

322. Pucci B, Bellincampi L, Tafani M, Masciullo V, Melino G, Giordano A. Paclitaxel induces apoptosis in Saos-2 cells with CD95L upregulation and Bcl-2 phosphorylation. Experimental cell research 1999;252(1):134-43.

323. Kim R, Tanabe K, Emi M, Uchida Y, Toge T. Death receptor-dependent and -independent pathways in anticancer drug-induced apoptosis of breast cancer cells. Oncology reports 2003;10(6):1925-30.

324. Vakifahmetoglu H, Olsson M, Zhivotovsky B. Death through a tragedy: mitotic catastrophe. Cell death and differentiation 2008;15(7):1153-62.

325. Jordan MA, Wendell K, Gardiner S, Derry WB, Copp H, Wilson L. Mitotic block induced in HeLa cells by low concentrations of paclitaxel (Taxol) results in abnormal mitotic exit and apoptotic cell death. Cancer research 1996;56(4):816-25.

326. Okada H, Mak TW. Pathways of apoptotic and non-apoptotic death in tumour cells. Nat Rev Cancer 2004;4(8):592-603.

327. Morse DL, Gray H, Payne CM, Gillies RJ. Docetaxel induces cell death through mitotic catastrophe in human breast cancer cells. Mol Cancer Ther 2005;4(10):1495-504.

328. Nabha SM, Mohammad RM, Dandashi MH, et al. Combretastatin-A4 prodrug induces mitotic catastrophe in chronic lymphocytic leukemia cell line independent of caspase activation and poly(ADP-ribose) polymerase cleavage. Clinical cancer research : an official journal of the American Association for Cancer Research 2002;8(8):2735-41.

329. Cenciarelli C, Tanzarella C, Vitale I, et al. The tubulin-depolymerising agent combretastatin-4 induces ectopic aster assembly and mitotic catastrophe in lung cancer cells H460. Apoptosis : an international journal on programmed cell death 2008;13(5):659-69.

330. Drayton S, Peters G. Immortalisation and transformation revisited. Curr Opin Genet Dev 2002;12(1):98-104.

196

331. Wright WE, Shay JW. Historical claims and current interpretations of replicative aging. Nat Biotechnol 2002;20(7):682-8.

332. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 1995;92(20):9363-7.

333. Klein LE, Freeze BS, Smith AB, 3rd, Horwitz SB. The microtubule stabilizing agent discodermolide is a potent inducer of accelerated cell senescence. Cell Cycle 2005;4(3):501-7.

334. Tierno MB, Kitchens CA, Petrik B, et al. Microtubule binding and disruption and induction of premature senescence by disorazole C(1). The Journal of pharmacology and experimental therapeutics 2009;328(3):715-22.

335. Ashkenazi A. Targeting death and decoy receptors of the tumour-necrosis factor superfamily. Nat Rev Cancer 2002;2(6):420-30.

336. Kerbel RS. Clinical trials of antiangiogenic drugs: opportunities, problems, and assessment of initial results. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2001;19(18 Suppl):45S-51S.

337. Lippert JW, 3rd. Vascular disrupting agents. Bioorganic & medicinal chemistry 2007;15(2):605-15.

338. Folkman J, Browder T, Palmblad J. Angiogenesis research: guidelines for translation to clinical application. Thrombosis and haemostasis 2001;86(1):23-33.

339. Pasquier E, Andre N, Braguer D. Targeting microtubules to inhibit angiogenesis and disrupt tumour vasculature: implications for cancer treatment. Curr Cancer Drug Targets 2007;7(6):566-81.

340. Belotti D, Vergani V, Drudis T, et al. The microtubule-affecting drug paclitaxel has antiangiogenic activity. Clinical cancer research : an official journal of the American Association for Cancer Research 1996;2(11):1843-9.

341. Vacca A, Iurlaro M, Ribatti D, et al. Antiangiogenesis is produced by nontoxic doses of vinblastine. Blood 1999;94(12):4143-55.

342. Vacca A, Ribatti D, Iurlaro M, et al. Docetaxel versus paclitaxel for antiangiogenesis. J Hematother Stem Cell Res 2002;11(1):103-18.

343. Hotchkiss KA, Ashton AW, Mahmood R, Russell RG, Sparano JA, Schwartz EL. Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): association with impaired repositioning of the microtubule organizing center. Molecular cancer therapeutics 2002;1(13):1191-200.

344. Hayot C, Farinelle S, De Decker R, et al. In vitro pharmacological characterizations of the anti-angiogenic and anti-tumor cell migration properties mediated by microtubule-affecting drugs, with special emphasis on the organization

of the actin cytoskeleton. International journal of oncology 2002;21(2):417-25.

197

345. Wang J, Lou P, Lesniewski R, Henkin J. Paclitaxel at ultra low concentrations inhibits angiogenesis without affecting cellular microtubule assembly. Anti-cancer drugs 2003;14(1):13-9.

346. Pasquier E, Carre M, Pourroy B, et al. Antiangiogenic activity of paclitaxel is associated with its cytostatic effect, mediated by the initiation but not completion of a mitochondrial apoptotic signaling pathway. Mol Cancer Ther 2004;3(10):1301-10.

347. Bocci G, Francia G, Man S, Lawler J, Kerbel RS. Thrombospondin 1, a mediator of the antiangiogenic effects of low-dose metronomic chemotherapy. Proc Natl Acad Sci U S A 2003;100(22):12917-22.

348. Damber JE, Vallbo C, Albertsson P, Lennernas B, Norrby K. The anti- tumour effect of low-dose continuous chemotherapy may partly be mediated by thrombospondin. Cancer Chemother Pharmacol 2006;58(3):354-60.

349. Shaked Y, Emmenegger U, Man S, et al. Optimal biologic dose of metronomic chemotherapy regimens is associated with maximum antiangiogenic activity. Blood 2005;106(9):3058-61.

350. Ferrara N, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors. Nature medicine 1999;5(12):1359-64.

351. Lau DH, Xue L, Young LJ, Burke PA, Cheung AT. Paclitaxel (Taxol): an inhibitor of angiogenesis in a highly vascularized transgenic breast cancer. Cancer Biother Radiopharm 1999;14(1):31-6.

352. Avramis IA, Kwock R, Avramis VI. Taxotere and vincristine inhibit the secretion of the angiogenesis inducing vascular endothelial growth factor (VEGF) by wild-type and drug-resistant human leukemia T-cell lines. Anticancer Res 2001;21(4A):2281-6.

353. Mabjeesh NJ, Escuin D, LaVallee TM, et al. 2ME2 inhibits tumor growth and angiogenesis by disrupting microtubules and dysregulating HIF. Cancer Cell 2003;3(4):363-75.

354. Murtagh J, Lu H, Schwartz EL. Taxotere-induced inhibition of human endothelial cell migration is a result of heat shock protein 90 degradation. Cancer Res 2006;66(16):8192-9.

355. Laohavinij S, Maoleekoonpairoj S, Cheirsilpa A, et al. Phase II study of paclitaxel and carboplatin for advanced non-small-cell lung cancer. Lung cancer 1999;26(3):175-85.

356. Escuin D, Kline ER, Giannakakou P. Both microtubule-stabilizing and microtubule-destabilizing drugs inhibit hypoxia-inducible factor-1alpha accumulation and activity by disrupting microtubule function. Cancer Res 2005;65(19):9021-8.

357. Semenza GL. Targeting HIF-1 for cancer therapy. Nature reviews. Cancer

2003;3(10):721-32.

198

358. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407(6801):249-57.

359. Ludford RJ. Factors determining the action of colchicine on tumour growth. British journal of cancer 1948;2(1):75-86.

