Poly(2-Oxazoline)S As New Drug Delivery Systems

POLY(2-OXAZOLINE)S AS NEW DRUG DELIVERY SYSTEMS

Zhijian He

A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Division of Molecular Pharmaceutics in the UNC Eshelman School of Pharmacy.

Chapel Hill 2015

Approved By

Alexander V. Kabanov

Elena V. Batrakova

Leaf Huang

William C. Zamboni

Lisa A. Carey

ABSTRACT

Zhijian He: Poly(2-oxazoline)s As New Drug Delivery Systems (Under the direction of Alexander V. Kabanov)

Taxane drugs, a class of important cancer chemotherapeutics, are often associated with excipient-related hypersensitivity, severe neurotoxicity, peripheral neuropathy, drug resistance and other limitations that influence the patients’ quality of life and treatment prognosis. In order to overcome these limitations, this dissertation research focuses on the new poly(2-oxazoline)s (POx) polymers as versatile nano-scale drug delivery systems. The synthesis and characterization of several new monomers including 2-benzyl-2-oxazoline, 2- methyl-2-oxazine, and a cationic monomer is summarized. In addition, the exploration of newly synthesized POx polymers using above monomers and new functionality groups (e.g. clickable initiator) are also briefly discussed.

The potential of a specific doubly amphiphilic POx triblock copolymer (previously termed P2) is further explored as a polymeric micelle system in delivering taxanes

(paclitaxel, the 3rd generation of toxoids, and multiple-drug combinations). The P2/paclitaxel micelle formulation is characterized by a facile preparation, up to 50 % wt. drug loading, excellent shelf stability, controllable sub-100 nm size, a very high maximum tolerated dose in mice, and a significant enhancement in efficacy when treating three different breast cancer

iii models. In addition to paclitaxel, a number of 3rd generation toxoids able to avoid common drug resistance mechanisms, are formulated in P2 micelles. An excellent solubilization of different 3rd generation taxoids is achieved irrespective of drug structures with up to 46 % wt. drug loading and less than 100 nm micelle size. Furthermore, a selected formulation with the new taxoid SB-T-1214 shows about one to two orders of magnitude more active in vitro than paclitaxel in LCC6-MDR, a multidrug resistant breast cancer cell line. It significantly inhibits the growth of LCC6-MDR orthotropic tumors, outperforming Taxol and Cremophor formulated SB-T-1214. Moreover, a number of chemotherapeutic drugs are simultaneously formulated in P2 micelles. The multi-drug loaded P2 micelles indicate a ratio-dependent synergistic activity against multiple cancer cells in vitro.

This dissertation also involves the study of a new and first cationic oxazoline monomer and corresponding polymer containing side-chain secondary amine groups. The cationic polymer is able to condense plasmid DNA into a sub 100 nm polyplex which is stable upon dilution in saline and thermal challenge. These polyplexes exhibit minimum serum protein binding and very low cytotoxicity in vitro compared to the polyplexes of DNA and commonly used poly(ethylene glycol)105-b-poly(L-lysine)51 (PEG-PLL). The in vitro transfection efficiency of the polypelxes is also studied in B16 murine melanoma cells as well as RAW264.7 macrophage cells. The cationic polymer represents a comparably safe and promising platform for delivering genes to macrophages and cancer cells.

In summary, POx polymers are versatile and promising platforms for taxane anticancer drugs delivery and gene delivery.

To my parents Fei He and Yulan Sun and my wife, Lin Zheng.

ACKNOWLEDGEMENT

First of all, I would like to express my utmost gratitude and appreciation to my supervisor,

Dr. Alexander Kabanov who offered me this great educational opportunity in his lab as a graduate student. In the past few years, he provided me constant support, priceless guidance and extraordinary wisdom throughout my PhD journey. He is a remarkable and exceptional mentor I am so blessed to have. I will be forever thankful to his mentorship, inspiration and sense of humor. I would also like to thank my committee members, Dr. Elena Batrakova, Dr.

Leaf Huang, Dr. William Zamboni and Dr. Lisa Carey for their precious guidance and advices throughout the years. They advised me through the obstacles in completion of my research work and reviewed all my progress and dissertation. In addition, I would like to extend my special appreciation to Dr. Rainer Jordan, Dr. Tantiana Bronich, Dr. Robert Luxenhofer, Dr.

Yingchao Han, Dr. Iwao Ojima, Dr. Anita Schulz, Dr. Devika Manickam, Dr. Marina Solkosky,

Dr. Daria Alakhova, Herdis Bludau and Xiaomeng Wan for their collaboration and as my external advisors.

Moreover, I want to express my gratitude to UNMC School of Pharmacy and UNC

Eshelman School of Pharmacy for allowing me pursuing higher education. I also like to acknowledge my colleagues and friends, Dr. Jing Tong, Dr. Yi Zhao, Dr. Xiang Yi, Dr. Jing Gao,

Matt Haney, Jubina Bregu, Nazar Filonov, Vivek Mahajan, Hemant Vishsaowa, Soo Kim,

vi Youngee Seo, Yuling Zhao, Shu Li, Yuhang Jiang, Dongfen Yuan, Dr. Yuanzeng Min, Dr. Zagit

Gaymalov, Lei Miao, Dr. Lei Peng, Duhyeong Hwang, and all past and present members of the kabanov Laboratory for their sincere friendship and kindly help throughout my graduate study.

Particularly, I will never forget the happiness, frustration, jokes, and parties we had in addition to the inspiring scientific conversations. I would like to acknowledge BRIC imaging core facility and Mouse Phase I Unit for their assistance especially Dr. Hong Yuan, Dr. Zibo Li, David Darr, and Charlene Santos.

I would particularly thank my parents, my wife Lin, my parents-in-law, brother-in-law and my sister for their unconditional support and love all along the journey. I cannot go this far without their understanding, patience, standing by my side, faith and love.

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TABLE OF CONTENTS

LIST OF TABLES…...……………………………………………………………………..………………...... xi

LIST OF FIGURES…………………………………………………………………………………………….…...... xii

LIST OF ABBREVATIONS AND SYMBOLS………………………………………………..…...... xvi

Chapter I Nanomedicine of Taxane Anticancer Drugs……………..…...... 1

1.1 Summary………………………………………………………………………………………………..1

1.2 Introduction……………………………………………………………………………………………2

1.3 Currently approved formulations of taxane………………………….………………..4

1.4 New taxane analogues in clinical investigation………………………………..……..7

1.5 Mechanism of action (MOA) of taxane drug……………………………………..…..11

1.6 PK and dose regimen of currently approved taxane formulations………….13

1.7 Limitations of taxane drug……………………………………………………………………..18

1.8 Taxane drug delivery using nanocarriers (nanomedicine)………………….……25

1.8.1 Lipsome formulation of taxanes in clinical trials...... ……..26

1.8.2 Conjugates and dendrimers for taxane delivery……………29

1.8.3 Taxane delivery using polymeric micelle………………….……33

1.8.4 Other taxane products in clinical trial………………….………..39

1.9 Conclusion……………………………………………………………………………………………..41

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Chapter II synthesis and characterization of new 2-oxazoline monomer and Poly(2-oxazoline)s polymer………………………………………………….…………..51 2.1 Summary……………………………………………………………………………………………..51

2.2 Introduction…………………………………………………………………………………………52

2.3 Initiator containing clickable functionality……………………………………………55

2.4 Sythesis of new monomer 2-Benzyl-2-Oxazoline

(BzOx) and BzOx containing polymer………………………………………….………58

2.5 Synthesis of new monomer 2-methyl-2-oxazine

(MeOz) for gradient polymerization………………….…………………………..…..62

2.6 Synthesis of new cationic POx monomer and polymer CPOx……………….64 Chapter III A Simple and Non-Smart Micellar Formulation of POx/Paclitaxel With Superior Safety and Efficacy In Vivo………………….…………………….77 3.1 Summary……………………………………………………………………………………………..77

3.2 Introduction…………………………………………………………………………………………78

3.3 Materials and Methods…………………………………………………………………….….81

3.4 Results and Discussion………………………………………..……………………...……….86

Chapter IV Other Applications of P2 POx Polymers (1): Preparation, In Vitro and In Vivo Evaluation of POx Micelles with High Capacity for 3rd Generation Taxoids………………………………………………………………………….………..111 4.1 Summary……………………………………………………………………………………..…….111

4.2 Introduction……………………………………………………………………………………….112

4.3 Materials and Methods………………………………………………………………………115

4.4 Results…………………………….……….………………………………..……………………...121

4.5 Discussion………………………………………………………………………………………....127

Chapter V Other Applications of P2 POx Polymers (2): Synergistic Combinations of Multiple Chemotherapeutic Agents in High Capacity POx Micelle……………………………………………………………………..………………………140 5.1 Summary……………………………………………………………………………………………140

5.2 Introduction……………………………………………………………………………………….141

5.3 Materials and Methods……………………………………………………………………...144

5.4 Results……………………………………………………………………………………………….149

5.5 Discussion………………………………………………………………………………………….154 Chapter VI: Poly(2-oxazolines) with Pending Cationic Side Chains as a Novel Non-Viral Platform for Gene Delivery With Low Protein Binding…………………………………………………………………………….……………………………………173 6.1 Summary……………………………………………………………………………………………173

6.2 Introduction……………………………………………………………………………………….174

6.3 Materials and Methods……………………………………………………………………..177

6.4 Results……………………………………………………………………………………………….186

6.5 Discussion………………………………………………………………………………………….194

Chapter VII: Summary and Future Experiments…………………………………………………….213

References……………………………………………………………………………………………………………217

LIST OF TABLES Table 1.1 Current approved taxane formulations……………...... 42

Table 1.2 New taxane analogues in clinical investigation…...... 43

Table 1.3 Liposomal taxanes in clinical investigation……..……………………………………….44

Table 1.4 Conjugates and dendrimers for taxanes in clinical investigation……………..45

Table 1.5 Polymeric micelles of taxanes in clinical investigation…………………………....46

Table 3.1 Drug concentration, loading efficiency (LE), loading capacity (LC), drug loaded micelle size and PDI of POx/PTX formulations………...... 92

Table 3.2 Size and PDI of two POx/PTX formulations before and after Lyophilization………………………………………………………………………………………………………….93

Table 3.3 Monitoring of animal death and weight in the MTD experiments...... 94

Table 3.4 Summary of cytotoxicity to liver Hep G2 and kidney LLC-PK1 cells...... 95

Table 3.5 Clinical chemistry parameters of Balb/c mice receiving POx and POx/PTX…………………………………………………………………………………………………….96

Table 4.1 Data of solubilization experiments……………………...... 132

Table 4.2 IC50 values of POx/SB-T-1214 micelles vs. other PTX formulations………….133

Table 5.1 Solution concentration, LE and LC of drugs in POx aqueous dispersions……………………………………………………………………………………………………………161

Table 5.2 Comparison of our results with others for drug formulation in LC and DL values………………………………………………………………………………………………..162

LIST OF FIGURES

Figure 1.1 MOA of taxane drugs (exemplified with PTX)….…...... 47

Figure 1.2 Mechanism of drug resistance to taxanes….………...... 48

Figure 1.3 Nanomedicine for taxane drug delivery…………………..……………………………..49

Figure 1.4 3rd generation of taxanes and POx micelle delivery systems….……………....50

Figure 2.1 Schematic demonstration of P2 synthesis………………………………………………68

Figure 2.2 Azide-Alkyne Huisgen Cycloadition………………………………………………………...69

Figure 2.3 Synthesis of Propargyl-P4 with clickable alkyne moiety…………………………..70

Figure 2.4 Synthesis of BzOx monomer and polymer HEZ007 and HEZ012………………71

Figure 2.5 Impact of PTX concentration on the solubilization of PTX in HEZ012 (10 g/L)………………………………………………………………………………………………………72

Figure 2.6 Monomer synthesis of MeOz………………………………………………………………….73

Figure 2.7 Synthesis of the new monomer, Boc-MeAmMeOx………………………………….74

1 Figure 2.8 H-NMR spectra (d3-MeOD, 300 K) of the novel monomer 2-(N-methyl,N-Boc-amino)-methyl-2-oxazoline with the assignment of all observed signals……………………………………………………………………………………..……..75

Figure 2.9 Block copolymerization of MeOx and Boc-MeAmMeOx using propargyl tosylate as the initiator and 5% K2CO3 as the terminating agent……………………………………………………………………………………………………….……..…..….76

Figure 3.1 Preparation and physicochemical properties………………………………………....97

Figure 3.2 SOP for small scale (1-5 mg) micelle production……………………………………..98

Figure 3.3 SOP for large scale (200 mg) micelle production……………………………………..99

Figure 3.4 POx/PTX 50/40 formulation………………………………………………………………….100

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Figure 3.5 Size of POx/PTX 50/40 micellar nanoformulation in water, PBS or 10mM nacl………………………………………………………………………………………………………..101

Figure 3.6 Zeta-potential of POx/PTX 50/40 micelles in water or 10 mM nacl by zeta sizer……………………………………………………………………………………………………102

Figure 3.7 Characterization of POx polymers…………………………………………………………103

Figure 3.8 MTD in nude mice and toxicology profiles…………………………………………….104

Figure 3.9 In vitro toxicity evaluation…………………………………………………………………….105

Figure 3.10 Cytotoxicity to LLC-PK1 normal pig kidney epithelial cells……………………106

Figure 3.11 Cytotoxicity to Hep G2 human liver hepatocellular carcinoma cells……………………………………………………………………………………………………………………….107

Figure 3.12 Efficacy of POx/PTX 50/40 in A2780 tumors………………………………………..108

Figure 3.13 Efficacy of POx/PTX 50/40 in A2780 tumors (treatment starting at later stage, tumor volume of ca. 400 mm3)…………………………………….……109

Figure 3.14 Efficacy of POx/PTX 50/40 in LCC6-MDR and T11 tumors...... 110

Figure 4.1 Preparation of POx/taxoid micellar nanoformulations………………………….134

Figure 4.2 Physicochemical properties of various POx/SB-T-1214 polymeric micelle formulations…………………………………………………………………………….135

Figure 4.3 In vitro cytotoxicity of various PTX and SB-T-1214 formulation in LCC6-MDR cells, LCC6-WT, and T11 cells…………………………………………………………...136

Figure 4.4 Establishment of the safe dose of SB-T-1214 in nude mice……………………137

Figure 4.5 Efficacy of various drug formulations in LCC6-MDR tumors…………………..138 xiv Figure 4.6 Efficacy of various drug formulations in T11 OST tumors………………………139

Figure 5.1 Chemical structure of amphiphilic POx copolymer, P(MeOx40-b-BuOx21-b-MeOx34), and schematic representation of multiple drug loaded micelles…………………………………………………………………….…….163

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Figure 5.2 Chemical structures of anti-cancer drugs………………………………………………164

Figure 5.3 Size distribution and stability studies of POx micelles loaded with single drugs……………………………….………………………………………………………………….165

Figure 5.4 Size distribution and stability studies of POx micelles co-loaded with two drugs………….………………………………………………………………………….166

Figure 5.5 Size distribution and stability studies of POx micelles co-loaded with three drugs………………………….……………………………………………………….167

Figure 5.6 CLSM images of MCF-7 cells incubated for 1 h with free PTX, POx copolymer alone and POx micelles co-loaded with PTX and 17-AAG…………………………………………………………………………………………………………….…..168

Figure 5.7 CLSM images of MCF-7 cells incubated for 4 h with free PTX, POx copolymer alone and POx micelles co-loaded with PTX and 17-AAG……………..169

Figure 5.8 Cytotoxicity of single drugs and binary drug combination, ETO/17-AAG solubilized in POx micelles, in MCF-7 cells and the corresponding CI vs. Fa plot………………………………………………………………………………....170