360. Hill SA, Lonergan SJ, Denekamp J, Chaplin DJ. The effect of vinca alkaloids on tumour blood flow. Advances in experimental medicine and biology 1994;345:417-22.

361. Hill SA, Lonergan SJ, Denekamp J, Chaplin DJ. Vinca alkaloids: anti- vascular effects in a murine tumour. European journal of cancer 1993;29A(9):1320- 4.

362. Thorpe PE. Vascular targeting agents as cancer therapeutics. Clin Cancer Res 2004;10(2):415-27.

363. Kageyama K, Yamada R, Otani S, et al. Cytotoxicity of docosahexaenoic acid and eicosapentaenoic acid in tumor cells and the dependence on binding to serum proteins and incorporation into intracellular lipids. Oncology reports 2000;7(1):79-83.

364. Natsume T, Watanabe J, Koh Y, et al. Antitumor activity of TZT-1027 (Soblidotin) against vascular endothelial growth factor-secreting human lung cancer in vivo. Cancer Sci 2003;94(9):826-33.

365. Segreti JA, Polakowski JS, Koch KA, et al. Tumor selective antivascular effects of the novel antimitotic compound ABT-751: an in vivo rat regional hemodynamic study. Cancer Chemother Pharmacol 2004;54(3):273-81.

366. Ding X, Zhang Z, Li S, Wang A. Combretastatin A4 phosphate induces programmed cell death in vascular endothelial cells. Oncol Res 2011;19(7):303-9.

367. Sheng Y, Hua J, Pinney KG, et al. Combretastatin family member OXI4503 induces tumor vascular collapse through the induction of endothelial apoptosis. Int J Cancer 2004;111(4):604-10.

368. Bohle AS, Leuschner I, Kalthoff H, Henne-Bruns D. Combretastatin A-4 prodrug: a potent inhibitor of malignant hemangioendothelioma cell proliferation. Int J Cancer 2000;87(6):838-43.

369. Merchan JR, Jayaram DR, Supko JG, He X, Bubley GJ, Sukhatme VP. Increased endothelial uptake of paclitaxel as a potential mechanism for its antiangiogenic effects: potentiation by Cox-2 inhibition. Int J Cancer 2005;113(3):490-8.

370. Prise VE, Honess DJ, Stratford MR, Wilson J, Tozer GM. The vascular response of tumor and normal tissues in the rat to the vascular targeting agent, combretastatin A-4-phosphate, at clinically relevant doses. International journal of oncology 2002;21(4):717-26.

199

371. Tozer GM, Prise VE, Wilson J, et al. Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability. Cancer research 2001;61(17):6413-22.

372. Canta A, Chiorazzi A, Cavaletti G. Tubulin: a target for antineoplastic drugs into the cancer cells but also in the peripheral nervous system. Current medicinal chemistry 2009;16(11):1315-24.

373. Argyriou AA, Koltzenburg M, Polychronopoulos P, Papapetropoulos S, Kalofonos HP. Peripheral nerve damage associated with administration of taxanes in patients with cancer. Critical reviews in oncology/hematology 2008;66(3):218-28.

374. Goa KL, Faulds D. Vinorelbine. A review of its pharmacological properties and clinical use in cancer chemotherapy. Drugs & aging 1994;5(3):200-34.

375. Rowinsky EK. The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annual review of medicine 1997;48:353-74.

376. Lysitsas DN, Katsouras CS, Papakostas JC, et al. Antirestenotic effects of a novel polymer-coated d-24851 eluting stent. Experimental data in a rabbit iliac artery model. Cardiovascular and interventional radiology 2007;30(6):1192-200.

377. Monzo M, Rosell R, Sanchez JJ, et al. Paclitaxel resistance in non-small-cell lung cancer associated with beta-tubulin gene mutations. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 1999;17(6):1786-93.

378. Aghajanian C, Burris HA, 3rd, Jones S, et al. Phase I study of the novel epothilone analog ixabepilone (BMS-247550) in patients with advanced solid tumors and lymphomas. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 2007;25(9):1082-8.

379. Lacey E. Mode of action of benzimidazoles. Parasitol Today 1990;6(4):112- 5.

380. Horton J. Albendazole: a broad spectrum anthelminthic for treatment of individuals and populations. Curr Opin Infect Dis 2002;15(6):599-608.

381. Merino G, Molina AJ, Garcia JL, Pulido MM, Prieto JG, Alvarez AI. Intestinal elimination of albendazole sulfoxide: pharmacokinetic effects of inhibitors. Int J Pharm 2003;263(1-2):123-32.

382. Velik J, Szotakova B, Baliharova V, et al. Albendazole repeated administration induces cytochromes P4501A and accelerates albendazole deactivation in mouflon (Ovis musimon). Res Vet Sci 2005;78(3):255-63.

383. Rawden HC, Kokwaro GO, Ward SA, Edwards G. Relative contribution of cytochromes P-450 and flavin-containing monoxygenases to the metabolism of albendazole by human liver microsomes. Br J Clin Pharmacol 2000;49(4):313-22.

384. Baliharova V, Skalova L, Maas RF, De Vrieze G, Bull S, Fink-Gremmels J.

The effects of benzimidazole anthelmintics on P4501A in rat hepatocytes and HepG2 cells. Res Vet Sci 2003;75(1):61-9. 200

385. Venkatesan P. Albendazole. J Antimicrob Chemother 1998;41(2):145-7.

386. Torrado S, Cadorniga R, Torrado JJ. Formulation parameters of albendazole solution. International Journal of Pharmaceutics 1996;140(1):45-50.

387. Kohri N, Yamayoshi Y, Xin H, et al. Improving the oral bioavailability of albendazole in rabbits by the solid dispersion technique. J Pharm Pharmacol 1999;51(2):159-64.

388. Garcia JJ, Bolas F, Torrado JJ. Bioavailability and efficacy characteristics of two different oral liquid formulations of albendazole. Int J Pharm 2003;250(2):351- 8.

389. Kalaiselvan R, Mohanta GP, Madhusudan S, Manna PK, Manavalan R. Enhancement of bioavailability and anthelmintic efficacy of albendazole by solid dispersion and cyclodextrin complexation techniques. Pharmazie 2007;62(8):604-7.

390. Mottier ML, Alvarez LI, Pis MA, Lanusse CE. Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni: correlation with their octanol- water partition coefficients. Exp Parasitol 2003;103(1-2):1-7.

391. Holland IB. ABC transporters, mechanisms and biology: an overview. Essays Biochem 2011;50(1):1-17.

392. Davidson AL, Dassa E, Orelle C, Chen J. Structure, function, and evolution of bacterial ATP-binding cassette systems. Microbiol Mol Biol Rev 2008;72(2):317- 64, table of contents.

393. Gillet JP, Gottesman MM. Advances in the molecular detection of ABC transporters involved in multidrug resistance in cancer. Curr Pharm Biotechnol 2011;12(4):686-92.

394. Merino G, Jonker JW, Wagenaar E, et al. Transport of anthelmintic benzimidazole drugs by breast cancer resistance protein (BCRP/ABCG2). Drug Metab Dispos 2005;33(5):614-8.

395. Barrowman MM, Marriner SE, Bogan JA. The binding and subsequent inhibition of tubulin polymerization in Ascaris suum (in vitro) by benzimidazole anthelmintics. Biochem Pharmacol 1984;33(19):3037-40.

396. Lacey E. Mode of action of benzimidazoles. Parasitology today 1990;6(4):112-5.

397. Chu SW, Badar S, Morris DL, Pourgholami MH. Potent inhibition of tubulin polymerisation and proliferation of paclitaxel-resistant 1A9PTX22 human ovarian cancer cells by albendazole. Anticancer research 2009;29(10):3791-6.