Figure 5.9 Cytotoxicity of single drugs and binary drug combinations, BTZ/17-AAG in POx micelles, in MCF-7 cells and the corresponding log CI vs. Fa plot…………………………………………………………………………………………………...171

Figure 5.10 IC50 values of micellar 17-AAG, BTZ and BTZ/17-AAG combination in PC3, MDA-MB-231, and HepG2 cancer cells and the CI vs. Fa plot………………………………………………………………………………………….…………172

Figure 6.1 Synthesis of the new monomer, Boc-MeAmMeOx………………………………..198

1 Figure 6.2 H-NMR spectra (d3-MeOD, 300 K) of the novel monomer 2-(N-methyl,N-Boc-amino)-methyl-2-oxazoline with the assignment of all observed signals……………………………………………………………………………………………199

Figure 6.3 Block copolymerization of MeOx and Boc-MeAmMeOx using propargyl tosylate as the initiator and 5% K2CO3 as the terminating agent…………………………………………………………………………………………………………….………200

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1 Figure 6.4 H-NMR spectra (400 MHz, d3-MeOD) of Boc-CPOx and the deprotected CPOx after single and repeated deprotection procedures, along with the respective structures and the peak assignments……………………………………………………………………………………………………….…201

Figure 6.5 Titration curves for CPOx copolymer and PEG-PLL in 0.1M aqueous NaCl using 0.1M HCl…………………………….……………………………………….………..202

Figure 6.6 pDNA condensation by CPOx assay by ethidium bromide exclusion and gel retardation assay of pDNA and polyplexes at various N/P ratios…....…………203

Figure 6.7 DLS measurements of the size distributions, particle size and PDI, And ζ-potential of CPOx/pDNA polyplexes as a function of N/P ratios…………………..204

Figure 6.8 Representative TEM image……………………………………………………………….…..205

Figure 6.9 Size and PDI of CPOx/pDNA polyplexes at various N/P ratios as a function of storage time………………………………………………………………………………………..206

Figure 6.10 Stability of CPOx/pDNA polyplexes upon dilution and heating……………207

Figure 6.11 SDS-PAGE and micro BCA assay of adsorbed plasma proteins on PEG-PLL/pDNA, PLL/pDNA, and CPOx/pDNA polyplexes……………………………………….208

Figure 6.12 In vitro cytotoxicity of the polymers and the polyplexes in RAW264.7 murine macrophages and B16 murine melanoma cells…………….…………209

Figure 6.13 Cellular uptake of CPOx/pDNA polyplex with N/P 5 in RAW264.7 macrophage cells…………………………….………………………………………………….210

Figure 6.14 Cellular uptake of CPOx/pDNA polyplex with N/P 10 in

RAW264.7 macrophage cells………………….…………………………………………………………….211

Figure 6.15 In vitro transfection of B16 murine melanoma and RAW264.7 murine macrophage cells…………………………………………………….……………..212

LIST OF ABBREVIATIONS AND SYMBOLS

α-TAS alpha-tocopheryl acid succinate ABC accelerated blood clearance ACN acetonitrile AFM atomic force microscope AIDS acquired immune-deficiency syndrome ALD adrenoleukodystrophy ALP alkaline phosphatase ALT alanine aminotransferase APTT activated partial thromboplastin time ASCO American Society of Clinical Oncology AUC area under the curve BBB blood brain barrier BC breast cancer BCA bicinchoninic acid BCL-2 B-cell lymphoma 2 BMS Bristol-Myer Squibb Boc tert butyloxycarbonyl BSA bovine serum albumin BTZ bortezomib BUN blood urea nitrogen CHK1 checkpoint kinase 1 CI combination index CLSM confocal laser scanning microscopy Cmax maximum concentration CMC critical micelle concentration CPOx cationic poly(2-oxazoline)s CrEL Cremophor EL xvi xvi

CTX cabazitaxel DC drug concentration DHA docosahexaenoic acid DLS dynamic light scattering DMEM Dulbecco’s Modified Eagle Medium DMF dimethylformamide DMSO dimethyl sulfoxide DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine DNA deoxyribonucleic acid DOPA 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine DOTAP N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride DSPC 1,2-distearoyl-sn-glycero-3-phosphocholine DTX docetaxel ECOP Eastern Cooperative Oncology Group ECM extracellular matrix EGFR epidermal growth factor receptor EPR enhanced permeability and retention EtBr ethidium bromide ETO etoposide FLT3 Fms-like tyrosine kinase 3 GC gastric carcinoma GEM genetically engineered mouse GOG Gynecologic Oncology Group GPC gel permeation chromatography GTP guanosine-5’-triphosphate h hours HCC hepatocellular carcinoma xvii

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid H&E hematoxylin & eosin HPLC high-performance liquid chromatography HRPC hormone refractory prostate cancer HPMA N-(2-hydroxypropyl)methacrylamide

IC50 half maximal inhibitory concentration IIT investigator initiated trial IP intraperitoneal IV intravenous LC loading capacity LCROP living cationic ring opening polymerization LDH lactate dehydrogenase LE loading efficiency LRP1 low-density lipoprotein receptor-related protein 1 MBC metastatic breast cancer mCRPC metastatic castration-resistant prostate cancer MDR multiple drug resistance MeOH methanol mg milligram min minute mL milliliter MOA mechanism of action mPEG-PLA methoxy poly(ethylene glycol)-b-poly(lactide) MS mass spectrum MTD maximum tolerated dose MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide MW molecular weight NCI National Cancer Institute xviii ng nanogram NMR nuclear magnetic resonance NSCLC non-small cell lung cancer OC ovarian cancer ORR overall response rate OST orthotopic syngeneic transplant PBS phosphate buffered saline pBuOx poly(2-butyl-2-oxazoline) pBzOx poly(2-benzyl-2-oxazoline) PD progressive disease PDI polydispersity index PEG poly(ethylene glycol) PEG-PLA methoxy poly(ethylene glycol)-block-poly(D,L-lactic acid) PEI polyethylenimine pEtOx poly(2-ethyl-2-oxazoline) PFS progression free survival P-gp P-glycoprotein PK pharmacokinetics PLA poly(D,L-lactic acid) PLGA poly(lactic-co-glycolic acid) PLL poly(L-Lysine) pMeOx poly(2-methyl-2-oxazoline) pNoOx poly(2-nonyl-2-oxazoline) POx poly(2-oxazoline)s PR partial response PSMA prostate specific membrane antigen PT prothrombin time PTX paclitaxel xix q1w every week q3w every three-weeks Ref. reference RES reticuloendothelial system RT room temperature SANS small angle neutron scattering SC subcutaneous SCCHNC squamous cell carcinoma of head and neck cancer SCID severe combined immune-deficiency SD stable disease SEC size exclusion chromatography SEM standard error of the mean siRNA small interfering RNA SPARC secreted protein acidic and rich in cysteine t1/2 half-life TAM tumor associated macrophage TEM transmission electron microscopy TFA trifluoroacetic acid TGA thermogravimetric analysis TGFβ transforming growth factor beta THF tetrahydrofuran TIC tumor initiating cells TNBC triple negative breast cancer μg microgram μL microliter USP United States Pharmacopeia VCR vincristine Vd volume of distribution xx

Vdss steady state volume of distribution VEGF vascular endothelial growth factor VRP verapamil vs. versus VTE venous thromboembolism 17-AAG 17-allylamino-17-demethoxygeldanamycin

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CHAPTER I: NANOMEDICINE OF TAXANE ANTICANCER DRUGS 1

1.1 Summary

Taxane anticancer drugs such as paclitaxel, docetaxel, and cabazitaxel, are one class of the most important chemotherapeutics in treating broad range of cancers such as ovarian, breast, non-small cell lung, head and neck, and prostate cancers. However, taxanes are associated with excipient-related hypersensitivity, severe neurotoxicity and peripheral neuropathy, drug resistance and other limitations that influence the patients’ quality of life and treatment prognosis. In order to overcome these limitations, a tremendous research effort is focused on new delivery strategies, especially nanomedicine of taxanes, and have already resulted in clinical trials or approvals. This review emphasizes current approved and clinical investigational taxanes, the updates of taxane mechanism of actions, the limitations of taxanes in clinical use, in particularly, sensory neuropathy and drug resistance; and provides an extensive overview of various clinical stage nanomedicine of taxanes such as liposomes, taxane-conjugates, dendrimers, polymeric micelles (e.g. poly(2-oxazoline)s micelles), and others for delivery of taxane drugs.

1 This chapter previously appeared as a manuscript soon to be submitted.

1.2 Introduction

Taxanes are diterpenes originally isolated from the plants of Taxus brevifolia (The bark of

Pacific Yews) [1]. It has been more than 50 years since U.S. National Cancer Institute (NCI) discovered first taxane - paclitaxel (PTX) [1] and later Dr. Susan Horwitz and colleagues found it can promote tubulin polymerization thus stabilize the microtubules [2]. The resulting deficiency of microtubule dynamics can arrest cells at G2-M phase during mitosis and ultimately cause apoptosis of rapid proliferating cells [3-5]. Current taxane agents are either natural-origins or semi-synthesized compounds including PTX [1], docetaxel (DTX) [6, 7], cabazitaxel (CTX) [8, 9], and other taxane derivatives [10]. They are widely used in medical oncology and made major impact in treating breast, ovarian, lung, head and neck, and other malignancies [11-14].

Taxanes are generally white crystalline powder with a melting point at approximately 200 oC (PTX, 215-217 oC; DTX, 187-192 oC; CTX, 170-180 oC) [15, 16] and their clinical applications have been critically limited by low water solubility [e.g. Taxol® approved by the Food and

Drug Administration (FDA) in 1992, where PTX is formulated in organic excipients of polyoxyethylated castor oil (Cremophor EL, CrEL) and dehydrated ethanol (50/50, v/v)].

However, the significant amount of toxic excipient CrEL often induces serious adverse reactions and severe hypersensitivity (sometimes fatal), requiring pre-management with corticosteroids, diphenhydramine, and H2 antagonist [17-19]. Furthermore, Taxol® exhibits non-linear pharmacokinetics (PK) in vivo which are less-predictable and undesirable [17, 20-

23]. Although improved solubility was achieved by modifying some groups on PTX, the new

2 taxanes including DTX and CTX are still considered insoluble [Defined in US Pharmacopeia

(USP)] and surfactants (other excipients) are necessary for the clinical formulations [24, 25].

Recent research efforts are focused on finding less toxic taxane derivatives, optimizing treatment regimens and discovering new delivery systems that reduce excipient-related toxicity while enhancing treatment outcome through tumor targeted delivery [24, 25].

Nanomedicines delivering taxanes to the tumor sites is one of the most intense research area encompassing liposomal formulations [26-31], polymer-taxane conjugates [32-35], polymeric micelles [36-43], dendrimers [44-47], and other nanocarriers [10, 25, 48]. It is proposed that these nanomedicines can avoid the usage of toxic excipients, improve the solubility of taxanes, extend their in vivo half-lives, deliver drugs preferentially to the tumor sites, adjust unwanted

PK and biodistribution, and circumvent drug resistance. Furthermore, nanocarriers are explored to deliver new taxane derivatives as well as to simultaneous incorporate multiple chemotherapeutic drugs in order to achieve synergistic antitumor effects [49-51]. This review attempts to update the current approved taxane formulations, their pharmacology, mechanism of action (MOA), PK and dose regimens, and limitations in clinical standpoint of view, and provide perspectives in the frontier of various taxane nanomedicines in preclinical or clinical stages.

1.3 Currently approved formulations of taxanes

PTX formulations

A summary of currently approved formulations of taxanes is presented in Table 1.1. The first three formulations are based on classical taxane member, namely PTX. PTX has a high molecular weight (MW) of 854 Da with formula of C47H51NO14, a very low aqueous solubility

(approximately 1 mg/L), and no ionizable groups to form salts to increase solubility through pH adjustment. Although the chemical structure of PTX was identified in 1960’s, it was until

1994 that two groups independently reported total synthesis procedures starting from a baccatin core structure containing the ABCD ring (Table 1.1 PTX structure), followed by addition of the "tail" part to the C13-hydroxyl group [52-54]. Anticancer activity of PTX is mainly due to the side chain, A ring, D oxetane ring and C2 benzoyl group. The C3’ amide acyl group in the C13 chain maintains the activity and the hydroxyl group at C2’ enhances its function [48, 55].

Taxol®, the first injectable dosage form of PTX, was approved by FDA in 1992 exclusively to American company Bristol-Myers Squibb (BMS). It is formulated in 50% CrEL and 50% dehydrated ethanol. The only clinically approved alternative to Taxol® in United States is

Abraxane®. It consists of lyophilized cakes of PTX-albumin nanoparticles in each 50 mL vial, containing 100 mg of paclitaxel and nearly 900 mg of human albumin. These novel nanoparticles are 130 nm in size prepared by high-pressure homogenization of PTX with human albumin to form colloidal nanosuspension [56-58].

Furthermore, Samyang Corporation (Seoul, Korea) has developed a new PTX formulation,

Genexol-PM®, which is approved in South Korea and other Asian countries. It is a methoxy- 4 poly(ethylene glycol)-b-poly(lactide) (mPEG--PLA) based polymeric micelle formulation of PTX, showing 3 fold increase in maximum tolerated dose (MTD), high levels of distribution of drug in various tissues including tumors, and significantly improved antitumor efficiency in tumor mice models compared to Taxol® [41, 43]. The high MTD of Genexol-PM® without additional toxicity in Phase I clinical trial may showcase a good alternative to Taxol® and Phase II trial is undergoing in United States for treating advanced breast cancer (BC) and non-small cell lung cancer (NSCLC) [40, 41]. Another approved PTX formulation is the liposome of PTX, Lipusu® injectables from Luye Pharma Group and was approved for Chinese market by Chinese FDA

[28]. The implementation of liposomes may solve the solubility problems of PTX while avoiding excipient-related side-effects. Lipusu® formulations demonstrated higher tolerated dose, enhanced efficacy and reduced side-effects to the blood pressure, the medulla, peripheral blood and the liver [28, 59].

DTX formulation

DTX (MW 807.9 Da) belongs to the second generation of taxane drugs. It is semi- synthesized from 10-deacetyl baccatin III (extracted from the needles of Taxus baccata L. that is renewable); therefore, DTX has a 10-position of hydroxyl group replacing an acetyl group in

PTX. Another chemical modification occurs at the C3’ position using tert-butyl carbamate ester group to replace benzyl amide in PTX. These two modifications convey DTX few times higher water solubility (5.0-6.0 mg/L) and enhanced binding affinity to tubulin than PTX [60,

61]. Indeed, DTX was reported to be more potent than PTX in P388, P388.10, SVras, J774.2

(murine cell lines) and Calcl8, HCT116, T24, N417, and KB cells (human cancer cell lines) [62].

The greater efficacy of DTX than PTX was also evidenced in vivo in B16 melanoma xenografts,

5 possibly due to higher binding affinity to microtubules [63]. Another explanation for enhanced efficacy is speculated from in vitro studies in P388 leukemia cells that DTX has 3 times higher intracellular concentration and lower efflux rate than PTX. Current only clinical formulation of

DTX is Taxotere® developed by Sanofi. Indications for Taxotere® include metastatic and recurrent breast cancer (MBC), gastric adenocarcinoma (GC), advanced and metastatic NSCLC, squamous cell carcinoma of head and neck cancer (SCCHNC), and hormone refractory prostate cancer (HRPC). One vial of Taxotere® contains single use of 80 mg DTX in polysorbate

80/dehydrated ethanol at 20 mg/mL concentration [6, 61].