398. Pourgholami MH, Woon L, Almajd R, Akhter J, Bowery P, Morris DL. In vitro and in vivo suppression of growth of hepatocellular carcinoma cells by albendazole. Cancer Lett 2001;165(1):43-9.

201

399. Pourgholami MH, Akhter J, Wang L, Lu Y, Morris DL. Antitumor activity of albendazole against the human colorectal cancer cell line HT-29: in vitro and in a xenograft model of peritoneal carcinomatosis. Cancer Chemother Pharmacol 2005;55(5):425-32.

400. Motoyama S, Minamiya Y, Saito S, et al. Hydrogen peroxide derived from hepatocytes induces sinusoidal endothelial cell apoptosis in perfused hypoxic rat liver. Gastroenterology 1998;114(1):153-63.

401. Locatelli C, Pedrosa RC, De Bem AF, Creczynski-Pasa TB, Cordova CA, Wilhelm-Filho D. A comparative study of albendazole and mebendazole-induced, time-dependent oxidative stress. Redox Rep 2004;9(2):89-95.

402. Khalilzadeh A, Wangoo KT, Morris DL, Pourgholami MH. Epothilone- paclitaxel resistant leukemic cells CEM/dEpoB300 are sensitive to albendazole: Involvement of apoptotic pathways. Biochem Pharmacol 2007;74(3):407-14.

403. Kuramoto K, Sakai A, Shigemasa K, et al. High expression of MCL1 gene related to vascular endothelial growth factor is associated with poor outcome in non- Hodgkin's lymphoma. Br J Haematol 2002;116(1):158-61.

404. Pourgholami MH, Cai ZY, Badar S, Wangoo K, Poruchynsky MS, Morris DL. Potent inhibition of tumoral hypoxia-inducible factor 1alpha by albendazole. BMC Cancer 2010;10:143.

405. Pourgholami MH, Khachigian LM, Fahmy RG, et al. Albendazole inhibits endothelial cell migration, tube formation, vasopermeability, VEGF receptor-2 expression and suppresses retinal neovascularization in ROP model of angiogenesis. Biochem Biophys Res Commun 2010;397(4):729-34.

406. Pourgholami MH, Yan Cai Z, Lu Y, Wang L, Morris DL. Albendazole: a potent inhibitor of vascular endothelial growth factor and malignant ascites formation in OVCAR-3 tumor-bearing nude mice. Clin Cancer Res 2006;12(6):1928-35.

407. Morris DL, Jourdan JL, Pourgholami MH. Pilot study of albendazole in patients with advanced malignancy. Effect on serum tumor markers/high incidence of neutropenia. Oncology 2001;61(1):42-6.

408. Pourgholami MH, Szwajcer M, Chin M, et al. Phase I clinical trial to determine maximum tolerated dose of oral albendazole in patients with advanced cancer. Cancer Chemother Pharmacol 2010;65(3):597-605.

409. Horton J. Albendazole: a review of anthelmintic efficacy and safety in humans. Parasitology 2000;121 Suppl:S113-32.

410. Horton J. Albendazole for the treatment of echinococcosis. Fundam Clin Pharmacol 2003;17(2):205-12.

411. D'Amato RJ, Lin CM, Flynn E, Folkman J, Hamel E. 2-Methoxyestradiol, an endogenous mammalian metabolite, inhibits tubulin polymerization by interacting at

202

the colchicine site. Proceedings of the National Academy of Sciences of the United States of America 1994;91(9):3964-8.

412. Rao PN, Engelberg J. Structural specificity of estrogens in the induction of mitotic chromatid non-disjunction in HeLa cells. Experimental cell research 1967;48(1):71-81.

413. Seegers JC, Aveling ML, Van Aswegen CH, Cross M, Koch F, Joubert WS. The cytotoxic effects of estradiol-17 beta, catecholestradiols and methoxyestradiols on dividing MCF-7 and HeLa cells. Journal of steroid biochemistry 1989;32(6):797- 809.

414. Gelbke HP, Knuppen R. The excretion of five different 2-hydroxyoestrogen monomethyl ethers in human pregnancy urine. Journal of steroid biochemistry 1976;7(6-7):457-63.

415. Berg D, Sonsalla R, Kuss E. Concentrations of 2-methoxyoestrogens in human serum measured by a heterologous immunoassay with an 125I-labelled ligand. Acta endocrinologica 1983;103(2):282-8.

416. Zhu BT, Conney AH. Is 2-methoxyestradiol an endogenous estrogen metabolite that inhibits mammary carcinogenesis? Cancer research 1998;58(11):2269-77.

417. Lakhani NJ, Lepper ER, Sparreboom A, Dahut WL, Venitz J, Figg WD. Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry. Rapid communications in mass spectrometry : RCM 2005;19(9):1176-82.

418. Pribluda VS, Gubish ER, Jr., Lavallee TM, Treston A, Swartz GM, Green SJ. 2-Methoxyestradiol: an endogenous antiangiogenic and antiproliferative drug candidate. Cancer metastasis reviews 2000;19(1-2):173-9.

419. Fotsis T, Zhang Y, Pepper MS, et al. The endogenous oestrogen metabolite 2-methoxyoestradiol inhibits angiogenesis and suppresses tumour growth. Nature 1994;368(6468):237-9.

420. Ireson CR, Chander SK, Purohit A, et al. Pharmacokinetics and efficacy of 2- methoxyoestradiol and 2-methoxyoestradiol-bis-sulphamate in vivo in rodents. British journal of cancer 2004;90(4):932-7.

421. Lakhani N, Sparreboom A, Venitz J, Dahut WL, Figg WD. Plasma protein binding of the investigational anticancer agent 2-methoxyestradiol. Anti-cancer drugs 2006;17(8):977-83.

422. Desai KG, Mallery SR, Schwendeman SP. Effect of formulation parameters on 2-methoxyestradiol release from injectable cylindrical poly(DL-lactide-co- glycolide) implants. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 2008;70(1):187-98.

203

423. Cushman M, He HM, Katzenellenbogen JA, Lin CM, Hamel E. Synthesis, antitubulin and antimitotic activity, and cytotoxicity of analogs of 2- methoxyestradiol, an endogenous mammalian metabolite of estradiol that inhibits tubulin polymerization by binding to the colchicine binding site. Journal of medicinal chemistry 1995;38(12):2041-9.

424. Edsall AB, Agoston GE, Treston AM, et al. Synthesis and in vivo antitumor evaluation of 2-methoxyestradiol 3-phosphate, 17-phosphate, and 3,17-diphosphate. Journal of medicinal chemistry 2007;50(26):6700-5.

425. Edsall AB, Mohanakrishnan AK, Yang D, et al. Effects of altering the electronics of 2-methoxyestradiol on cell proliferation, on cytotoxicity in human cancer cell cultures, and on tubulin polymerization. Journal of medicinal chemistry 2004;47(21):5126-39.

426. LaVallee TM, Burke PA, Swartz GM, et al. Significant antitumor activity in vivo following treatment with the microtubule agent ENMD-1198. Molecular cancer therapeutics 2008;7(6):1472-82.

427. Verenich S, Gerk PM. Therapeutic promises of 2-methoxyestradiol and its drug disposition challenges. Molecular Pharmaceutics 2010;7(6):2030-9.

428. Seegers JC, Lottering ML, Grobler CJ, et al. The mammalian metabolite, 2- methoxyestradiol, affects P53 levels and apoptosis induction in transformed cells but not in normal cells. The Journal of steroid biochemistry and molecular biology 1997;62(4):253-67.

429. Attalla H, Makela TP, Adlercreutz H, Andersson LC. 2-Methoxyestradiol arrests cells in mitosis without depolymerizing tubulin. Biochemical and biophysical research communications 1996;228(2):467-73.

430. Mukhopadhyay T, Roth JA. Induction of apoptosis in human lung cancer cells after wild-type p53 activation by methoxyestradiol. Oncogene 1997;14(3):379- 84.

431. Bu S, Blaukat A, Fu X, Heldin NE, Landstrom M. Mechanisms for 2- methoxyestradiol-induced apoptosis of prostate cancer cells. FEBS letters 2002;531(2):141-51.

432. Cushman M, He HM, Katzenellenbogen JA, et al. Synthesis of analogs of 2- methoxyestradiol with enhanced inhibitory effects on tubulin polymerization and cancer cell growth. J Med Chem 1997;40(15):2323-34.

433. Hughes RA, Harris T, Altmann E, et al. 2-Methoxyestradiol and analogs as novel antiproliferative agents: analysis of three-dimensional quantitative structure- activity relationships for DNA synthesis inhibition and estrogen receptor binding. Molecular pharmacology 2002;61(5):1053-69.

434. Li L, Bu S, Backstrom T, Landstrom M, Ulmsten U, Fu X. Induction of apoptosis and G2/M arrest by 2-methoxyestradiol in human cervical cancer HeLaS3 cells. Anticancer research 2004;24(2B):873-80.

204

435. Carothers AM, Hughes SA, Ortega D, Bertagnolli MM. 2-Methoxyestradiol induces p53-associated apoptosis of colorectal cancer cells. Cancer letters 2002;187(1-2):77-86.

436. Mukhopadhyay T, Roth JA. Superinduction of wild-type p53 protein after 2- methoxyestradiol treatment of Ad5p53-transduced cells induces tumor cell apoptosis. Oncogene 1998;17(2):241-6.

437. Kumar AP, Garcia GE, Orsborn J, Levin VA, Slaga TJ. 2-Methoxyestradiol interferes with NF kappa B transcriptional activity in primitive neuroectodermal brain tumors: implications for management. Carcinogenesis 2003;24(2):209-16.

438. Gao N, Rahmani M, Dent P, Grant S. 2-Methoxyestradiol-induced apoptosis in human leukemia cells proceeds through a reactive oxygen species and Akt- dependent process. Oncogene 2005;24(23):3797-809.

439. Shimada K, Nakamura M, Ishida E, Kishi M, Konishi N. Roles of p38- and c-jun NH2-terminal kinase-mediated pathways in 2-methoxyestradiol-induced p53 induction and apoptosis. Carcinogenesis 2003;24(6):1067-75.

440. Schulze-Osthoff K, Ferrari D, Los M, Wesselborg S, Peter ME. Apoptosis signaling by death receptors. European journal of biochemistry / FEBS 1998;254(3):439-59.

441. Tsukamoto A, Kaneko Y, Yoshida T, Han K, Ichinose M, Kimura S. 2- Methoxyestradiol, an endogenous metabolite of estrogen, enhances apoptosis and beta-galactosidase expression in vascular endothelial cells. Biochem Biophys Res Commun 1998;248(1):9-12.

442. Lleonart ME, Artero-Castro A, Kondoh H. Senescence induction; a possible cancer therapy. Molecular cancer 2009;8:3.

443. Pasquier E, Sinnappan S, Munoz MA, Kavallaris M. ENMD-1198, a new analogue of 2-methoxyestradiol, displays both antiangiogenic and vascular- disrupting properties. Mol Cancer Ther 2010;9(5):1408-18.

444. Sattler M, Quinnan LR, Pride YB, et al. 2-methoxyestradiol alters cell motility, migration, and adhesion. Blood 2003;102(1):289-96.

445. Harbeck N, Schmitt M, Paepke S, Allgayer H, Kates RE. Tumor-associated proteolytic factors uPA and PAI-1: critical appraisal of their clinical relevance in breast cancer and their integration into decision-support algorithms. Critical reviews in clinical laboratory sciences 2007;44(2):179-201.

446. Klauber N, Parangi S, Flynn E, Hamel E, D'Amato RJ. Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2- methoxyestradiol and taxol. Cancer research 1997;57(1):81-6.

447. Kang SH, Cho HT, Devi S, et al. Antitumor effect of 2-methoxyestradiol in a rat orthotopic brain tumor model. Cancer research 2006;66(24):11991-7.

205

448. Yue TL, Wang X, Louden CS, et al. 2-Methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and Fas expression. Molecular pharmacology 1997;51(6):951-62.

449. Ricker JL, Chen Z, Yang XP, Pribluda VS, Swartz GM, Van Waes C. 2- methoxyestradiol inhibits hypoxia-inducible factor 1alpha, tumor growth, and angiogenesis and augments paclitaxel efficacy in head and neck squamous cell carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 2004;10(24):8665-73.

450. Cicek M, Iwaniec UT, Goblirsch MJ, et al. 2-Methoxyestradiol suppresses osteolytic breast cancer tumor progression in vivo. Cancer research 2007;67(21):10106-11.

451. Ganapathy M, Ghosh R, Jianping X, et al. Involvement of FLIP in 2- methoxyestradiol-induced tumor regression in transgenic adenocarcinoma of mouse prostate model. Clinical cancer research : an official journal of the American Association for Cancer Research 2009;15(5):1601-11.

452. Schumacher G, Kataoka M, Roth JA, Mukhopadhyay T. Potent antitumor activity of 2-methoxyestradiol in human pancreatic cancer cell lines. Clinical cancer research : an official journal of the American Association for Cancer Research 1999;5(3):493-9.

453. Lin HL, Yang MH, Wu CW, et al. 2-Methoxyestradiol attenuates phosphatidylinositol 3-kinase/Akt pathway-mediated metastasis of gastric cancer. International journal of cancer. Journal international du cancer 2007;121(11):2547- 55.

454. Azab SS, Salama SA, Hassan MH, et al. 2-Methoxyestradiol reverses doxorubicin resistance in human breast tumor xenograft. Cancer chemotherapy and pharmacology 2008;62(5):893-902.

455. Kataoka M, Schumacher G, Cristiano RJ, Atkinson EN, Roth JA, Mukhopadhyay T. An agent that increases tumor suppressor transgene product coupled with systemic transgene delivery inhibits growth of metastatic lung cancer in vivo. Cancer research 1998;58(21):4761-5.

456. Sato F, Fukuhara H, Basilion JP. Effects of hormone deprivation and 2- methoxyestradiol combination therapy on hormone-dependent prostate cancer in vivo. Neoplasia 2005;7(9):838-46.

457. Azab SS, Salama SA, Abdel-Naim AB, Khalifa AE, El-Demerdash E, Al- Hendy A. 2-Methoxyestradiol and multidrug resistance: can 2-methoxyestradiol chemosensitize resistant breast cancer cells? Breast cancer research and treatment 2009;113(1):9-19.

458. Li L, Da J, Landstrom M, Ulmsten U, Fu X. Antiproliferative activity and toxicity of 2-methoxyestradiol in cervical cancer xenograft mice. International journal of gynecological cancer : official journal of the International Gynecological Cancer Society 2005;15(2):301-7. 206

459. Li L, Yu F, Wu X, Cheng J, Ulmsten U, Fu X. Effects of 2-methoxyestradiol on endometrial carcinoma xenografts. Journal of cancer research and clinical oncology 2007;133(5):315-20.

460. Sutherland TE, Schuliga M, Harris T, et al. 2-methoxyestradiol is an estrogen receptor agonist that supports tumor growth in murine xenograft models of breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2005;11(5):1722-32.

461. Arlen PM, Gulley JL, Parker C, et al. A randomized phase II study of concurrent docetaxel plus vaccine versus vaccine alone in metastatic androgen- independent prostate cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 2006;12(4):1260-9.