CTX formulation

Another example of second-generation taxanes is CTX (previously as XRP-6258), a 7, 10- dimethyloxy derivative of DTX with MW of 854 Da and formula of C45H57NO14. It can also be semi-synthesized from natural source of yew tree needles. CTX is practically insoluble in water and requires solubilization excipients for clinical formulation. For patients with metastatic castration-resistant prostate cancer (mCRPC), DTX is the initial standard treatment; however, once they developed DTX refractory there are not many further options to extend progression-free survival (PFS). It remains no options until June 2010 that FDA approved CTX formulation (Jevtana®, Sanofi) as a new therapeutic drug for treating HRPC patients who were previously treated with and became resistant to DTX [8, 9, 25, 64].

1.4 New taxane analogues in clinical investigation

There are many taxane drugs under clinical investigation. Here we mainly focus on new taxane analogues, which are chemically modified on traditional PTX or DTX. Most of new taxane analogues undergoing clinical trials are listed in Table 1.2. For example, larotaxel

(XRP9881) is a promising agent for multidrug resistance (MDR) BC treatment. It is a new semi- synthesized taxoid originated from 10-deaceyl baccatin III and also capable to cross blood- brain barrier (BBB) possibly due to minimal recognition by P-glycoprotein (Pgp) efflux pumps.

Phase I and II trials identified dose regimen of larotaxel in solid tumors as 90 mg/m2, intraveneous (IV) infusion for 1 h every three-weeks (q3w). Using this regimen larotaxel exhibited good efficacy against taxane-pretreated MBC patients while dose-limiting toxicities include neutropenia and neutropenic complications [65, 66].

Milataxel (TL139) is a novel analog to DTX that overcomes Pgp involved resistance. One advantage is that milataxel does not require polysorbate 80 or CrEL to formulate which avoids excipients-related hypersensitivity reactions [67]. A phase II study reported in 2008 for colorectal cancer treatment (35 mg/m2 IV q3w) demonstrated no clinical significance while six patients suffered neutropenic sepsis and two patients were deceased [68]. Another Phase II study in treating platinum-refractory NSCLC patients showed tolerability of the milataxel at a dose of 35 mg/m2, nevertheless peripheral neuropathy was the most frequent non- haematological toxicity [69].

Furthermore, TPI-287 [70], TL310 [71], and Ortataxel (BAY-59-8862; IDN-5109) are third- generation taxane analogues in order to overcome resistance related with overexpressed Pgp and/or mutant tubulin. In the Phase I trial, TPI-287 appears to be well tolerated up to 127.5

7 mg/m2 every week (q1w) for 3 weeks with 1-week rest. Drug induced toxicities included nausea, vomiting, diarrhoea, fatigue, anorexia, rash, and anaemia while dose limiting toxicity is still grade 3 peripheral neuropathy [72]. Currently, TPI-287 are studied in several Phase II clinical trials (total 17 hits in clinicaltrials.gov website using TPI-287 as a keyword to search) in treating mostly brain malignancies such as glioblastoma, recurrent neuroblastoma and medulloblastoma, and brain metastasis originated from BC, as well as melanoma. 3 trials were completed while 4 were terminated or withdrawn. Up to date there is no more report from

Pubmed search. In contrast, clinical data for TL310 is very limited both in Pubmed and clinicaltrials.gov. In a Phase I study, patients with advanced refractory solid tumors were administered orally up to 160 mg/m2 TL310 on days 1, 8 and 15 of a 28-day cycle. One out of five patients experienced grade 5 neutropenic sepsis, grade 4 neutropenia and grade 4 thrombocytopenia. A partial response (PR) was observed in one GC patient dosed at 80 mg/m2 of TL310 and disease were stable for more than two treatment cycles in six patients (3 cases of oesophageal, 1 melanoma, 1 ovary, 1 cervix cancer) within total 18 patients [71]. Ortataxel

(BAY-59-8862; IDN-5109) is a semisynthetic analogue to DTX synthesized originally by Dr. Iwao

Ojima group, and was further developed by Indena in collaboration with Bayer. It showed efficacy in preclinical studies in multi-drug resistant tumors and a superior and durable effects on reduction of the tumor microvessel density against HRPC compared to PTX. Phase I and II studies were performed in 1 h IV infusion q3w of 75 mg/m2. A 1 h IV infusion of 45 mg/m2 q1w was also tested and recommended. The dose-limiting toxicities observed in these studies were grade 4 febrile neutropenia and grade 4 neutropenia while peripheral neuropathy were also reported [73]. Phase II studies demonstrated activity against taxane-resistant MBC and

NSCLC. The oral bioavailability were reported as 19 - 31% although no clinical trials were performed on oral administration of the drug to date [74]. Dr. Ojima’s research group also developed a library of third generation taxanes aiming lower toxicity and higher activity against resistance cancer cells than PTX. In section 1.8.3, we will discuss these new taxanes in combination with polymeric micelle delivery systems.

Another oral formulation of new taxane is Tesetaxel (DJ-927). A Phase I study was performed in patients with advanced malignancies using a single oral dose ranging from 1.5 to 40 mg/m2 with a q3w regimen and MTD was identified as 27 mg/m2. However, Tesetaxel showed modest to low (oral dose 27 mg/m2 q3w) activity in patients with advanced GC and other cancers, while similar toxicities to current therapies such as neutropenia, sepsis, diarrhoea and peripheral neuropathy were observed [75]. Therefore, Daiichi Pharmaceutical

(Daiichi Sankyo) has discontinued development of Tesetaxel (Scrip Daily Online, 7 Nov 2006,

S00939534).

Last but not least, BMS developed several taxane analogues investigated clinically including BMS-275183, BMS-184476, and BMS-188797. BMS-275183 is an oral formulation of

C4 methyl carbonate analogue of PTX with additional modifications to the side chain and was shown active against PTX resistant tumors in vitro and in vivo. BMS-275183 has oral bioavailability of 24% in human, considerably higher than PTX, and Phase I studies determined oral dose up to 200 mg/m2 q1w was tolerable but twice a week regimen of 100 mg/m2 appeared less toxic in terms of neuropathy, fatigue, diarrhoea and neutropenia. It was shown to be partial responsive for q1w 200 mg/m2 oral administration in 9 patients of 38 total (23.7%) including NSCLC (4 of 13 patients), prostate carcinoma (2 of 2 patients), primitive

9 neuroectodermal tumor (1 of 1 patient), cholangio carcinoma (1 of 2 patients) and undifferentiated sarcoma (1 of 1 patient) [76].

BMS-184476 is also a new taxane analogue with C7-hydroxyl group replaced by a 7- methylthiomethyl ether group compared to PTX. The substitution increases the solubility and potency over PTX and DTX in vitro and in vivo [77]. A Phase I study of BMS-184476 indicated that a 1 h IV infusion at 60 mg/m2 showed no grade 3 or 4 peripheral neuropathy, which is a recommended dose for Phase II trial [78]. Another Phase I trial also tested BMS-184476 q1w for 3 weeks followed by one week break as a treatment cycle and found neutropenia was the dose-limiting toxicity and later recommended 50 mg/m2 on days 1 and 8 every 21 days. Two patients died of neutropenia-related complications [79, 80]. A Phase II study for BMS-184476 as second line treatment of NSCLC suggested that the 60 mg/m2 was well tolerable and can achieve antitumor activity in previously treated patients [81]. No recent report on further

Phase II or III trials on this drug since 2005.

The third drug candidate is BMS-188797, a synthetic analogue to PTX with a C4 carbon formed 4-desacetyl-4-methylcarbonate. It has very low solubility (using the same excipient as

Taxol®) and approximately twice potent over PTX [78, 82]. The recommended Phase II dose was 50 mg/m2 q1w or 175 mg/m2 q3w IV. The dose-limiting toxicity was febrile neutropenia.

A combination trial of BMS-188797 with cisplatin determined a recommended dose of 110 mg/m2 (1 h infusion, q3w) followed by cisplatin 75 mg/m2 [82-85]. All above three analogues have no recent updates and seems that BMS discontinued the further development of these drugs.

1.5 MOA of taxane drugs

Taxol® was originally as second line treatment for ovarian cancer (OC) in 1980's but soon be applied as first line treatment due to great antitumor responses in nearly 26% of OC patients that intrinsically resistance to platinum class agents [86]. The general antitumor mechanism for taxane drugs is to bind tubulin and interfere tubulin polymerization steps, thus in turn stabilize microtubules and cause cell cycle arrest at G2-M phase and ultimately induce apoptosis [87]. Microtubules are assembled from a 100kDa protein tubulin which is the heterodimer of α-tubulin and β-tubulin proteins (50kDa each). The dimer formation requires binding to a guanosine-5'-triphosphate (GTP) molecule at the nucleotide exchangeable site (E site of β-tubulin) and the non-exchangeable site (N-site of α-tubulin). The binding of taxanes occurs at the E site of β-tubulin heterodimers where GTP-binding sites locate, and therefore disrupt assemble and disassemble dynamics of microtubules [88, 89]. The microtubule dynamics is crucial for chromosome segregation during mitosis. A recent study found that levels of PTX in primary breast tumors are well below the concentration required for retaining cells in mitotic arrest. The authors further identified that cells progressed through mitosis under lower concentrations of PTX can separate their chromosomes on multipolar spindles which led to chromosome missegregation followed by cell death [90] (Fig. 1.1).

The MOA of DTX is not fully understood except for most intense research focus of stabilizing microtubules. Increasing reports suggest that DTX outperform PTX as an anticancer drug; for example, DTX has shown about 2.5 folds higher potency than PTX to inhibit cell proliferation in vitro. Proposed explanation is that DTX has higher binding affinity to tubulin than PTX, and it can also interfere cell mitosis in S phase in addition to G2/M phase of the cell

11 cycle [63, 91]. Further in vivo evidence demonstrated that DTX has a longer retention time in tumor cells than PTX due to greater uptake, slower efflux and longer terminal elimination half- life in tumor tissue [92, 93]. The major drawbacks of taxane drugs including PTX and DTX are low solubility and drug resistance. For solubility issues, by structural modification, DTX has already shown improved water solubility as several times higher (5.0-6.0 mg/L) than PTX, although it is still considered insoluble [94]. For HRPC treatment, patients often develop resistance to DTX, resulting in therapy failure [95].

Superior to PTX and DTX, CTX is a new tubulin-binding taxane drug but with much less affinity to Pgp due to the presence of extra methyl groups at C7 and C10 positions. The poor affinity to Pgp significantly reduces the risk to develop drug resistance. It has demonstrated greater efficacy in preclinical, phase I, II and III clinical trials in DTX-resistant tumors [66, 96,

97]. Furthermore, the extra methyl groups in CTX also enhance their capability to cross BBB where the clinical significance needs to be further justified [96].

1.6 PK and dose regimen of currently approved taxane formulations

Taxol®

After IV injection of Taxol®, the elimination of PTX follows a rapid and biphasic feature and average distribution and elimination half-life is 0.34 and 5.8 h respectively. Facilitated by CYP

(CYP2C8 and 3A4) metabolism and biliary excretion, PTX is rapidly eliminated from circulation.

The steady-state volume of distribution (Vdss) is large for PTX due to its high plasma protein binding. Taxol® has a nonlinear PK in which reported cause is the formulation vehicle CrEL.

When administering at high dose, the large amount of CrEL in the formulation may alter the hepatic transport function and biliary excretion rates of PTX. Another explanation is that PTX is incorporated in CrEL micelles and lead to altered erythrocyte accumulation [22, 23, 87].

In clinic, Taxol® is administered IV over a period of 3-24 h after dilution to a concentration of 1 mg/mL. The most commonly prescribed dosage regimen is 135 mg/m2 or 175 mg/m2 q3w.

More specifically, to treat OC, premedication (e.g. dexamethasone, diphenhydramine, H2 blockers) are needed to prevent hypersensitivity reactions. For previously untreated patients, dose regimen is 175 mg/m² IV over 3 hours (h) q3w (follow with cisplatin), or 135 mg/m² IV over 24 h q3w (follow with cisplatin) while previously treated patients will receive various regimens in the range of 135-175 mg/m² IV over 3 h q3w. For node positive BC adjuvant chemotherapy, a 175 mg/m² IV over 3 h q3w for 4 times is used in a doxorubicin-containing regimen while MBC uses the same dose. Dose regimen for NSCLC is 135 mg/m² IV over 24 h q3w (follow with cisplatin) while treating acquired immune deficiency syndrome (AIDS)- related Kaposi's Sarcoma (2nd-line therapy) uses much lower dose (e.g. 135 mg/m² IV over 3 h q3w or 100 mg/m² IV over 3 h q2w. In addition, an off-label investigational usage of Taxol®

13 to treat pancreatic cancer is 125 mg/m² IV in combination with gemcitabine [98].

Abraxane®

The PK of PTX plasma concentrations after IV administration of Abraxane® showed a biphasic profile that the initial rapid decline indicates drug distribution from the circulation to the peripheral compartment followed by a slower drug elimination phase. The PK data of 260 mg/m2 of Abraxane® over 30 min infusion was compared to the PK of 175 mg/m2 Taxol® IV over 3 h infusion. Clearance and Vd was 43% and 53% larger for Abraxane® than PTX injection respectively while elimination half-lives were almost the same [56-58]. For MBC, 260 mg/m2

IV over 30 min q3w while for NSCLC recommended dose regimen of Abraxane® is 100 mg/m2

IV over 30 min on days 1, 8, and 15 along with the administration of carboplatin (immediately after Abraxane®) on day 1 of each 21-day cycle. Furthermore, in treating pancreatic adenocarcinoma, dose regimen follows 125 mg/m2 IV over 30-40 min on days 1, 8 and 15 of each 28-day cycle; as combination therapy regimen, gemcitabine needs to be administered on days 1, 8 and 15 of each 28-day cycle immediately after Abraxane® [99].

Genexol-PM®

PK parameters of Genexol-PM® was reported upon 13 patients receiving 135-390 mg/m2 dose in a phase I clinical trial. Maximum concentration (Cmax) was between 1.5 and 3.32 h of the infusion and PK profile indicated a biphasic model. The area under the curve at infinity

(AUCinf) and Cmax of Genexol-PM® appears to be lower than equivalent doses of Taxol®. The terminal plasma elimination half-life is between 11 to 12.7 h. In addition, the researchers compared those patients who did or did not experienced grade 3-4 neuropathy or myalgia in each group and reported a correlation between AUCinf and Cmax versus the neuromuscular

14 toxicities; however, the AUCinf and Cmax parameters between two groups of patients are not statistically significant different. The phase I trial found major dose limiting toxicity were neuromuscular toxicity and myelosuppression, and identified MTD of Genexol-PM® as 390 mg/m2, relatively higher than Abraxane® at 300 mg/m2. The following phase II trial used a dose regimen of 230 mg/m2 IV infusion for 3 h along with cisplatin 60 mg/m2 on day 1 through a 3-week cycle for treating advanced NSCLC patients. If no pre-specified toxicity observed after first treatment cycle, then doses for following cycles were escalated to 300 mg/m2. A recent report on another phase II trial on NSCLC appears to use Genexol-PM® at 230 mg/m2 on day 1 combined with gemcitabine 1,000 mg/m2 on day 1 and day 8 in a 3-week cycle regimen [40, 100-102].

Lipusu®

Following IV administration, PK profile of Lipusu® in both rats and dogs indicated a biphasic model that a rapid distribution phase in the first hour was observed followed by a slow elimination phase. The rapid distribution phase is thought due to clearance of liposomal nanoparticles by reticuloendothelial system (RES) out of circulation. Biodistribution results also confirmed that Lipusu® was mainly located at RES organs including liver and spleen following IV bolus injection. For example, spleen has highest drug concentration followed by liver and PTX concentration decreased slowly in liver. In addition, very limited amount of PTX penetrated BBB to the brain and drug concentration fell below the detection limit at 4 h post injection.

The dose regimen for Lipusu® is 175 mg/m2 (q3d or q2w) IV to patients with advanced

NSCLC, GC, BC, and squamous cell carcinoma of the head and neck cancer (SCCHNC). A

15 premedication is recommended including IV administration of methylprednisolone 40 mg at least 30 min prior to Lipusu®, or oral administration of dexamethasone 2.25-3 mg 2 h before

Lipusu® [28, 59].

Taxotere®

Usually DTX is administered via IV and PK of DTX follows a three-compartment model in which a rapid initial decline phase (average half-life: 4.5 min) representing the distribution of

DTX from the central to peripheral compartments; second phase (half-life of 38.3 minutes) and a relatively slow decline phase (average half-life: 12.2 h) representing efflux of DTX from the peripheral compartments. Excretion of DTX and its metabolites are mainly through renal and biliary following oxidation by metabolism enzyme CYP3A4. Approximately 94% of DTX bound to α1-acid glycoprotein, albumin or lipoproteins in vitro; however, polysorbate 80 as excipients in clinical formulation compete with serum proteins and thus changing PK of DTX by modifying protein binding profile. Further studies identified that unbound DTX is associated with severe hematological toxicity. For oral administration, bioavailability of DTX is very poor mainly because of the existence of Pgp proteins in the apical epithelial of the gastrointestinal tract as an efflux pump. Also due to high levels of Pgp in the BBB, the penetration of DTX into the brain tissue is very limited [91-93, 103-107].

The Taxotere® injectable formulation is a 20 mg/mL DTX solution using polysorbate

80/dehydrated ethanol (50/50, v/v). It requires dilution to final concentration of 0.3-0.74 mg/mL in either saline or 5% dextrose solution. Premedication with oral corticosteroids is also needed for all patients. Taxotere® is usually administered at a dose of 60 to 100 mg/m2 q3w

IV over 1 h. More specifically, a 60 mg/m2 to 100 mg/m2 single agent regimen is used for

16 locally advanced or MBC or 75 mg/m2 administered 1 h after doxorubicin 50 mg/m2 and cyclophosphamide 500 mg/m2 q3w for 6 cycles as an adjuvant therapy for BC. For the rest indications DTX is normally administered at 75 mg/m2. For instance, treating chemotherapy- naive NSCLC, it will be followed by cisplatin 75 mg/m2, while in case of platinum therapy failure

DTX is used as a single agent. In HRPC, medication of 5 mg prednisone twice a day continuously is also required. A dose regimen of 75 mg/m2 DTX followed by cisplatin 75 mg/m2 (both on day 1 only) followed by fluorouracil 750 mg/m2 per day as a 24 h IV (days 1 - 5), starting at the end of cisplatin infusion is used for GC. Furthermore, two regimens are recommended for

SCCHNC: first is the same as GC dose regimen for 4 cycles; second is DTX 75 mg/m2 followed by cisplatin 100 mg/m2 IV (day 1), followed by fluorouracil 1000 mg/m2 per day as a 24 h IV

(days 1 - 4) for 3 cycles [108].

Jevtana®

The PK profile of CTX also exhibit a triphasic model in which half-life of α, β, and γ phase are 4 min, 2 h, and 95 h respectively and a drug concentration peaked at 1 h following IV infusion. 89 - 92% of CTX bound to serum albumin and lipoproteins and free drug is heavily metabolized by CYP3A4/5 enzymes in the liver and only partially (10 - 20%) by CYP2C8. CTX and metabolites are mainly excreted into feces and urine [95, 109, 110]. For treating HRPC that previously treated with DTX-containing regimen, JEVTANA® is administered at 25 mg/m2 as a 1-h IV infusion q3w in combination of oral prednisone 10 mg daily throughout treatment [111].

1.7 Limitations of taxane drugs Taxol®

General issue with taxane drugs is the low solubility. Excipients are needed to deliver the drug in clinical formulations. Taxol®, the first approved PTX formulation, is characterized by very low drug load (ca. 1% wt. of PTX), and the rest 99% of formulation are excipients,

CrEL/ethanol. It is extensively reported that CrEL causes severe allergic, hypersensitivity, anaphylactic reactions, and neurotoxicity such as ganglionopathy, axonopathy and demyelination in both animals and humans, which become dose limiting toxicity for clinical intervention. A standard premedication of corticosteroid (e.g., dexamethasone) and H2 blockers before Taxol® infusion is required to reduce hypersensitivity reactions although 40% of patients still develop minor hypersensitivity reactions. In addition, as discussed in section

1.4, CrEL is also reported to cause nonlinear, unpredictable and undesirable PK profile of PTX

[11, 19, 58, 98].

Abraxane®

The clinical demand for alternative formulations resulted in the approval of Abraxane®,

130 nm human serum albumin (HSA) nanoparticles carrying ca. 10% wt. PTX. Abraxane® became popular in current cancer therapy. It was proposed that albumin-bound PTX can transcytose endothelial cells into the interstitial space through binding to gp60, a 60 kDa glycoprotein receptor [56]. More recent report indicated that preferential intratumoral distribution of albumin-bound PTX is due to the albumin affinity to SPARC (secreted protein, acidic and rich in cysteine), a matrix-associated protein overexpressed in approximately 50-

60% of BC [112]. Pre- and clinical studies have demonstrated that the albumin-bound form of

PTX has many advantages over Taxol® including: 1) Avoid CrEL related toxicity; 2) Shorter 18 period of infusion time (30 min) without the need of special IV tubing; 3) Approximately 74% increase of response rates in MBC patients; 4) Significantly higher MTD in human; 5) Lower incidence of grade 4 neutropenia [56-58]. Abraxane® was approved by the FDA in 2005 for treating BC and 2012 for NSCLC.

However, in the phase III clinical trial CA-031 in advanced NSCLC patients, weekly albumin- bound PTX plus carboplatin demonstrated only 10% improvement of PFS (p = 0.21) and overall survival (p = 0.27) compared to Taxol® plus carboplatin which were not statistically significant even though albumin-bound PTX had decreased toxicity [113]. In addition, Abraxane® dosing at 260 mg/m2 displayed more predictable linear PK profile, nevertheless the AUC (p = 0.52) and half-life (p = 0.48) were not significantly improved in comparison with 175 mg/m2 Taxol®.

Last not least, Abraxane® treatment is also associated with peripheral neuropathies which will be discussed separately [114].

Lipusu®

No significant improvement in efficacy was observed in the clinical data as compared to

Taxol® but the toxicity associated with Taxol® including anaphylaxis, peripheral neurotoxicity, nausea and vomiting etc. was much lower or rare seen for Lipusu®. Furthermore, intrapleural administration of Lipusu® showed more mild and manageable symptoms of diarrhea, anemia, neutropenia, thrombocytopenia, hepatotoxicity and chest pain than Taxol® [28, 29, 115].

Taxotere®

Polysorbate 80, the excipient in Taxotere®, was reported to not only impact DTX PK profile but cause haemolysis and cholestasis. 25% of patients in a phase II clinical trial developed dose-limiting toxicity such as myelosuppression and impairment of cardiac, renal, and

19 neurologic function following the first IV administration of DTX at 100mg/m2 and total of 34% of the patients cannot complete the 6 courses therapy in the trial [116, 117]. To minimize toxicity without compromising therapeutic outcome, an alternative lower dose but more frequent schedule was tested. For example, a phase II clinical study of DTX used weekly dose of 25mg/kg IV administration with 12 consecutive weeks of therapy (cumulative dose of

300mg/kg), very mild side-effects (1 of the 6 patients) and enhanced clinical responses

(prolonged progression free intervals from 99 to 332days) were observed [117, 118]. Another strategy is to locally administer (e.g. intraperitoneal, IP) Taxotere® which causes longer retention of the drug in the peritoneal cavity in contrast to IV route. Therefore, the same 100 mg/m2 dose of DTX administered IP to eight patients showed no dose-limiting toxicities [119].

DTX-induced neuropathies will be discussed separately in detail.

Jevtana®

The major dose-limiting toxicity is fatal febrile neutropenia, which requires immediate

Granulocyte-colony stimulating factor (G-CSF, Filgrastim®, Pegfilgrastim®) treatment. It is critical to maintain neutrophil count >1500/mm otherwise Jevtana® dose needs to be adjusted. Special attention should be paid to patients ≥ 65 years of age because they have higher incidence of febrile neutropenia and other toxicities. In addition, severe hypersensitivity reactions such as hypotension, bronchospasm, and generalized rash/erythema were also observed which requires immediate discontinue of infusion. Severe hypersensitivity reactions have been reported and pretreatment with H2 blocker and corticosteroids is needed [111].

Peripheral neuropathy, a serious problem for taxanes

Sensory neuropathy is a very common, hard to quantify, but serious toxicity occurred during cancer treatment with taxane-containing regimens including Taxol®, Abraxane® and

Taxotere®. Distal symmetrical paresthesias are the typical sign of taxane-related neuropathy which can be partially and gradually reversed taking months to years if stop the taxane treatment. When pre-conditions (e.g. long-standing diabetes mellitus) exist or co-treatment with other known neurotoxic drugs, patients can experience other adverse symptoms including autonomic and motor changes. Distal fingertip paresthesia is a frequent event while neurogenic damage from discogenic root compression is rare, different from cisplatin-induced neuropathies [120, 121]. When receiving taxanes, other tubulin poisons, and platinum drugs, most susceptible neurons are distal ones that transmit pain and touch sensations.

Although mechanism is still not fully understood, the taxane-induced neuropathy may ascribe to following reasons: 1) Taxanes promote microtubule polymerization in the soma of sensory neurons and nerve axons thus interfering with axonal transport process [122-124]; 2)

Microglial activation within the spinal cord and macrophage activation in the dorsal root ganglia and peripheral nerve may initiate taxane-induced peripheral neuropathy by signal transduction pathways [125]; 3) Taxane-damaged cells include axons, neurons and Schwann cells.

Kudlowitz and Muggia [120] provided comprehensive and insightful review on defining risk of neuropathy in cancer patients from large randomized clinical trials. The authors compared different treatment schedules for the relative neurotoxic potential such as q3w vs. q1w for the same taxane Taxol® in 3 type of cancers; Taxol® vs. Abraxane® in BC and NSCLC;

21 and two different taxanes combined with platinum therapy in ovarian and NSCLC. First, when compared different schedules in the Eastern Cooperative Oncology Group (ECOP) trial, Taxol®

80 mg/m2 weekly seems to have better 5 year survival vs. 175 mg/m2 Taxol® q3w schedules

(89.7% vs. 86.5%; P=0.01). More incidence of grade 2 – 4 sensory neuropathy were observed in weekly regimen; however, grades 3 and 4 sensory neuropathy in weekly of Taxol® is similar to DTX [126]. The data on Abraxane® are inconsistent and difficult to interpret. For example, clinical trials revealed an increased peripheral neuropathy by 260 mg/m2 Abraxane® as compared to 175 mg/m2 Taxol® and a case of superficial keratopathy uniquely associated with

Abraxane® [114]. A more recent randomized phase III clinical trial of weekly 90 mg/m2 Taxol® compared to weekly 150 mg/m2 Abraxane® in combination with Bevacizumab as the first-line therapy for locally recurrent MBC, indicates that Abraxane® offers no benefits to PFS compared to Taxol®, but inducing greater hematologic toxicity and sensory neuropathy. The results were presented by Rugo at the American Society of Clinical Oncology (ASCO) 2012 meeting and is awaiting full publication [127]. Since formulation requires HSA, the cost of

Abraxane® is considerably high while the improvement in efficacy remains marginal.

Considering the lower cost, equivalent if not better efficacy, pre-manageable hypersensitivity and lower toxicity of peripheral neuropathy, oncologists recommend to give Taxol® rather than

Abraxane® to MBC patients as part of first line therapy regimen. On the contrary, Abraxane® was studied in NSCLC trials where carboplatin was paired with Abraxane® 100 mg/m2 q1w vs.

Taxol® 200 mg/m2 q3w. Results showed similar overall survival and PFS but Abraxane® regimen had less severe neuropathy and hematologic toxicity events than Taxol® [128]. These results are controversial to the Rugo and colleagues’ MBC trial. However, there clearly remains

22 a clinical demand for a formulation of PTX with improved therapeutic outcome and less severe sensory neuropathy and other neurotoxicities.

The Gynecologic Oncology Group (GOG) trials compared combination of Taxol® with carboplatin or cisplatin to treat OC. For example, GOG104 trial found longer Taxol® infusion such as 96 h had less neuropathy while unchanged efficacy compared to 24 h infusion time when combining with cisplatin [129]. Following GOG158 trial demonstrated noninferiority of carboplatin + Taxol® over cisplatin + Taxol® regarding efficacy and neuropathy although carboplatin has reported less neurotoxicity than cisplatin [130]. Moreover, a Phase III trial by

SCOTROC group compared carboplatin + Taxol® vs. carboplatin + Taxotere® and identified that patients under Taxol®-containing q3w regimen had significantly higher rates of grades 2-4 neurotoxicity than Taxotere® study arm [131].

In addition, the sensory neuropathy is also reported in Taxotere®, exemplified in a phase

II clinical trial that 20 of the 41 patients dosed with cumulative 150-500 mg/m2 Taxotere® developed mild neuropathy, while 15 patients exhibited moderate or severe predominant sensory neuropathy after the cumulative dose exceeding 600 mg/m2 [132, 133]. The DTX induced neurotoxic effects also include paraesthesias and numbness in hands and feet with early sign of lacking tendon reflexes and vibratory perception; motor weakness (found in half of the patients) [134]; and predominant proximal muscle weakness [121].

Hematological toxicity for taxanes

Clinical investigations identified PTX-induced leukopenia and neutropenia are dose and schedule dependent; for example, longer infusion (24 h) had higher rates of hematologic toxicity than 3 h infusions. Anaemia induced by PTX is thought to be 1) the direct toxicity to

23 bone marrow, 2) the renal impairment with a secondary deficiency to generate erythropoietin, or 3) stress-induced erythrocyte death (eryptosis). In a phase II clinical study, the most serious side effects of hematologic toxicity induced by Taxol® include grade IV granulocytopenia in 20 cases (48%), grade III-IV anaemia in 13 cases (31%) and grade III/IV thrombocytopenia in 14 cases (33%) [135].

MDR, another problem for Taxanes

Other than low water solubility, side effects associated with excipients, and peripheral neuropathy, another major limitation generally for taxane drugs is the development of MDR.

MDR of Taxanes has been attributed mainly to: 1) increased drug efflux by Pgp protein encoded by ABCB1 gene; 2) cells containing mutations in tubulin subunits that taxanes cannot bind and function; 3) intrinsic alterations in the levels or activity of proteins that regulates cell cycle and apoptosis (Fig. 1.2). More specifically, in many ovarian and breast tumor cells, ATP- dependent Pgp located on the plasma membrane can efflux taxanes out of the cell, leading to decreased intracellular drug concentration. Secondly, from the parent OC cell line 1A9,

Giannakakou et al. developed two sub-lines 1A9-PTX10 and 1A9-PTX22 carrying mutations of tubulin that resistant to PTX. The authors measured the polymerized fraction of tubulin in the two resistant cell lines along with the parent 1A9 cell line and found that tubulin in the PTX resistant cell lines did not polymerize under same conditions allowing the polymerization of tubulin in the parent line. Sequence analysis identified that mutations of Phe270Val and

Ala346Thr respectively on tubulin are responsible for the resistance [136]. Other mechanism of resistance involves the regulation of tumor suppressor protein p53 [137, 138], the pro- apoptotic protein B-cell lymphoma 2 (BCL2) [139], or the enzymes that metabolize the taxanes

24 such as the cytochrome P450 family CYP1B1 and CYP3A4 in liver cells [140, 141].