462. Josefsson E, Tarkowski A. Suppression of type II collagen-induced arthritis by the endogenous estrogen metabolite 2-methoxyestradiol. Arthritis and rheumatism 1997;40(1):154-63.

463. Brahn E, Banquerigo ML, Lee JK, Park EJ, Fogler WE, Plum SM. An angiogenesis inhibitor, 2-methoxyestradiol, involutes rat collagen-induced arthritis and suppresses gene expression of synovial vascular endothelial growth factor and basic fibroblast growth factor. The Journal of rheumatology 2008;35(11):2119-28.

464. Huerta-Yepez S, Baay-Guzman GJ, Garcia-Zepeda R, et al. 2- Methoxyestradiol (2-ME) reduces the airway inflammation and remodeling in an experimental mouse model. Clinical immunology 2008;129(2):313-24.

465. Liu D, Bachmann KA. An investigation of the relationship between estrogen, estrogen metabolites and blood cholesterol levels in ovariectomized rats. The Journal of pharmacology and experimental therapeutics 1998;286(1):561-8.

466. Turner RT, Evans GL. 2-Methoxyestradiol inhibits longitudinal bone growth in normal female rats. Calcified tissue international 2000;66(6):465-9.

467. Sweeney C, Liu G, Yiannoutsos C, et al. A phase II multicenter, randomized, double-blind, safety trial assessing the pharmacokinetics, pharmacodynamics, and efficacy of oral 2-methoxyestradiol capsules in hormone-refractory prostate cancer. Clin Cancer Res 2005;11(18):6625-33.

468. Chou TC. Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer research 2010;70(2):440-6.

469. Jia J, Zhu F, Ma X, Cao Z, Li Y, Chen YZ. Mechanisms of drug combinations: interaction and network perspectives. Nature reviews. Drug discovery 2009;8(2):111-28.

470. Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacological reviews 2006;58(3):621-81.

471. Greco WR, Bravo G, Parsons JC. The search for synergy: a critical review from a response surface perspective. Pharmacological reviews 1995;47(2):331-85. 207

472. Johnson MD, MacDougall C, Ostrosky-Zeichner L, Perfect JR, Rex JH. Combination antifungal therapy. Antimicrobial agents and chemotherapy 2004;48(3):693-715.

473. Peterson JJ, Novick SJ. Nonlinear blending: a useful general concept for the assessment of combination drug synergy. Journal of receptor and signal transduction research 2007;27(2-3):125-46.

474. Chou TC. Drug combinations: from laboratory to practice. The Journal of laboratory and clinical medicine 1998;132(1):6-8.

475. Chou TC, Talalay P. Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55.

476. Mallick S, Pattnaik S, Swain K, De PK. Current perspectives of solubilization: potential for improved bioavailability. Drug Dev Ind Pharm 2007;33(8):865-73.

477. Shinde A. Solubilization of poorly soluble drugs: A review. Pharmaceutical Reviews.

478. Strickley RG. Solubilizing excipients in oral and injectable formulations. Pharm Res 2004;21(2):201-30.

479. Lee YC, Zocharski PD, Samas B. An intravenous formulation decision tree for discovery compound formulation development. Int J Pharm 2003;253(1-2):111- 9.

480. Loftsson T, Brewster ME, Másson M. Role of cyclodextrins in improving oral drug delivery. American Journal of Drug Delivery 2004;2(4):261-275.

481. Davis ME, Brewster ME. Cyclodextrin-based pharmaceutics: past, present and future. Nat Rev Drug Discov 2004;3(12):1023-35.

482. Loftsson T, Brewster ME. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J Pharm Sci 1996;85(10):1017-25.

483. Antlsperger G, Schmid G. Toxicological comparison of cyclodextrins. In; 1996; 1996. p. 149-156.

484. Olivier P, Verwaerde F, Hedges AR. Subchronic toxicity of orally administered Beta-cyclodextrin in rats. International Journal of Toxicology 1991;10(4):407.

485. Bellringer ME, Smith TG, Read R, Gopinath C, Olivier P. beta-Cyclodextrin: 52-week toxicity studies in the rat and dog. Food Chem Toxicol 1995;33(5):367-76.

486. Perrin JH, Field FP, Hansen DA, Mufson RA, Torosian G. beta-Cyclodextrin as an aid to peritoneal dialysis. Renal toxicity of beta-cyclodextrin in the rat. Res Commun Chem Pathol Pharmacol 1978;19(2):373-6.

208

487. Frank DW, Gray JE, Weaver RN. Cyclodextrin nephrosis in the rat. Am J Pathol 1976;83(2):367-82.

488. Irie T, Uekama K. Pharmaceutical applications of cyclodextrins. III. Toxicological issues and safety evaluation. J Pharm Sci 1997;86(2):147-62.

489. Stella VJ, Rajewski RA. Cyclodextrins: their future in drug formulation and delivery. Pharm Res 1997;14(5):556-67.

490. Rasheed A, CK AK, VVNSS S. Cyclodextrins as drug carrier molecule: a review. Sci Pharm 2008;76:567-598.

491. Loftsson T, Konradsdottir F, Masson M. Influence of aqueous diffusion layer on passive drug diffusion from aqueous cyclodextrin solutions through biological membranes. Pharmazie 2006;61(2):83-9.

492. Tinwalla AY, Hoesterey BL, Xiang TX, Lim K, Anderson BD. Solubilization of thiazolobenzimidazole using a combination of pH adjustment and complexation with 2-hydroxypropyl-beta-cyclodextrin. Pharm Res 1993;10(8):1136-43.

493. Tommasini S, Calabro ML, Raneri D, Ficarra P, Ficarra R. Combined effect of pH and polysorbates with cyclodextrins on solubilization of naringenin. J Pharm Biomed Anal 2004;36(2):327-33.

494. Li P, Tabibi SE, Yalkowsky SH. Combined effect of complexation and pH on solubilization. J Pharm Sci 1998;87(12):1535-7.

495. Hadgraft J, Valenta C. pH, pK(a) and dermal delivery. Int J Pharm 2000;200(2):243-7.

496. Sridevi S, Diwan PV. Effect of pH and complexation on transdermal permeation of gliquidone. Pharmazie 2002;57(9):632-4.

497. Wang AZ, Langer RS, Farokhzad OC. Nanoparticle Delivery of Cancer Drugs. Annual review of medicine 2011.

498. Shekhar C. Lean and mean: nanoparticle-based delivery improves performance of cancer drugs. Chemistry & biology 2009;16(4):349-50.

499. Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res 1986;46(12 Pt 1):6387-92.

500. Bangham AD. Liposomes: the Babraham connection. Chem Phys Lipids 1993;64(1-3):275-85.

501. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 2005;4(2):145-60.

502. Hofheinz RD, Gnad-Vogt SU, Beyer U, Hochhaus A. Liposomal encapsulated anti-cancer drugs. Anti-cancer drugs 2005;16(7):691-707.

209

503. John TA, Vogel SM, Tiruppathi C, Malik AB, Minshall RD. Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer. American journal of physiology. Lung cellular and molecular physiology 2003;284(1):L187-96.

504. Schubert W, Frank PG, Razani B, Park DS, Chow CW, Lisanti MP. Caveolae-deficient endothelial cells show defects in the uptake and transport of albumin in vivo. J Biol Chem 2001;276(52):48619-22.

505. Desai N, Trieu V, Yao Z, et al. Increased antitumor activity, intratumor paclitaxel concentrations, and endothelial cell transport of cremophor-free, albumin- bound paclitaxel, ABI-007, compared with cremophor-based paclitaxel. Clin Cancer Res 2006;12(4):1317-24.

506. Hawkins MJ, Soon-Shiong P, Desai N. Protein nanoparticles as drug carriers in clinical medicine. Adv Drug Deliv Rev 2008;60(8):876-85.

507. Minelli C, Lowe SB, Stevens MM. Engineering nanocomposite materials for cancer therapy. Small 2010;6(21):2336-57.

508. Baker JR, Jr. Dendrimer-based nanoparticles for cancer therapy. Hematology Am Soc Hematol Educ Program 2009:708-19.

509. Kim SH, Jeong JH, Chun KW, Park TG. Target-specific cellular uptake of PLGA nanoparticles coated with poly(L-lysine)-poly(ethylene glycol)-folate conjugate. Langmuir 2005;21(19):8852-7.

510. Gu F, Zhang L, Teply BA, et al. Precise engineering of targeted nanoparticles by using self-assembled biointegrated block copolymers. Proc Natl Acad Sci U S A 2008;105(7):2586-91.

511. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plus adjuvant chemotherapy for operable HER2-positive breast cancer. The New England journal of medicine 2005;353(16):1673-84.

512. Yang JC, Haworth L, Sherry RM, et al. A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. The New England journal of medicine 2003;349(5):427-34.

513. Hurwitz HI, Fehrenbacher L, Hainsworth JD, et al. Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer. J Clin Oncol 2005;23(15):3502-8.

514. Coiffier B, Lepage E, Briere J, et al. CHOP chemotherapy plus rituximab compared with CHOP alone in elderly patients with diffuse large-B-cell lymphoma. The New England journal of medicine 2002;346(4):235-42.

515. Druker BJ, Talpaz M, Resta DJ, et al. Efficacy and safety of a specific inhibitor of the BCR-ABL tyrosine kinase in chronic myeloid leukemia. The New England journal of medicine 2001;344(14):1031-7.

210

516. Wang AZ, Gu F, Zhang L, et al. Biofunctionalized targeted nanoparticles for therapeutic applications. Expert Opin Biol Ther 2008;8(8):1063-70.

517. Jamshidi M, Mohraz M, Zangeneh M, Jamshidi A. The effect of combination therapy with albendazole and praziquantel on hydatid cyst treatment. Parasitol Res 2008;103(1):195-9.

518. Maizels RM, Denham DA. Diethylcarbamazine (DEC): immunopharmacological interactions of an anti-filarial drug. Parasitology 1992;105 Suppl:S49-60.

519. Molyneux DH, Zagaria N. Lymphatic filariasis elimination: progress in global programme development. Ann Trop Med Parasitol 2002;96 Suppl 2:S15-40.

520. Skehan P, Storeng R, Scudiero D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990;82(13):1107-12.

521. Chou TC, Motzer RJ, Tong Y, Bosl GJ. Computerized quantitation of synergism and antagonism of taxol, topotecan, and cisplatin against human teratocarcinoma cell growth: a rational approach to clinical protocol design. J Natl Cancer Inst 1994;86(20):1517-24.

522. Giannakakou P, Villalba L, Li H, Poruchynsky M, Fojo T. Combinations of paclitaxel and vinblastine and their effects on tubulin polymerization and cellular cytotoxicity: characterization of a synergistic schedule. Int J Cancer 1998;75(1):57- 63.

523. Sackett DL, Giannakakou P, Poruchynsky M, Fojo A. Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents. Cancer Chemother Pharmacol 1997;40(3):228-32.

524. Bassani VL, Krieger D, Duchene D, Wouessidjewe D. Enhanced water- solubility of albendazole by hydroxypropyl- -cyclodextrin complexation. Journal of Inclusion Phenomena and Macrocyclic Chemistry 1996;25(1):149-152.

525. Marriner SE, Morris DL, Dickson B, Bogan JA. Pharmacokinetics of albendazole in man. Eur J Clin Pharmacol 1986;30(6):705-8.

526. Jung H, Medina L, Garcia L, Fuentes I, Moreno-Esparza R. Absorption studies of albendazole and some physicochemical properties of the drug and its metabolite albendazole sulphoxide. J Pharm Pharmacol 1998;50(1):43-8.

527. Jung H, Hurtado M, Sanchez M, Medina MT, Sotelo J. Clinical pharmacokinetics of albendazole in patients with brain cysticercosis. J Clin Pharmacol 1992;32(1):28-31.

528. Yepez-Mulia L, Morales-Hurtado R, Viveros-Guzman N, et al. Evaluation of albendazole prodrugs in experimental trichinellosis. Arch Med Res 1999;30(5):368- 74.

211

529. Mingjie W, Shuhua X, Junjie C, et al. Albendazole-soybean oil emulsion for the treatment of human cystic echinococcosis: evaluation of bioavailability and bioequivalence. Acta Trop 2002;83(2):177-81.

530. Redondo PA, Alvarez AI, Garcia JL, Villaverde C, Prieto JG. Influence of surfactants on oral bioavailability of albendazole based on the formation of the sulphoxide metabolites in rats. Biopharm Drug Dispos 1998;19(1):65-70.

531. Wen H, New RR, Muhmut M, et al. Pharmacology and efficacy of liposome- entrapped albendazole in experimental secondary alveolar echinococcosis and effect of co-administration with cimetidine. Parasitology 1996;113 ( Pt 2):111-21.

532. Palomares-Alonso F, Gonzalez CR, Bernad-Bernad MJ, et al. Two novel ternary albendazole-cyclodextrin-polymer systems: dissolution, bioavailability and efficacy against Taenia crassiceps cysts. Acta Trop;113(1):56-60.

533. Torrado S, Torrado JJ, Cadórniga R. Preparation, dissolution and characterization of albendazole solid dispersions. International Journal of Pharmaceutics 1996;140(2):247-250.

534. Ceballos L, Elissondo C, Moreno L, et al. Albendazole treatment in cystic echinococcosis: pharmacokinetics and clinical efficacy of two different aqueous formulations. Parasitol Res 2008;103(2):355-62.

535. Daniel-Mwambete K, Torrado S, Cuesta-Bandera C, Ponce-Gordo F, Torrado JJ. The effect of solubilization on the oral bioavailability of three benzimidazole carbamate drugs. Int J Pharm 2004;272(1-2):29-36.

536. Casulli A, Morales MA, Gallinella B, Turchetto L, Pozio E. 2- Hydroxypropyl-beta-cyclodextrin improves the effectiveness of albendazole against encapsulated larvae of Trichinella spiralis in a murine model. J Antimicrob Chemother 2006;58(4):886-90.

537. Rigter IM, Schipper HG, Koopmans RP, et al. Relative bioavailability of three newly developed albendazole formulations: a randomized crossover study with healthy volunteers. Antimicrob Agents Chemother 2004;48(3):1051-4.

538. Kalaiselvan R, Mohanta GP, Manna PK, Manavalan R. Inhibition of albendazole crystallization in poly(vinylpyrrolidone) solid molecular dispersions. Pharmazie 2006;61(7):618-24.

539. Challa R, Ahuja A, Ali J, Khar RK. Cyclodextrins in drug delivery: an updated review. AAPS PharmSciTech 2005;6(2):E329-57.

540. Arun R, Ashok KCK, Sravanthi V. ChemInform Abstract: Cyclodextrins as Drug Carrier Molecule: A Review. ChemInform 2009;40(14).

541. Gould S, Scott RC. 2-Hydroxypropyl-beta-cyclodextrin (HP-beta-CD): a toxicology review. Food Chem Toxicol 2005;43(10):1451-9.

212

542. Pourgholami MH, Wangoo KT, Morris DL. Albendazole-cyclodextrin complex: enhanced cytotoxicity in ovarian cancer cells. Anticancer Res 2008;28(5A):2775-9.