1.8 Taxane drug delivery using nanocarriers (nanomedicine)

Nanoformulations to deliver taxane agents have been investigated in the past few decades and several nanomedicines have been approved in clinic such as Abraxane®, Genexol-PM® and Lipusu® while more of them are reaching or undergoing Phase III clinical trials. Compared to traditional formulations such as CrEL or polysorbate 80, nanomedicine have many advantages overcoming the limitations we discussed in section 1.7. First, most nanomedicines utilize either liposomes or polymers as drug carriers that are biodegradable or biocompatible materials, thus avoiding excipient-related toxicity (e.g. hypersensitivity) [38, 39, 50, 142, 143].

Second, nanomedicine can improve the PK profiles of taxanes if the delivery vehicles are long circulating types. The classical hydrophilic PEG chains make nanomedicines less visible to the

RES system and thereby increase circulation time. We will also cover some PEG alternatives later in this section [144-146]. Third, the vasculature of solid tumors is misarranged with increased permeability and decreased lymphatic drainage. The pore size of tumor blood vessels is about 200 nm to 1200 nm compared to the < 2nm of pore size in normal vasculature endothelium. Nanomedicines with the size range between 20 - 200nm can extravasate into tumor sites but not into normal tissues with tight endothelial junctions, which is referred to as enhanced permeability and retention (EPR) effect (a passive targeting mechanism) [147-

149]. Although still under evaluation, active targeting is proposed to further enhance the tumor accumulation and internalization of nanomedicines. The surface chemistry of nanomedicine enables covalent or non-covalent conjugation of targeting groups that actively bind to certain receptors overexpressed on tumor cells and therefore “homing” nanomedicine

25 at tumor sites (e.g. Folate receptor) [150-155]. Last but not least, nanomedicine allows the co- delivery of multiple anticancer drugs simultaneously in order to achieve synergistic anticancer effects [50, 156, 157]. In this section, we provide an extensive overview of various nanomedicine of taxanes such as liposomes, taxane-polymer conjugates, dendrimers and polymeric micelles that are mostly under clinical investigation (Fig. 1.3 a, b, c).

1.8.1 Lipsomal formulation of taxanes in clinical trials

Liposomes, first discovered by Bangham and colleagues, became popular vehicles to deliver numerous drugs [158]. They are lipid bilayer vesicles with interior aqueous compartment. The bilayer is normally made of various amphiphilic phospholipids and capable of carrying hydrophobic drugs while the aqueous cavity can house hydrophilic drugs. Several liposome drugs are already reached clinic while more liposomal formulations are in clinical investigation. The size and surface charge of liposomes can be tailored by extruding liposomes through designated pore-size membranes and by adjusting recipe of phospholipids respectively [143]. In addition, PEGylated liposomes showed extended circulation time due to evading RES system. Targeting groups or other functions can be realized at the terminal of PEG.

Previously we have discussed Lipusu® as the first approved liposomal PTX commercialized in

China and it is undergoing clinical trials in US (clinicaltrials.gov). Here we will focus on the taxane liposomal formulations as single agent currently in clinical trials (Table 1.3) [159]

(Pubmed and clinicaltrials.gov.).

There are two PTX-containing liposomal formulations reached Phase II clinical trials, LEP-

ETU (developer: NeoPharm) and EndoTAG®-1 (developer: Medigene). The former LEP-ETU was prepared by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-

26 glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cardiolipin, alpha-tocopheryl acid succinate (α-TAS) and cholesterol. The average size of produced liposomes is approximately 150 nm [30, 160]. The MTD, PK, dose regimen and toxicity as well as efficacy of LEP-ETU were determined in a Phase I trial on 25 patients with advanced solid tumors (breast, colon, and ovarian). The patients were treated at 135 - 375 mg/m2 dose over 90 min IV infusion q3w for 6 cycles. The 375 mg/m2 dose of LEP-ETU was associated with febrile neutropenia and peripheral neuropathy and MTD was identified as 325 mg/m2 while most common side effects observed were fatigue, nausea, anemia, hypoesthesia, and neutropenia. Antitumor efficacy was evidenced in 3 PR and 11 stable disease (SD) for the

25 patients [161, 162], enabling LEP-ETU into the phase II clinical trial to treat MBC. A multicenter, open-label trial performed in 35 India MBC patients at the dose of 275 mg/m2 q3w found: 15 patients had PR, 1 patient had complete response, and 10 patients had SD while 9 patients had progression of the disease [163]. Currently NeoPharm is planning the

Phase III trial.

The latter PTX liposome formulation is EndoTag®-1, a cationic liposome appears to target tumor vasculature in addition to deliver PTX chemotherapeutics to the tumor [164, 165].

EndoTag®-1 can induce tumor endothelial cell apoptosis and intra-tumoral thrombosis which may provide tremendous advantages when combined with PTX chemotherapy [164-169].

EndoTAG®-1 was prepared by encapsulating PTX in cationic lipids DOPC/N-[1-(2,3-

Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) to obtain 180 – 200 nm liposomes and the drug loading can reach 5% (molar ratio, 45/50/5). It was evaluated in phase

II clinical trials to treat various solid tumors as well as a combination therapy with gemcitabine

27 to treat pancreatic cancer [169, 170]. A recent phase II trial in advanced triple-negative breast cancer (TNBC) patients indicated a benefit PFS rate at week 16 was 59.1%] when combining

EndoTAG®-1 (22 mg/m2) and Taxol® (70 mg/m2) weekly over single drug treatment (PFS rate

= 34.2% on EndoTAG®-1 or 48.0% on Taxol®) [171]. The phase I/II study in non-resectable therapy-refractory SCCHNC patients also demonstrated the safety of EndoTAG®-1 but efficacy needs to be further evaluated [169]. Another Phase II trial of EndoTAG®-1 combined with gemcitabine showed 30% improvement in median survival as compared to gemcitabine alone

[170]. A case study reported that EndoTAG®-1 prevented tumor progression for 9 months in a patient with progressive hepatocellular carcinoma (HCC) [172]. There are 4 completed studies in clinicaltrial.gov up to date when using EndoTAG®-1 as a keyword. In the ASCO 2013 Annual

Meeting, Medigene reported positive results from an Investigator Initiated Trial (IIT) with neoadjuvant EndoTAG®-1 in high-risk HER2-negative BC. SynCore Biotechnology Co. received the Medigene license in 2013 and committed to continue the planned Phase III clinical trial with EndoTAG®-1 for TNBC treatment (http://www.medigene.com/products- pipeline/development-projects/endotag-1).

In addition to PTX liposomal formulations, DTX based liposomes are also under evaluation.

For example, NeoPharm also developed LE-DT which is the DTX liposome formulation that utilizes similar lipid components DOPC, cardiolipin, α-TAS and cholesterol to produce 100 nm liposomes. In Phase I study, LE-DT showed impressive efficacy results and safety profile in treating advanced solid tumors. LE-DT was well tolerated and expected toxicities include neutropenia, anemia, and fatigue, but no peripheral neuropathy or edema. Clinical benefits such as PR and SD were detected in 41% of the patients and the recommended dose of LE-DT

28 for phase II trial is 85 mg/m2 or 110 mg/m2 in combination with G-CSF in a q3w IV regimen

[173]. In 2010, NeoPharm started to enroll patients in the planned Phase II clinical trial for LE-

DT in the treatment of locally advanced or metastatic pancreatic cancer patients based on its success in Phase I trial. Currently only one report of Phase I data can be found from Pubmed search.

Another DTX liposome named ATI-1123 is under development by Azaya therapeutics, a

Texas-based biotechnology company. ATI-1123 utilizes protein stabilizing liposome technology to incorporate DTX which is believed to be a safer and more effective strategy than Taxotere®.

The ATI-1123 liposomes were prepared from phospholipids, cholesterol, HSA, and sucrose with a size around 90 nm. Azaya recently completed the Phase I study of ATI-1123 in 29 patients suffering from advanced solid malignancies. The trial identified a linear PK and 90 mg/m2 as the MTD dose where grade 3 stomatitis and febrile neutropenia occurred. One lung cancer patient had PR and 75% of all rest patients showed SD [174]. Currently Azaya planned the Phase II clinical studies for ATI-1123 to treat NSCLC, GC, PC, and soft-tissue sacoma at multiple centers (http://www.azayatherapeutics.com/pipeline/ati-1123).

1.8.2 Conjugates and dendrimers for taxane delivery

To overcome limitations of taxanes (e.g. solubility issues), many researchers investigated the conjugation of taxanes to biodegradable polymers (water-soluble or amphiphilic block copolymers) or fatty acid through ester or amide bonds, or stimuli-responsive linkers. Most common conjugation sites are the C2’ and/or C7 hydroxyl groups while both hydroxyl groups are critical for taxane activity [175]. Possibly because of less steric hindrance, the C2’ hydroxyl group is more reactive than C7 hydroxyl group and thus most often used for taxane-conjugates.

These taxane-conjugates can not only enhance taxanes’ solubility, but extend circulation time through evading the RES, improve PK profile, and passively target tumor tissues by EPR effect.

Taxane-conjugates revealed relatively higher drug content and stability as well as controlled drug release over polymeric micelles or liposomes. Here we provide overview of taxane- conjugates currently under clinical evaluation including opaxio, taxoprexin, GRN1005,

PNU166945, NKTR-105 and DEPTM-DTX dendrimer conjugates (Table 1.4).

Opaxio (Paclitaxel poliglumex) developed by Cell Therapeutics, a US firm based at Seattle, is the α-poly-L-glutamic acid conjugated to PTX via C2’ hydroxyl group. It was shown to increase PTX solubility, enhance drug exposure to tissues including tumors, and overcome Pgp induced MDR [176, 177]. The clinical studies revealed Opaxio’s clinical efficacy without causing alopecia, which subsequently results in the phase II and III trials for treating OC and

NSCLC. A phase II trial [178] in patients with persistent or recurrent ovarian, fallopian, or primary peritoneal cancers, Opaxio at dose of 175 mg/m2 resulted in 16% PR, a 2.8 months

PFS and an 15.4 months OS, while neutropenia and peripheral neuropathy were major toxicities. However, the Phase III study for NSCLC has not demonstrated significant improvement in efficacy or safety profile [25].

Another PTX conjugate is Taxoprexin®, a natural fatty acid docosahexaenoic acid (DHA) conjugated through an ester bond to PTX using the C2’ hydroxyl group. Taxoprexin formulation still contains small amount of CrEL (equivalent to 20% of CrEL in Taxol®). A Phase I clinical trial in 23 cancer patients tested 2 h IV infusion q1w at dose between 200 - 1100 mg/m2) and recommended 1100 mg/m2, approximately 4.6 times of the Taxol® dose, for Phase II clinical studies. The clinical studies identified grade 3 hyperbilirubinemia as the dose limiting toxicity

30 and grade 1 sensory neuropathy as toxicity at 1100 mg/m2. It was then tested in a Phase II trial as the first-line treatment in patients with stage IIIB or IV NSCLC. The dose was adjusted to 900 mg/m2 due to toxicities observed in the first 13 patients. Efficacy results revealed that

Taxoprexin has moderate activity in advanced NSCLC patients that PR and SD was only 40.1%

(18 patients) while the dose limiting toxicity was myelosuppression [179]. Another Phase II study of Taxoprexin in previously untreated locally advanced or metastatic GC or esophageal adenocarcinoma also indicated a modest activity when administer 1100 mg/m2 IV for 2 h q3w, however, 93% of patients were associated with grade 3-4 neutropenia and 17% of patients febrile with neutropenia. The hematological toxicity was comparable to Taxol® and Taxotere®

[180]. In a Phase II study in malignant melanoma patients, treatment of Taxoprexin induced grade 3-4 toxicities of neutropenia (10%) and musculoskeletal pain (10%) [181]. A recent

Phase III study found that 900 mg/m2 IV of Taxoprexin was not superior to dacarbazine 1000 mg/m2 IV both at q3w regimen to treat metastatic melanoma [182].

GRN1005 (ANG1005, Angiochem Inc.) is a PTX-peptide conjugate that three PTX molecules conjugated via the C2’ hydroxyl group to one molecule of a novel Angiopep-2 peptide, a 19 amino-acid peptide targeting low-density lipoprotein receptor-related protein 1 (LRP1) receptor. LRP1 targeting will facilitate PTX to cross BBB to treat brain disease such as brain tumors. Preclinical studies found GRN1005 exhibited improved antitumor efficacy and survival benefit in several cancer mouse models [183]. In a Phase I clinical studies 56 patients with solid tumors (41 with brain metastases) were treated with GRN1005 and under MTD dose of

650 mg/m2 of GRN1005 demonstrated promising efficacy with no detection of anti-GRN1005 antibodies. 11% of previous taxane-treated patients had SD for at least 4 months and 25%

31 patients experienced overall PR (BC, n = 2; NSCLC, n = 2; and OC, n = 1) and shrinking of brain tumors (17 – 50%) although major toxicity is myelosuppression [32]. In addition, GRN1005 achieved therapeutic activity in recurrently gliomas and showed similar toxicity to Taxol® in a

Phase I trial, and currently entered phase II clinical trials [184].

Furthermore, PNU166945 is a HPMA conjugated through an amino acid chain at the 2' position of PTX. Meerum Terwogt et al. [185] reported that dose limiting toxicity not observed at dose up to 196 mg/m2. However, the clinical trial was discontinued due to severe neurotoxicity [185]. A PEG-DTX conjugate NKTR-105 is under developing by Nektar Thera- peutics and has entered Phase I study on patients with solid tumors [94]. Currently no report can found in Pubmed or Nektar website about NKTR-105 clinical data.

A unique type of taxane-conjugation uses dendrimers instead of linear polymers.

Dendrimers are highly-branched and well-defined macromolecules with a center molecule as a starting point followed by repeated adding branching lysine units layer by layer (or

“generation-by-generation”) to procure the desired dendrimer structures. Currently there are two dendrimer-taxane products reached Phase I stage, both are DTX conjugates. One is reported to be developed and under the Phase I trial by Sylvania Platinum Ltd; however, no clinical data is currently reported for this study. Another dendrimer-DTX product DEPTM-DTX entered the Phase I clinical trial in 2014 and under development by StarPharma, an Australian company. Preclinical studies of DEPTM-DTX showed many advantages over Taxotere® including: 1) enhanced solubility therefore polysorbate 80 are not required in the formulation which reduces risk of anaphylactic reactions and removes premedication with corticosteroids;

2) extended circulation time and plasma half-life is about 40-60 h; 3) eliminated neutropenia

32 toxicity which is dose limiting toxicity with Taxotere®; 4) improved efficacy and preferential distribution to tumor tissue and lower toxicity compared to Taxotere® alone.

(http://www.starpharma.com/drug_delivery/dep_docetaxel).

1.8.3 Taxane delivery using polymeric micelles

The ideal taxane delivery system is to achieve superior efficacy while minimizing toxicity and overcoming taxane limitations that we discussed in section 1.7. To solubilize taxanes and avoid CrEL or polysorbate 80 usage is one of the goals. Furthermore, to modify taxane PK profile and biodistribution thereby drugs can be preferentially accumulated at tumor sites is highly desirable. One of such delivery system is polymeric micelles, a colloidal dispersion formed spontaneously by amphiphilic block copolymers. The “amphiphilic” is defined that these polymers contain both hydrophobic and hydrophilic blocks. Thus in aqueous solutions above critical micelle concentration (CMC) they are able to form core-shell architectured nanoparticles (micelles) with 10 to 100 nm size range, capable of incorporating insoluble drugs.

The Hydrophobic blocks become particle cores and loading space to accommodate various poorly soluble drugs. The hydrophilic blocks constitute brush-shaped shells that stabilize the nanoparticles in aqueous solution. In this regard, PEG is most common protecting shell used in various nanoparticles in nanomedicines due to its high solubility, low toxicity and non- immunogenicity. PEG is believed to prevent particle opsonization and allow them evading from the RES to increase nanoparticle half-life, which is often referred to “stealth” property.

Currently several polymer micelle systems are approved or under clinical evaluation including

NK105 (micelle based on PEG-polyaspartate copolymers modified with 4-phenyl-1-butanol), and Genexol (based on monomethoxy-PEG-b-poly(D,L-lactide, mPEG-PLA) block copolymer;

33 approved in South Korea), and others (Table 1.5).

The first polymeric micelle formulation of PTX is Genexol-PM®/Cynviloq®, developed by

Samyang pharmaceuticals and approved in Korea and several other countries (section 2). It is also under Phase II/III clinical investigation in US carried out by Sorrento therapeutics for treating MBC, NSCLC, PC, Bladder cancer, and OC. The polymer used in Genexol-PM® is mPEG-

PLA, which PEG is the hydrophilic shell and PLA is the hydrophobic core. Genexol-PM® has a particle size about 24 nm measured by dynamic light scattering (DLS) with narrow size distribution (polydispersity index, PDI = 0.08). The surface charge of Genexol polymeric micelles is almost neutral (Zeta-potential = -8.1 mV). In vitro release experiment of PTX from the Genexol-PM® using phosphate buffered saline (PBS) and 37 oC sink conditions showed a first-order controlled release behavior that 65% of PTX was released at 24 h while 95% at 48 h [43]. The Phase I clinical trial identified MTD of Genexol-PM® as 390 mg/m2 while at this dose, patients developed grade 4 neutropenia or grade 3 peripheral neuropathy. Acute hypersensitivity reactions were not observed during this study and common toxicities also included myalgia [100]. The Phase II trials in US were performed to treat NSCLC [40], MBC

[102], and advanced GC [186] where Genexol-PM® revealed significant efficacy in all studies.

For example, 69 patients with advanced NSCLC received Genexol-PM® 230 – 300 mg/m2 and cisplatin 60 mg/m2 on day 1 of a q3w as the first-line therapy. Overall response rate (ORR) was

37.7% and the median survival time was 21.7 months. The major toxicities included grade 3/4 neutropenia (29.0% and 17.4%, respectively), grade 3 peripheral neuropathy (13.0%) and grade 3 arthralgia (7.3%) while 4 patients (5.8%) developed grade 3/4 hypersensitivity reactions. The CrEL-free, polymeric micelle formulation has higher MTD thus allowing 2-3

34 times higher dose of PTX administered compared to Taxol® without significant increased toxicity. Another Phase II trial in advanced NSCLC patients was reported recently where 43 patients received Genexol-PM® at 230 mg/m2 on day 1 and gemcitabine 1,000 mg/m2 on day

1 and day 8 of q3w regimen for total 6 cycles. The ORR was 46.5% while median OS was 14.8 months. The most common toxicities included anemia (n = 29, 67%), asthenia (n = 17, 40%), myalgia (n = 16, 37%), peripheral neuropathy (n = 15, 35 %), diarrhea (n = 12, 30%), grade 3/4 neutropenia (n = 7, 16%) and pneumonia (n = 5, 12%) and 2 patients were deceased due to pneumonia and dyspnea. Now Genexol-PM® entered a phase III trial for treating MBC [40].

Another polymeric micelle formulation of PTX is NK105, in which PTX is entrapped in the micelle core of amphiphilic block copolymers consisting of PEG and 4-phenyl-1-butanol modified polyaspartate (hydrophobic segment) [36]. This formulation is under development by Nippon Kayaku and NanoCarrier Inc. NK105 has about 23% wt. of PTX content and around

85 nm in micelle size. It can be IV injected without causing hypersensitivity. The Phase I and

Phase II clinical trials revealed its improved efficacy and less toxicities compared to Taxol®. A randomized, open-label, multi-national phase III trial (clinicaltrials.gov Identifier:

NCT01644890) to treat metastatic or recurrent BC patients is ongoing to evaluate survival benefit [36, 37].

A patented technology XR-17 developed by the Sweden company Oasmia Pharmaceutical

AB was used as drug delivery system. Oasmia researched PTX and DTX loaded XR-17 (vitamin

A derivatives micelles) as their novel pipelines. Current product Paclical (PTX in XR-17) has completed its Phase III clinical trial against OC and is in registration/approval stage in Russia and Commonwealth of Independent States (CIS). The micelle size appears around 20-60 nm

35 while drug loading and clinical data were not reported.

The fourth example of polymeric micelles reached clinical stage is a DTX formulation named BIND-014 currently under development by BIND therapeutics. It is a targeted PEG- poly(D,L-lactide-co-glycolide) (PLGA) micelles using an RNA aptamer Accurin as targeting moiety that binds to the extracellular domain of the prostate-specific membrane antigen

(PSMA) on the surface of prostate cancer cells [187-191]. BIND-014 has been shown to have enhanced cellular uptake compared to their non-targeted counterparts in vitro and in vivo.

The micelles have a particle size about 90 nm and drug loading close to 11%. The BIND-014 was selected through a combinatorial screening optimization of formulation regarding size, targeting group density, PEG shell and PLGA core MW, surface charge, drug loading, initial release rate, as well as encapsulation process [192]. After validation for PK, tolerability, tumor accumulation and efficacy, the best candidate formulation was selected (namely BIND-014).

It was evaluated in NSCLC and mCRPC. Currently BIND therapeutics released preliminary data from a Phase II trial at the 26th the European Organization for Research and Treatment of

Cancer, the National Cancer Institute and the American Association for Cancer Research

(EORTC-NCI-AACR) meeting.

(http://bindtherapeutics.com/pdfs/posters/2014%2011%20EORTC-NCI-AACR.pdf).

The clinical results indicated that BIND-014 was well tolerated and had promising antitumor activity in 40 patients with advanced or metastatic NSCLC at a dose of 60 mg/m 2 q3w.

This dose, lower than conventional Taxotere®, also showed activity in patients with KRAS mutant tumors, which normally poorly respond to Taxotere® treatment. Five patients (13%,

N=40) achieved a PR with a median PFS of 2.7 months and 2 of 9 confirmed KRAS mutant

36 patients experienced an objective response (22%) with median PFS of 2.7 months. In addition,

BIND-014 treated patients experienced substantially reduced toxicities such as neutropenia, anemia, neuropathy, and alopecia commonly associated with Taxotere®.

Although PEG was used as a gold standard for micelle shells exemplified in most of above discussed polymeric micelles, PEG also has some limitations: 1) Nearly 25 % of patients never treated with PEG drugs (PEG used extensively in cosmetics and food additives) pre-exist anti-

PEG antibodies, which may result in accelerated blood clearance (ABC) and treatment failure after multiple dosing of PEG products [193, 194]. 2) PEG is prone to oxidative degradation

[195, 196] and may be responsible for the reported cases of PEG-mediated complement activation [197]. In contrast, a class of polymers named poly(2-oxazoline)s (POx) show potential to overcome drawbacks of PEG, meanwhile exhibit advantages encompassing biocompatibility, stealth behavior, high functionalization possibilities, and robust and versatile chemistry [198-201]. POx are prepared by the living cationic ring-opening polymerization

(LCROP) of 2-substituted-2-oxazolines firstly reported in 1960’s independently by four research groups. The polymerization is a highly living process, resulting in defined degrees of polymerization and low molar mass distribution (Ð = Mw/Mn from 1.01-1.3). Noteworthy, the application of microwave synthesizers has tremendously reduced the time of POx polymerization and made structure–property relationship studies feasible as the results of high-throughput combinatorial polymer synthesis.

The hydrophilicity of polymer, depending on the 2-substitution of the 2-oxazoline monomers, can be fine-tuned by the gradual increase of aliphatic side chain length ranging from highly hydrophilic [poly(2-methyl-2-oxazoline)s, (pMeOx)] to highly hydrophobic [e.g.

37 poly(2-nonyl-2-oxazoline) (pNOx)]. Hydrophilic pMeOx and poly(2-ethyl-2-oxazoline) (pEtOx) are reported, similar to PEG, to be non-toxic, stealth and protein repelling [198, 202].

Therefore, pMeOx or pEtOx are proposed to be new alternatives for PEG as the polymeric micelle shell or liposome surface decoration. Block copolymerization of two or more 2- oxazoline monomers can give amphiphilic block copolymers. For example, recently amphiphilic POx systems with diblock structure (pMeOx-pBuOx) or triblock structure (pMeOx-

-pBuOx-pMeOx) was investigated for oncology drug delivery. The triblock copolymers P2

(polymer code from previous report [38]) with pBuOx core achieved unprecedentedly high drug loading (e.g. ca. 48% paclitaxel loading), high stability as well as superior safety and efficacy (data not shown). As a novel high drug loading drug delivery system, POx micelles was applied to incorporate various 3rd generation taxanes developed by Dr. Ojima research group, the same group synthesized ortataxel which has advanced to the Phase II clinical trial discussed in section 1.3. These micelles showed high loading capacity to incorporate all 3rd generation taxanes (Fig. 1.4) and superior antitumor activity both in vitro and in vivo. It is hypothesized that superior efficacy is due to combining the advantages of polymeric micelle delivery system and the activity of new taxane analogues that can overcome MDR [203]. Other preclinical studies for POx micelles include combination therapy [50]. POx micelles can load a large amount of multiple drug combinations simultaneously which can achieve synergistic antitumor activity in vitro. The in vivo evaluation for 2-in-1 or 3-in-1 micelles is undergoing.

The promising preclinical data on POx micelles provide a robust basis to translate into clinical trials.

Altogether, polymeric micelles not only avoid toxic excipient and may provide higher drug

38 loading and dose, but also enable the simultaneous delivery of multiple water-insoluble cancer drugs in combination. It may increase antitumor efficacy by packaging two or three complementary drugs that act synergistically against various cancers. Recently new start-up companies begin to develop multidrug loaded taxane-containing micelle formulations (e.g.

Co-D therapeutics).

1.8.4 Other taxane products in clinical trials

Other taxane products can be categorized as nanomedicine but not belongs to any of above categories such as liposomes, polymer conjugates or taxane conjugates. For example,

OncoGel is a PTX loaded ReGel ™ system, a thermo-sensitive controlled-release delivery system. ReGel is a triblock copolymer consists of PLGA-PEG-PLGA, which can transit from a solution (2-15 oC) to a viscous biodegradable controlled-release gel at body temperature. This formulation, as a drug depot, is ideal for local target delivery of PTX to solid tumors while avoiding systemic exposure and toxicities. OncoGel can continuously release PTX to the tumor tissues for 6 weeks [204-206]. Up to date two clinical trials on Oncogel were completed: a

Phase I trial in patients with superficially-accessible advanced solid cancers and a Phase IIa trial as adjuvant therapy to radiotherapy in patients with esophageal cancer ineligible for surgery [207, 208]. OncoGel administered at doses up to 2.0 mg PTX/cm3 tumor volume was well tolerated in both clinical studies and no serious adverse events and hematological toxicities were observed, due to prolonged release of paclitaxel from OncoGel with low systemic concentrations via PK parameters. The endoscopic ultrasound (EUS) was used to guide OncoGel injection and measuring tumor response. The Phase IIa study of 11 patients indicated that 2 (18%) patients had PR, 6 (55%) patients had SD, while 2 (18%) patients had

39 progressive disease (PD) and 1 patient developed tumor invasion of the lumen thus EUS cannot measure. However, the following Phase IIb study for OncoGel did not show any improvement on the primary endpoint of overall tumor response as an adjuvant therapy to radiotherapy in patients with esophageal cancers, thus BTG plc discontinued further development of this product.

Another example is TOCOSOL™, a 100 nm oil-in-water nano-emulsion of PTX composed of vitamin E, TPGS, and poloxamer 407. TOCOSOL™ showed significant improvement in antitumor efficacy and safety profile compared to Taxol® in both B16 and HCT-15 cancer mouse models [209]. The following phase I clinical trial also demonstrated TOCOSOL™ with well tolerance and less toxicity compared to Taxol® [210]. However, the Phase III clinical trials of TOCOSOL™ in MBC patients were terminated due to similar objective response rate to

Taxol® [211].

1.9 Conclusion

Taxanes remain the major chemotherapeutics in the management of malignancies while many limitations has hindered its clinical use. Although new taxane analogues showed improved solubility and enhanced activity against MDR tumors, major issues still exists including excipients related toxicities, undesirable PK and systemic neuro- or hemato-toxicities.

In the past decades, nanomedicine shed lights in overcoming these issues and currently some nanomedicine succeed to reach clinic such as Abraxane®, Genexol-PM® and Lipusu®. More liposomal PTX are under clinical investigation, but other polymer-based taxane formulations starts to thrive and may represent future medicine for cancer treatment. Polymeric micelles, as new such delivery systems, become major category of taxane nanomedicine. They generally have higher capacity to carry taxane drugs (NK105, Genexol-PM®) than liposome, and flexible to be modified with active targeting groups that specifically direct taxanes to the tumor tissues exemplified by BIND-014. There are numerous new targeted taxane formulations undergoing preclinical research and expected to advance in the clinical evaluation soon (e.g. SER-207 by

Serina therapeutics). In addition to targeting, polymeric micelles have the ability to delivery multiple anticancer drugs simultaneously may impact greatly to future cancer treatment. We also believe the POx micelle systems holds the potential to efficiently deliver conventional and new taxanes and applicable to targeted delivery and multidrug combination therapy.

Figure 1.1 MOA of taxane drugs (exemplified with PTX). PTX (chemical structure and red color coded modeling structure is shown) binds to tubulin (grey for tubulin assembles), and forms PTX-tubulin complexes. The MOA of PTX can be explained as following: 1) a dividing cell (green color for microtubules, blue for chromosomes) coule be arrested in mitosis phase followed by apoptosis; or 2) may progress through mitosis, under the exposure to low concentration of PTX, but results in a multipolar spindle cell, which subsequently undergoing apoptosis.

Figure 1.2 Mechanism of drug resistance to taxanes: 1) PTX (green) entered the cell and may be effluxed out of the cell by Pgp efflux pump; 2) The cell contains tubulin mutations that PTX can not bind and function on; 3) Deregulation of intracellular pathway components (e.g. p53, BCL2) that inhibits apoptosis; and 4) Upregulation of drug metabolism enzymes (e.g.CYP3A4).

c b

Figure 1.3 Nanomedicine for taxane drug delivery. (a) liposomal formulation of PTX (red color for PTX, and yellow for lipids); (b) dendrimer of DTX (blue color for dendrimers, and green for DTX molecule); (c) polymeric micelle formulation of DTX (blue for hydrophilic corona and dark brown for hydrophobic core of amphiphilic polymers, and green for DTX molecules).

Figure 1.4 3rd generation of taxanes and POx micelle delivery systems. Chemical structures of 3rd-generation toxoids (Top panel). Schematic showing of formation of POx/taxoid micelles formed through self-assembly by thin film-rehydration method (bottom panel).

CHAPTER II: SYNTHESIS AND CHARACTERIZATION OF NEW 2-OXAZOLINE MONOMER AND POLY(2-OXAZOLINE)S POLYMERS 1

2.1 Summary

The previous chapter discussed potential of POx polymers as new drug delivery systems which may overcome drawbacks of PEG. POx polymers exhibit high functionalization possibilities and versatile chemistry [198-201]. Our previous research reported amphiphilic triblock copolymers P2 as a high drug loading nanocarrier; however, only one terminal secondary amine group on P2 is available for further conjugation. In this chapter, I will discuss the new chemistry of POx that also contains alkyne functional group that amenable to “click” chemistry. In addition, novel POx polymers obtained from new monomers such as aromatic ring containing POx and cationic POx will be also analyzed.

1 2-benzyl-2-oxazoline monomer and polymer data of this chapter previously appeared as part of a manuscript submitted to Polymer Advanced Technology.

2.2 Introduction

Poly(2-oxazoline)s (POx)

Polymeric micelles, a colloidal dispersion formed spontaneously by amphiphilic block copolymers, have been implemented in drug delivery field (i.e. oncology drugs). The

“amphiphilic” is defined that these polymers contain both hydrophobic and hydrophilic blocks.