543. Pourgholami MH, Akhter J, Wang L, Lu Y, Morris DL. Antitumor activity of albendazole against the human colorectal cancer cell line HT-29: in vitro and in a xenograft model of peritoneal carcinomatosis. Cancer chemotherapy and pharmacology 2005;55(5):425-32.

544. Castillo JA, Palomo-Canales J, Garcia JJ, Lastres JL, Bolas F, Torrado JJ. Preparation and characterization of albendazole beta-cyclodextrin complexes. Drug Dev Ind Pharm 1999;25(12):1241-8.

545. Evrard B, Chiap P, DeTullio P, et al. Oral bioavailability in sheep of albendazole from a suspension and from a solution containing hydroxypropyl-beta- cyclodextrin. J Control Release 2002;85(1-3):45-50.

546. Mehlen P, Puisieux A. Metastasis: a question of life or death. Nat Rev Cancer 2006;6(6):449-58.

547. Folkman J, Browder T, Palmblad J. Angiogenesis research: guidelines for translation to clinical application. Thromb Haemost 2001;86(1):23-33.

548. Dvorak HF, Nagy JA, Dvorak JT, Dvorak AM. Identification and characterization of the blood vessels of solid tumors that are leaky to circulating macromolecules. Am J Pathol 1988;133(1):95-109.

549. Frijlink HW, Franssen EJ, Eissens AC, Oosting R, Lerk CF, Meijer DK. The effects of cyclodextrins on the disposition of intravenously injected drugs in the rat. Pharm Res 1991;8(3):380-4.

550. Sideris EE, Koupparis MA, Macheras PE. Effect of cyclodextrins on protein binding of drugs: the diflunisal/hydroxypropyl-beta-cyclodextrin model case. Pharm Res 1994;11(1):90-5.

551. Diaz D, Bernad Bernad MJ, Gracia Mora J, Escobar Llanos CM. Complexation and solubility behavior of albendazole with some cyclodextrins. Pharm Dev Technol 1998;3(3):395-403.

552. Mayer LD, Janoff AS. Optimizing combination chemotherapy by controlling drug ratios. Mol Interv 2007;7(4):216-23.

553. Giannakakou P, Fojo T. Discodermolide: just another microtubule-stabilizing agent? No! A lesson in synergy. Clin Cancer Res 2000;6(5):1613-5.

554. Dayan AD. Albendazole, mebendazole and praziquantel. Review of non- clinical toxicity and pharmacokinetics. Acta Trop 2003;86(2-3):141-59.

555. Dollery CT, Boobis AR. Therapeutic drugs: Churchill Livingstone; 1999.

213

556. McGarry HF, Plant LD, Taylor MJ. Diethylcarbamazine activity against Brugia malayi microfilariae is dependent on inducible nitric-oxide synthase and the cyclooxygenase pathway. Filaria J 2005;4:4.

557. Gonzalez Canga A, Sahagun Prieto AM, Diez Liebana MJ, Fernandez Martinez N, Sierra Vega M, Garcia Vieitez JJ. The pharmacokinetics and interactions of ivermectin in humans--a mini-review. AAPS J 2008;10(1):42-6.

558. Duce IR, Scott RH. Actions of dihydroavermectin B1a on insect muscle. British journal of pharmacology 1985;85(2):395-401.

559. Geary TG, Sims SM, Thomas EM, et al. Haemonchus contortus: ivermectin- induced paralysis of the pharynx. Exp Parasitol 1993;77(1):88-96.

560. FURUSAWA S, SHIBATA H, NISHIMURA H, et al. Potentiation of Doxorubicin Induced Apoptosis of Resistant Mouse Leukaemia Cells by Ivermectin. Pharmacy and Pharmacology Communications 2000;6(3):129-134.

561. Sharmeen S, Skrtic M, Sukhai MA, et al. The antiparasitic agent ivermectin induces chloride-dependent membrane hyperpolarization and cell death in leukemia cells. Blood 2010;116(18):3593-603.

562. Hamel E. Antimitotic natural products and their interactions with tubulin. Medicinal research reviews 1996;16(2):207-31.

563. Ireland CM, Gull K, Gutteridge WE, Pogson CI. The interaction of benzimidazole carbamates with mammalian microtobule protein. Biochem Pharmacol 1979;28(17):2680-2.

564. Lacey E, Watson TR. Structure-activity relationships of benzimidazole carbamates as inhibitors of mammalian tubulin, in vitro. Biochem Pharmacol 1985;34(7):1073-7.

565. Clement MJ, Rathinasamy K, Adjadj E, Toma F, Curmi PA, Panda D. Benomyl and colchicine synergistically inhibit cell proliferation and mitosis: evidence of distinct binding sites for these agents in tubulin. Biochemistry 2008;47(49):13016-25.

566. Spagnuolo PA, Hu J, Hurren R, et al. The antihelmintic flubendazole inhibits microtubule function through a mechanism distinct from Vinca alkaloids and displays preclinical activity in leukemia and myeloma. Blood 2010;115(23):4824-33.

567. Chen CH, Lee WJ, Chang TC, Chen RJ, Chien CH, Chow SN. Antiproliferative effects of 2-methoxyestradiol alone and in combination with chemotherapeutic agents on human endometrial cancer cells. European journal of gynaecological oncology 2009;30(3):275-80.

568. Han GZ, Liu ZJ, Shimoi K, Zhu BT. Synergism between the anticancer actions of 2-methoxyestradiol and microtubule-disrupting agents in human breast cancer. Cancer research 2005;65(2):387-93.

214

569. Clark EA, Hills PM, Davidson BS, Wender PA, Mooberry SL. Laulimalide and synthetic laulimalide analogues are synergistic with paclitaxel and 2- methoxyestradiol. Molecular Pharmaceutics 2006;3(4):457-67.

570. Seeger H, Diesing D, Guckel B, Wallwiener D, Mueck AO, Huober J. Effect of tamoxifen and 2-methoxyestradiol alone and in combination on human breast cancer cell proliferation. The Journal of steroid biochemistry and molecular biology 2003;84(2-3):255-7.

571. James J, Murry DJ, Treston AM, et al. Phase I safety, pharmacokinetic and pharmacodynamic studies of 2-methoxyestradiol alone or in combination with docetaxel in patients with locally recurrent or metastatic breast cancer. Invest New Drugs 2007;25(1):41-8.

572. Escuin D, Burke PA, McMahon-Tobin G, et al. The hematopoietic-specific beta1-tubulin is naturally resistant to 2-methoxyestradiol and protects patients from drug-induced myelosuppression. Cell Cycle 2009;8(23):3914-24.

573. Chu SW, Badar S, Morris DL, Pourgholami MH. Potent inhibition of tubulin polymerisation and proliferation of paclitaxel-resistant 1A9PTX22 human ovarian cancer cells by albendazole. Anticancer Res 2009;29(10):3791-6.

574. Gokmen-Polar Y, Escuin D, Walls CD, et al. beta-Tubulin mutations are associated with resistance to 2-methoxyestradiol in MDA-MB-435 cancer cells. Cancer Res 2005;65(20):9406-14.

575. Gokmen-Polar Y, Escuin D, Walls CD, et al. beta-Tubulin mutations are associated with resistance to 2-methoxyestradiol in MDA-MB-435 cancer cells. Cancer research 2005;65(20):9406-14.

576. Djavaheri-Mergny M, Wietzerbin J, Besancon F. 2-Methoxyestradiol induces apoptosis in Ewing sarcoma cells through mitochondrial hydrogen peroxide production. Oncogene 2003;22(17):2558-67.