Thus in aqueous solutions above CMC they are able to form core-shell architectured nanoparticles (micelles) with 10 to 100 nm size range, capable of incorporating insoluble drugs.

PEG is believed to prevent particle opsonization and allow them evading from the RES to increase particle half-life, which is often referred to “stealth” property. However, PEG has some limitations: 1). Nearly 25 % of patients never treated with PEG drugs (PEG used extensively in cosmetics and food additives) pre-exist anti-PEG antibodies, which may result in accelerated blood clearance (ABC) and treatment failure after multiple dosing of PEG products[193, 194]. 2). PEG is prone to oxidative degradation [195, 196] and may be responsible for the reported cases of PEG-mediated complement activation [197].

In contrast, POx show potential to overcome drawbacks of PEG, meanwhile exhibit advantages encompassing biocompatibility, stealth behavior, high functionalization possibilities, and robust and versatile chemistry [198-201]. The hydrophilicity of POx polymers,

52 depending on the 2-substitution of the 2-oxazoline monomers, can be fine tuned by the gradual inscrease of aliphatic side chain length ranging from highly hydrophilic [poly(2- methyl-2-oxazoline)s, (pMeOx)] to highly hydrophobic [e.g. poly(2-nonyl-2-oxazoline) (pNOx)].

Hydrophilic pMeOx and poly(2-ethyl-2-oxazoline) (pEOx) are reported, similar to PEG, to be non-toxic, stealth and protein repeling. Block copolymerization of two or more 2-oxazoline monomers can give amphiphilic block copolymers. For example, we recently investigated amphiphilic POx systems with diblock structure (pMeOx-pBuOx) or triblock structure (pMeOx-

-pBuOx-pMeOx) for oncology drug delivery. We found triblock copolymers P2 (polymer code from our previous report [38]) with pBuOx core achieved best drug loading (e.g. ca. 48% paclitaxel loading), high stability as well as superior safety and efficacy (data shown in Chapter

III-V).

The typical synthesis procedure of P2 triblock copolymers is shown in Fig. 2.1. Briefly, a cationic initiator methyl triflate (MeTf) will react with first monomer MeOx to form a cationic initiating salt, then second MeOx monomer amino- group can attack the initiating salt via nucleophilic reaction to open the first monomer ring, and subsequently the polymer chains can propagate as such until the MeOx monomers are all consumed, assuming the whole process is living without early termination under controlled condition. After first block being produced, we add in second type of monomers BuOx to polymerize as second block, and followed by another MeOx block polymerization as third block. Finally the polymer chain will be terminated by nucleophiles (e.g. piperazine). In P2 polymer we used boc-protected piperazine as terminating agent and subsequently de-protect boc group in order to release functional secondary amine group [38]. In this Chaper, the focus is the synthsis and

53 characterization of new monomer and POx polymers with new functionalities. The exploration of newly synthesized POx as versatile drug delivery platforms will also be briefly discussed.

2.3 Initiator containing clickable functionality

POx systems can be functionalized with amino, aldehyde mercapto, hydroxyl and carboxyl, alkene, and alkyne groups by selection of the initiator or terminating agent [212]. We previously reported piperazine as terminator in P2 polymer where the secondary amine group is availble for protein or other targeting group (e.g. Folate) conjugation, whereas initiation site is usually a methyl group unable to be functionalized. This section will dicuss the application of initiators containing “clickable” alkyne groups.

Initiators

To obtain a well-defined products, triflate or tosylate-based initiators can ensure rapid and quantitative initiation reactions. Other initiators are also found in the literature including alkylhalides, nosylates, lewis acids (e.g. boron trifluoride), zirconium/tris(pentafl uorophenyl)borate, and trihalogenobismuthine [198, 213, 214].

Clickable initiators

Among all the possibilities of functionalization of POx, reactions with high selectivity and effeciency are of our most interest (e.g. “click” reactions). There have already many reports on thiol-ene and thiolyne reactions, and the Huisgen's cycloaddition catalyzed by the copper

(CuAAC) reaction [215] (Fig. 2.2) where POx contains double, triple bonds, or azido groups

[212]. I therefore utilized propargyl tosylate as an initiator and polymerized diblock POx polymers with the alkyne functional group avaivable for click reaction (Huisgen’s cycloaddition) in order to efficiently and selectively attach small molecule ligands, antibody

55 targeting groups or fluorescence tracers.

Synthesis procedure and results

Under dry and inert condition 361.6mg (1.72mmol, 1eq) of Propargyl tosylate (initiator) and 10.246g (120.4mmol, 70eq) of 2-methyl-2-oxazoline (MeOx) were dissolved in 30mL dry acetonitrile at room tempreture (RT). The mixture was subjected to microwave-assisted synthesizer (150W maximum, 100°C) for 2.5 h. After cooling to RT, the monomer for the second block, 2-butyl-2-oxazoline (BuOx, 4.372g, 34.4mmol, 20eq) were added and the mixture was reacted under the same way as the first block at 100°C for 1hour. Finally the polymer propagyl-P4 (P4 was coded in ref. [38] for diblock copolymers) was terminated by an excess of Ammonia (5.16mmol, 3eq). The solvent was then removed by rotary evaporation and the residue was re-dissolved in about 100mL ethanol:chloroform (3:1, v:v). After precipitation from cold diethylether (approx. 10 times the amount of polymer solution) the product was isolated by centrifugation. The precipitation was repeated 2 more times and the polymer (propagyl-P4) was obtained as colorless powder after lyophilization from water.

Approximately 11g propargyl-P4 polymer were synthesized (73% yield, Mth=8.7kg/mol).

GPC (DMAc): Mn=9.7kg/mol (Ð = 1.34); 1H NMR (CDCl3, 298 K):TM 3.47(br, 236H, N-CH2-

CH2); 2.4−2.0 (m, 176H, CO-CH3, CO-CH2); 1.56 (br, 16H, -CH2-CH2-CH2-); 1.32 (br, 16H, -CH2-

CH3); 0.90 ppm (br, 24H, -CH3butyl). Via tensiometry method, the CMC for propargyl-P4 was identified about 8.8mg/L (1µM).

The further application of propargyl-P4 is under investigation by our collaroators. Certainly, this diblock copolymer has potential that initiation site could be clicked with targeting ligands

56 while terminating site piperazine group can be conjugated with oligonucleotides, peptides or proteins. (Fig.2.3)

2.4 Sythesis of new monomer 2-Benzyl-2-Oxazoline (BzOx) and BzOx containing polymers

Previous research indicated that polymeric micelles containing aromatic moieties in the polymer chains show better drug loading and retention compared to those without aromatic units. This could be due to π-π stacking and hydrophobic interaction between aromatic groups of polymers and drugs [216]. The aromatic moieties have also been introduced into POx amphiphiles by use of 2-phenyl-2-oxazoline (PhOx) [217, 218]. However, this monomer polymerizes extraordinarily slow, thus copolymerization of PhOx with BuOx as hydrophobic core can be expected to yield pseudo-blockcopolymers. In this part, a new monomer 2-benzyl-

2-Oxazoline (BzOx) was synthesized and copolymerized with BuOx as the central block of triblock copolymers analogue to P2.

Monomer synthesis and characterization

Benzyl nitrile 50g (426.8mmol, 1eq) and Cadmium acetate dihydrate (catalyst; 2.844g,

10.67mmol, 0.025eq) were mixed with 2-aminoethanol (31.3g, 512.2mmol, 1.2eq) and heated to 130°C for two days with stirring. When the reaction n was completed, the reaction mixture was cooled to RT and CH2Cl2 was added. The organic layer was washed 4 times with water and 1 time with brine and dried over MgSO4. Filtration and rotary evaporation of the solvent CH2Cl2 gives a crude product, which was further purified by two times distillation (Fig.

2.4 a).

24g of monomer 2-benzyl-2-Oxazoline (35% yeild) was synthesized and structure of the monomer was confirmed by IR: 1664(C=N str)(s), and NMR spectrum 1H NMR (CDCl3, 298

K):TM 7.27(m, 5H, phenyl); 4.16 (t, 2H, CH2-O); 3.7 (t, 2H, CH2-N); 3.57 (s, 2H, CH2-phenyl).

Polymer synthesis and characterization (1)

Under dry and inert condition 42.1mg (0.2mmol, 1eq) of Propargyl toluenesulfonate

(initiator) and 599mg (7mmol, 35eq) of 2-methyl-2-oxazoline (MeOx) were dissolved in 1.5mL dry acetonitrile and 1.5mL chlorobenzene at RT. The mixture was subjected to microwave- assisted synthesizer (150W maximum, 130°C) for 5 minutes. After cooling to RT, the monomer for the second block, 2-butyl-2-oxazoline (BuOx, 256.4mg, 2mmol, 10eq) and 2-benzyl-2-

Oxazoline (BzOx, 334.4mg, 2mmol, 10eq) were added and the mixture was reacted under the same way as the first block at 130°C for 5minutes. Finally, 600mg (7mmol, 35eq) of 2-methyl-

2-oxazoline (MeOx) were added as the third block under the same polymerization condition and ultimate polymer was terminated by an excess of Ammonia (0.6mmol, 3eq). The solvent was then removed by rotary evaporation and the residue was re-dissolved in about 10mL ethanol:chloroform (3:1, v:v). After precipitation from cold diethylether (approx. 10 times the amount of polymer solution) the product was isolated by centrifugation. The precipitation was repeated 2 more times and the polymer (Code: HEZ007) was obtained as colorless powder after lyophilization from water. (Fig. 2.4 b).

Approximately 1.76g HEZ007 polymer were synthesized (98% yield, Mth=8.9kg/mol). GPC

(DMAc): Mn=7.3kg/mol (Ð = 1.35); 1H NMR (CDCl3, 298 K):TMδ= 7.23(m, 40H, Phenyl); 3.47

(br, 220H, N-CH2CH2); 2.33 −1.9 (m, 137H, CO-CH3, CO-CH2); 1.55 (br, 12H, -CH2-CH2-CH2-);

1.31 (br, 12H, -CH2-CH3); 0.89ppm (br, 18H, -CH3butyl). Via tensiometry method, the CMC for

HEZ007 was identified about 5.9mg/L (0.81µM).

Polymer synthesis and characterization (2)

Under dry and inert condivtion 242.3mg (1.14mmol, 1eq) of Propargyl toluenesulfonate 59

(initiator) and 3.415mg (39.9mmol, 35eq) of 2-methyl-2-oxazoline (MeOx) were dissolved in

15mL dry acetonitrile and 15mL chlorobenzene at RT. The mixture was subjected to microwave-assisted synthesizer (150W maximum, 100°C) for 2.5 h. After cooling to RT, the monomer for the second block, 2-butyl-2-oxazoline (BuOx, 2.1835g, 17.1mmol, 15eq) and 2- benzyl-2-Oxazoline (BzOx, 920.4mg, 5.7mmol, 5eq) were added and the mixture was reacted under the same way as the first block at 100°C for 1hour. Finally, 3.364g (39.9mmol, 35eq) of

2-methyl-2-oxazoline (MeOx) were added as the third block under the same polymerization condition and ultimate polymer (code: HEZ012) was terminated by an excess of Ammonia

(3.42mmol, 3eq). The solvent was then removed by rotary evaporation and the residue was re-dissolved in about 100mL ethanol:chloroform (3:1, v:v). After precipitation from cold diethylether (approx. 10 times the amount of polymer solution) the product was isolated by centrifugation. The precipitation was repeated 2 more times and the polymer (Code: HEZ012) was obtained as colorless powder after lyophilization from water. (Fig. 2.4 c).

Approximately 1.5g HEZ012 polymer were synthesized (83% yield, Mth=8.9kg/mol). GPC

(DMAc): Mn=7.9kg/mol (Ð = 1.40). Unfortunately, we cannot obtain molecular formular by

NMR since the initiator signal (as a reference) is not identified from the spectrum. Via tensiometry method, the CMC for HEZ012 was identified about 35mg/L (4.4µM).

Micelle formulation and drug release

We hypothesized that solubilization of PTX may be further improved by introduction of aromatic moieties in the side chains of the hydrophobic block as additional interactions with the aromatic residues of PTX could occur. To evaluate this hypothesis, BzOx moieties were

60 incorporated into the hydrophobic domain. The effect of the change of the PTX concentration on the solubilization profile was evaluated while keeping the POx concentration constant at

10 g/L. The drug concentration (DC) and loading capacity (LC) values of HEZ012-based formulatios linearly increased with the increase of PTX concentration up to 6 g/L (Fig. 2.5 a).

However, above 6 g/L of PTX, rapid drop in loading efficiency (LE) and LC values was observed.

At 10 g/L of PTX, LC and LE were below 2 % (w/w). Contrary to P2 the z-averaged particle size of PTX loaded HEZ012 micelles did not show direct correlation to PTX concentration and remained below 50 nm until the maximal loading was reached (Fig.2.5 b). At 8 g/L PTX and above a rapid increase in particles size to 100 nm and increase in PDI was observed as well.

Drug release profile showed no changes when BzOx components were incorporated in the central block compared to P2 micelles (data not shown).

2.5 Synthesis of new monomer 2-methyl-2-oxazine (MeOz) for gradient polymerization

We have identified that P2 triblock copolymers with pMeOx-pBuOx-pMeOx structure acheived best drug solubilization and resulting P2/PTX micelles exhibit superior drug exposure and efficacy against breast tumors (Chapter III). However, synthesis of triblock copolymers could be time consuming and costly due to adding monomers sequencially for polymerization in a block-by-block manner. Furthermore, multi-step synthesis also enhance the chance that living polymer chains are early terminated by contamination (e.g. water or other nucleophiles).

Therefore a simple one-pot method to polymerize while remaining tri-block architecture and similar property of P2 would be interesting advancement. This can be realized by gradient polymerization which generates pseudo-triblock copolymers if reaction kinetics of monomers for each block are significantly different. More specifically, I may utilize 2-methyl-2-oxazine

(MeOz) as the third block monomer giving the reaction kinetics of three monomers were reported as MeOx > BuOx > MeOz. Therefore, the gradient copolymer structure is expected to be pMeOx-pBuOx-pMeOz [219].

Synthesis of the MeOz monomer and results

Acetylnitrile 100g (2.44mol, 2eq) and Cadmium acetate dihydrate (catalyst; 9.75g, 0.03eq) were mixed with 2-amino-propanol (91.63g, 1.22mol, 1eq) and heated to 90°C reflux for eight days with stirring. When the reaction was completed, the reaction mixture was cooled to RT and purified by two times distillation.

35g of monomer MeOz (29.2% yield) was synthesized and structure of the monomer was confirmed by IR and NMR spectrum. Although the MeOz monomer was successfully

62 synthesized, I have not further explored its potential in gradient polymerization due to other experimental commitment. (Fig. 2.6)

2.6 Synthesis of new cationic POx monomer and polymer CPOx

There have been attempts to develop gene delivery systems via partially hydrolyzing

PEtOx into linear PEI-POx diblock polymers.[220, 221] One major drawback of this approach is its complexity - the polymer first, has to be hydrolyzed, and second, coupled to unhydrolyzed chain to obtain desired block copolymers. Moreover, this approach does not allow introducing other functional side chains. The direct synthesis of cationic POx by LCROP has been challenging because the growing polymer chains are very sensitive to and could be terminated by nucleophiles.[198] Cesana et al.[222] previously synthesized the first cationic

2-oxazoline monomers bearing tert-butyloxycarbonyl (Boc) protected primary amines.