577. Stander BA, Marais S, Vorster CJ, Joubert AM. In vitro effects of 2- methoxyestradiol on morphology, cell cycle progression, cell death and gene expression changes in the tumorigenic MCF-7 breast epithelial cell line. The Journal of steroid biochemistry and molecular biology 2010;119(3-5):149-60.

578. Carrier RL, Miller LA, Ahmed I. The utility of cyclodextrins for enhancing oral bioavailability. J Control Release 2007;123(2):78-99.

579. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. Journal of immunology 1984;133(4):1710-5.

580. Giannakakou P, Villalba L, Li H, Poruchynsky M, Fojo T. Combinations of paclitaxel and vinblastine and their effects on tubulin polymerization and cellular cytotoxicity: characterization of a synergistic schedule. International journal of cancer. Journal international du cancer 1998;75(1):57-63.

215

581. Hudes GR, Nathan FE, Khater C, et al. Paclitaxel plus estramustine in metastatic hormone-refractory prostate cancer. Seminars in oncology 1995;22(5 Suppl 12):41-5.

582. Hudes GR, Greenberg R, Krigel RL, et al. Phase II study of estramustine and vinblastine, two microtubule inhibitors, in hormone-refractory prostate cancer. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 1992;10(11):1754-61.

583. Seidman AD, Scher HI, Petrylak D, Dershaw DD, Curley T. Estramustine and vinblastine: use of prostate specific antigen as a clinical trial end point for hormone refractory prostatic cancer. The Journal of urology 1992;147(3 Pt 2):931-4.

584. Dieras V, Fumoleau P, Kalla S, Misset JL, Azli N, Pouillart P. Docetaxel in combination with doxorubicin or vinorelbine. European journal of cancer 1997;33 Suppl 7:S20-2.

585. Knick VC, Eberwein DJ, Miller CG. Vinorelbine tartrate and paclitaxel combinations: enhanced activity against in vivo P388 murine leukemia cells. Journal of the National Cancer Institute 1995;87(14):1072-7.

586. Garcia P, Braguer D, Carles G, Briand C. Simultaneous combination of microtubule depolymerizing and stabilizing agents acts at low doses. Anti-cancer drugs 1995;6(4):533-44.

587. Tahir SK, Kovar P, Rosenberg SH, Ng SC. Rapid colchicine competition- binding scintillation proximity assay using biotin-labeled tubulin. Biotechniques 2000;29(1):156-60.

588. Laclette JP, Guerra G, Zetina C. Inhibition of tubulin polymerization by mebendazole. Biochem Biophys Res Commun 1980;92(2):417-23.

589. Gupta K, Bishop J, Peck A, Brown J, Wilson L, Panda D. Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin. Biochemistry 2004;43(21):6645-55.

590. Lin CM, Singh SB, Chu PS, et al. Interactions of tubulin with potent natural and synthetic analogs of the antimitotic agent combretastatin: a structure-activity study. Molecular pharmacology 1988;34(2):200-8.

591. Bryan J, Wilson L. Are cytoplasmic microtubules heteropolymers? Proceedings of the National Academy of Sciences of the United States of America 1971;68(8):1762-6.

592. Wilson L, Jordan MA. Microtubule dynamics: taking aim at a moving target. Chemistry & biology 1995;2(9):569-73.

593. Panda D, Chakrabarti G, Hudson J, et al. Suppression of microtubule dynamic instability and treadmilling by deuterium oxide. Biochemistry

2000;39(17):5075-81.

216

594. Banerjee A, Luduena RF. Kinetics of colchicine binding to purified beta- tubulin isotypes from bovine brain. The Journal of biological chemistry 1992;267(19):13335-9.

595. Storey S. Targeting apoptosis: selected anticancer strategies. Nat Rev Drug Discov 2008;7(12):971-2.

596. Kim M, Liao J, Dowling ML, et al. TRAIL inactivates the mitotic checkpoint and potentiates death induced by microtubule-targeting agents in human cancer cells. Cancer research 2008;68(9):3440-9.

597. Cryns V, Yuan J. Proteases to die for. Genes Dev 1998;12(11):1551-70.

598. Salvesen GS, Dixit VM. Caspases: intracellular signaling by proteolysis. Cell 1997;91(4):443-6.

599. Sheikh MS, Fornace AJ, Jr. Death and decoy receptors and p53-mediated apoptosis. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 2000;14(8):1509-13.

600. Nimmanapalli R, Perkins CL, Orlando M, O'Bryan E, Nguyen D, Bhalla KN. Pretreatment with paclitaxel enhances apo-2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of prostate cancer cells by inducing death receptors 4 and 5 protein levels. Cancer research 2001;61(2):759-63.

601. Huisman C, Ferreira CG, Broker LE, et al. Paclitaxel triggers cell death primarily via caspase-independent routes in the non-small cell lung cancer cell line NCI-H460. Clinical cancer research : an official journal of the American Association for Cancer Research 2002;8(2):596-606.

602. Keane MM, Ettenberg SA, Nau MM, Russell EK, Lipkowitz S. Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. Cancer research 1999;59(3):734-41.

603. Pourgholami MH, Cai ZY, Wang L, Badar S, Links M, Morris DL. Inhibition of cell proliferation, vascular endothelial growth factor and tumor growth by albendazole. Cancer Invest 2009;27(2):171-7.

604. Becker CM, Rohwer N, Funakoshi T, et al. 2-methoxyestradiol inhibits hypoxia-inducible factor-1{alpha} and suppresses growth of lesions in a mouse model of endometriosis. The American journal of pathology 2008;172(2):534-44.

605. Chen N, Chen X, Huang R, et al. BCL-xL is a target gene regulated by hypoxia-inducible factor-1{alpha}. The Journal of biological chemistry 2009;284(15):10004-12.

606. Erler JT, Cawthorne CJ, Williams KJ, et al. Hypoxia-mediated down- regulation of Bid and Bax in tumors occurs via hypoxia-inducible factor 1-dependent and -independent mechanisms and contributes to drug resistance. Molecular and cellular biology 2004;24(7):2875-89.

217

607. Burns RG. Alpha-, beta-, and gamma-tubulins: sequence comparisons and structural constraints. Cell motility and the cytoskeleton 1991;20(3):181-9.

608. Ikeda Y, Kimura K, Hirayama F, Arima H, Uekama K. Controlled release of a water-soluble drug, captopril, by a combination of hydrophilic and hydrophobic cyclodextrin derivatives. J Control Release 2000;66(2-3):271-80.

609. Badawy SIF, Marshall AL, Ghorab MM, Adeyeye CM. A study of the complexation between danazol and hydrophilic cyclodextrin derivatives. Drug Development and Industrial Pharmacy 1996;22(9-10):959-966.

610. Dollo G, Le Corre P, Chollet M, et al. Improvement in solubility and dissolution rate of 1, 2-dithiole-3-thiones upon complexation with beta-cyclodextrin and its hydroxypropyl and sulfobutyl ether-7 derivatives. J Pharm Sci 1999;88(9):889-95.

611. Moyano J, Gines J, Arias M, Rabasco A. Evaluation of dissolution characteristics of oxazepam-2-hydroxypropyl-[beta]-cyclodextrin binary systems. Pharmaceutica Acta Helvetiae 1994;69(2):81-90.

612. Fernandes CM, Ramos P, Falcao AC, Veiga FJ. Hydrophilic and hydrophobic cyclodextrins in a new sustained release oral formulation of nicardipine: in vitro evaluation and bioavailability studies in rabbits. J Control Release 2003;88(1):127-34.

613. Jain AC, Adeyeye MC. Hygroscopicity, phase solubility and dissolution of various substituted sulfobutylether beta-cyclodextrins (SBE) and danazol-SBE inclusion complexes. Int J Pharm 2001;212(2):177-86.

218