Polymerization of this monomer followed by deprotection of Boc groups generated POx with pendant cationic groups. However, Boc-protected pendant primary amine monomers provided difficulties for direct polymerization due to the interference from protected-primary amine (nucleophile) to the ring opening process. Hartlieb et al.[223] applied a very similar synthetic approach to Cesana’s and obtained hydrogel scaffolds to capture or enrich DNAs intended to be applied in bioanalytical systems such as gene chips. More recently, Rinkenauer et al.[224] has explored a number of cationic 2-oxazoline polymers by polymer-analogue modification of POx side chains with primary or tertiary amines. They found that 1) long hydrophobic side chain induced high cytotoxicity of the resulting polymer and 2) primary amines with at least 40 mol. % amine content were required for efficient transfection.

In this section, I will discuss the synthesis of a new monomer containing secondary amine group and the cationic POx polymerized from this monomer. It is the first cationic POx polymer synthesized with secondary amine side chain that can be potentially applied in siRNA,

DNA, and protein delivery. In addition, the alkyne group incoporated directly from initators allows this cationic POx clickable to attach targeting moieties or other functions.

Synthesis of Boc-MeAmMeOx monomer and characterization

Boc-MeAmMeOx monomer was synthesized by ring-closing reaction (Fig. 2.7) following the general procedure of Levy and Litt and a newer version of Weberskirch et al..[225, 226]

Using a triple-necked flask with a thermometer and dropping funnel, 21.06 g (111.7 mmol) N-

Boc-sarcosine were dissolved in 350 mL tetrahydrofuran (THF) and 15.45 mL (111.7 mmol) triethylamine were added, the mixture was cooled to 3 °C. Isobutylchloroformiate (14.5 mL,

111.7 mmol) were added dropwise under vigorous stirring. Subsequently, 12.95 g (111.7 mmol) chloroethylamine hydrochloride were dissolved in 50 mL dimethylformamide (DMF) and added dropwise followed by 15.45 mL triethylamine. The yellowish solution was stirred at RT for 1 h. The reaction mixture was filtered and concentrated. After dilution with dichloromethane, the solution was extracted thrice each with 10% aqueous soda and brine.

The combined aqueous phases were extracted with dichloromethane. From the combined organic phases, all volatiles were removed the residue dissolved in 150 mL methanol (MeOH) and 22.505 g (162.8 mmol) of K2CO3 were added. The mixture was stirred under inert atmosphere overnight at RT and then refluxed for 5 h. Volatiles were removed at reduced pressure and the residue was dried at 0.002 mbar. From the yellowish solid the product was obtained by distillation at 3.6 x 10-3 mbar. At 76-83 °C, the product was fractionated and the product was obtained as a colorless liquid (13.683 g, 28.8%).

1 H-NMR (d3-MeOD, 300 MHz, 295 K): 4.256 (t, 2H, N-CH2-CH2-O), 3.954 (s, 2H, C-CH2-

N(Me)(Boc)), 3.735 (t, 2H, N-CH2-CH2-O), 2.818 (s, 3H, N-CH3), 1,35 (d, 9H, O-C(CH3)3) ppm.

(Fig. 2.8)

13 C-NMR (d3-MeOD, 75 MHz, 295 K): 166.2/4 (C-CH2-N(Me)(Boc)), 156.1/155.7 (N-C(O)-

O-C(CH3)3), 80.1 (N-C(O)-O-C(CH3)3), 68.0 (N-CH2-CH2-O), 53.2 (N-CH2-CH2-O), 45.7/44.9 (C-

CH2-N(Me)(Boc)), 34.1 (N-CH3), 27.2 (N-C(O)-O-C(CH3)3) ppm.

Synthesis of Propargyl-P(MeOx50-b-MeAmMeOx16) (CPOx)

The polymer was synthesized following published methods [38, 198, 227] and its reaction strategy is depicted in Fig. 2.9. Briefly, under dry and inert condition 63.1 mg (0.3 mmol, 1eq) of propargyl toluenesulfonate (initiator, 1) and 1276.5mg (15 mmol, 50eq) of 2-methyl-2- oxazoline (MeOx, 2) were dissolved in 3 mL dry acetonitrile at RT. The mixture was heated

(microwave-assisted synthesizer, 150W maximum, and 130 °C) for 5 min. After cooling to RT, the cationic monomer for the second block, Boc-MeAmMeOx (1011.4 mg, 4.7 mmol, 16 eq,

3) was added and the mixture was reacted at 70°C overnight. Finally the polymer was terminated by an excess of 5% aq. K2CO3. The solvent was then removed and the residue was re-dissolved in 5 mL methanol: chloroform (3:1, v:v). After precipitation from cold diethylether, the product was isolated by centrifugation. The precipitation was repeated two more times and the polymer (4) was obtained as colorless powder after lyophilization of an aqueous solution in water. De-protection of Boc group was performed in trifluoroacetic acid

(TFA):H2O:triisobutylsilane (TIBS) (95:2.5:2.5) and the mixture was dried and redissolved in water followed by dialysis against water for two days. De-protected product CPOx (5) was obtained by lyophilization of an aqueous solution in water. The yield of CPOx was 91.5% from

66 deprotection of Boc-CPOx.

1 H-NMR (CDCl3, 298K): δ=4.1-3.9 (br, 37H, -N-CO-CH2-N-Boc, a); 3.47(br, 249H, N-CH2-

CH2, b); 2.87 (br, 63H, boc-N-CH3, c); 2.05 (m, 147, -N-CO-CH3, d); 1.4 (s, 180, tertbutyl, e).

Synthesis of carboxyrhodamine 110 fluorescence labeled polymers (F-CPOx)

4.15 mg (3 equivalent, eq) Carboxyrhodamine110-Azide (Click Chemistry Tools) and 20 mg (1 eq) propargyl-P(MeOx50-b-MAMeOx20) were dissolved in 1 mL mixture of water and methanol(1:1, v:v). Then 60 μg (0.1 eqv.) of copper (II) sulfate pentahydrate were added supplemented with 208.6 μg (0.2 eq) Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA,

1mg/mL stock solution in water/methanol) in water/methanol solution as the stabilizing ligand of copper. The above mixture was bubbled with Argon gas to remove oxygen and supplemented with 200 μL water/methanol solution containing sodium ascorbate (475.4 μg, from 2mg/mL stock solution, 1 eq) drop wise. The reaction was allowed to proceed for 24 h at RT, and reaction mixture was loaded onto LH-20 column to remove unreacted small molecules followed by addition of 10-fold molar excess of EDTA disodium salt and stirred at

RT for 1 h. Finally the reaction mixture was purified with NAP10 size exclusion column, dialyzed against deionized water for 2 days with MW cutoff of 3500, (GE healthcare,

Piscataway, NJ) and freeze-dried to give Carboxyrhodamine 110-labeled polymers F-CPOx (Fig.

2.9, compound 6).[228] Column purification was repeated twice to completely remove free fluorescence dye. The conversion rate (fluorescence labeling efficiency) was about 72% determined from the calibration of free fluorescence dye standard. The detailed gene delivery potential such as cytotoxicity, cellular uptake, and transfection will be discussed in Chapter VI.

Figure 2.1 Schematic demonstration of P2 synthesis

Figure 2.2 Azide-Alkyne Huisgen Cycloadition

Figure 2.3 Synthesis of Propargyl-P4 with clickable alkyne moiety

Figure 2.4 Synthesis of BzOx monomer and polymer HEZ007 and HEZ012

Figure 2.5 Impact of PTX concentration on the solubilization of PTX in HEZ012 (10 g/L). (a): DC, LE, and LC values; (b) z-averaged particles size and PDI. Data are presented as mean ± S.D. (n=3).

Figure 2.6 Monomer synthesis of MeOz

Figure 2.7 Synthesis of the new monomer, Boc-MeAmMeOx (3). i) Triethylamine, isobutylchloroformate, 3 °C, THF, ii) Triethylamine, 2-chloroethylammonium chloride, DMF, iii)

MeOH, K2CO3, reflux.

1 Figure 2.8 H-NMR spectra (d3-MeOD, 300 K) of the novel monomer 2-(N-methyl,N-Boc- amino)-methyl-2-oxazoline with the assignment of all observed signals.

Figure 2.9 Block copolymerization of MeOx (2) and Boc-MeAmMeOx (3) using propargyl tosylate (1) as the initiator and 5% K2CO3 as the terminating agent. The deprotection of the pendant amino group of (4) was performed with TFA to obtain Propargyl-p(MeOx50-b- MAMeOx16) or CPOx, (5). Fluorescence labeled CPOx (F-CPOx), (6) was produced by click reaction of carboxyrhodamine 110-Azide with CPOx.

CHAPTER III: A SIMPLE AND NON-SMART MICELLAR FORMULATION OF POX/PACLITAXEL WITH SUPERIOR SAFETY AND EFFICACY IN VIVO 1

3.1 Summary

The solubility of PTX, a potent antineoplastic compound, has been hampering safe administration of the drug and has been a major roadblock in the development of clinically suitable formulation of the drug. Taxol® and Abraxane® (albumin-bound PTX), several other formulation and drug conjugates of PTX are in clinical trials. Here, we report on POx micellar formulation of PTX, which is characterized by a facile preparation, up to 50 % wt. drug loading, excellent shelf stability and controllable, sub-100 nm size. The specific POx used in this study was doubly amphiphilic triblock copolymer previously reported as “P2”. In addition, we observe very high maximum tolerated dose compared to both Abraxane® and Taxol® and improved therapeutic outcome in A2780 ovarian xenograft, LCC6-MDR orthotopic model as well as particularly faithful and aggressive T11 orthotopic, syngeneic transplants.

Pharmacokinetic, biodistribution and safety profiles indicate dependencies on the drug loading. The promising preclinical data on POx/PTX nanoformulation showcase the need to investigate new excipients and is a robust basis to translate into clinical trials.

1 This chapter previously appeared as a manuscript soon to be submitted.

3.2 Introduction

PTX [1], has demonstrated promising antineoplastic effects against MBC, NSCLC, advanced OCs, head and neck cancer and other malignancies [48]. By interfering with tubulin polymerization, thus perturbing microtubule dynamics, PTX leads to chromosome missegregation on multipolar spindles [5, 11, 90]. Apart from excellent potency, PTX is characterized by an extremely low solubility in aqueous media (<1 mg/L) [16], demanding delivery vehicles for parenteral administration. Three formulations are currently clinically approved, two of which by the FDA. Both are blockbusters and make PTX the best-selling chemotherapeutic in history [90].

The first clinical formulation of PTX was Taxol®. It is characterized by very low drug load

(ca. 1% wt. of PTX), thus, the amount of the excipient, Cremophor EL/ethanol, necessary to deliver effective doses of PTX is substantial; for example, excipient plasma concentration can reach 0.4% and persist above 0.1% for over 24 h [20]. It is well documented that Cremophor

EL in Taxol® formulation causes severe allergic, hypersensitivity, anaphylactic reactions, nephro- and neuro-toxicity in both animals and humans, which significantly limits dosing and impedes clinical intervention [18, 19, 21, 23, 56, 112].

The clinical demand for alternative formulations led to the development of Abraxane®, a

130 nm nanoparticle formulation comprising HSA and ca. 10% wt. PTX. Evidenced advantages such as increased antitumor activity and tumor accumulation in several mice xenograft models, significantly higher MTD in human, as well as approximately 74% increase of response rates in MBC patients ultimately led to clinical approval [56, 112, 229]. However, clinical trials revealed an increased peripheral neuropathy as compared to Taxol® [57, 58]. Moreover, a

78 recent randomized phase III clinical trial of weekly Taxol® compared to weekly Abraxane® in combination with Bevacizumab as first-line therapy for locally recurrent or MBC, indicates that

Abraxane® offers no benefits to PFS compared to Taxol®, while inducing greater hematologic toxicity and sensory neuropathy [127]. Thus, there clearly remains a clinical demand for a formulation of paclitaxel with improved safety profile and therapeutic outcome.

Such efforts have been directed towards nanospheres [230], liposomes [31], cyclodextrin complexes [231], water-soluble prodrugs [232] and polymeric micelle formulations. For example, Genexol-PM® [42], a formulation comprising a block copolymer of PEG and PLA is clinically approved in Korea. NK105, a formulation of PTX using a block copolymer of PEG and modified polyaspartate recently successfully passed a phase II clinical study [37]. However, even these developmental formulation can only overcome the common limitations of PTX formulations in part. The MTD of Genexol-PM® was identified as 50 and 60 mg/kg in non- tumor bearing female SPF C3H/HeNcrj mice and nude mice respectively, which is only 2-3x compared to Taxol® [42]. NK105 could be safely administered at 100 mg/kg in balb/c female nude mice [36]. A superior dosage form which exhibits high drug loading, desirable PK and tumor accumulation, and low toxicity while increasing therapeutic efficacy remains elusive.

Self-assembled nano-sized polymeric micelle formulation based on doubly amphiphilic

POx block copolymers [38, 50, 198, 199] fulfill unmet needs in formulation of PTX. We have recently reported on the change of morphology in dependence of drug loading; when drug loading exceeds 10% wt., small angle neutron scattering data suggest formation of nano- raspberries with hydrodynamic radii < 50 nm [39]. Drug delivery systems in this size regime are discussed to be particularly useful in cancer chemotherapy, due to the EPR [233] and

79 because such small sized entities exhibit superior tumor penetration, particularly in poorly permeable tumors [234].

Notably, polymer design proved to be of major importance in our studies. The hydrophilic block comprises pMeOx, which imposes stealth properties similar if not superior to PEG [202,

212, 214, 235-237]. The hydrophobic block comprising pBuOx was screened as an ideal environment for solubilizing unprecedentedly high amount of PTX with outstanding formulation stability [38, 50, 238]. Moreover, triblock structure proved critically superior as compared to diblock structure [42, 238]. This type of amphiphilic triblock copolymers was previously reported as “P2”. In following chapters, POx represents P2 unless otherwise specified.

3.3 Materials and Methods

Preparation of POx/PTX polymeric micelles

Two batches of amphiphilic triblock copolymers P(MeOx33-b-BuOx26-b-MeOx45), Mn =

10.0 kg/mol, ĐM (Mw/Mn) = 1.14 and P(MeOx47-b-BuOx21-b-MeOx36), Mn = 9.9 kg/mol, ĐM

(Mw/Mn) = 1.19 were synthesized as described in the previous study [38, 50]. POx/PTX micelles were prepared by a thin film method. Briefly, pre-determined amounts of POx and PTX (stock solution 10-20 g/L in ethanol) were mixed with small amount of ethanol followed by complete removal of ethanol. We tested and optimized small (1-5 mg scale, air flow at 40 °C) and large scale (200mg scale, rotary evaporator) production methods to control the thin film formation process. Appropriate amounts of deionized (DI) water or normal saline was used to rehydrate the dried thin-film under heating at 50-60 °C for up to 20 min in order to obtain drug loaded polymeric micelles. The resulting micelle formulation was stored as aqueous solution in refrigerator for up to 2 weeks or as lyophilized powder. Polymeric micelle characterization methods, drug loading and release profiles are described in the supplementary materials.

In vivo studies

All animal procedures were performed in compliance with federal animal welfare regulations, and protocols were approved by the Institutional Animal Care and Use committee.

All animals used in PK, biodistribution, MTD, toxicology and efficacy studies were allowed to acclimate for at least 72 h in the animal facilities before experimentations. Animals were exposed to a 12 h light/dark cycle and received food and water ad libitum throughout the studies. Dosages of POx/PTX micelle formulations or commercial drugs Taxol® and Abraxane®

81 are expressed as the quantity of PTX administered.

MTD studies

MTDs for Taxol®, Abraxane® and POx/PTX micelles were determined in a dose escalation method in female nude mice (tumor-free 6-8 weeks of age). Animals (n=3 per group) received i.v. injections of POx/PTX micelles (20, 40, 60, 90, 120 and 150 mg/kg), Taxol® (20, 25, and 30 mg/kg), Abraxane® (30, 60, 90 and 120 mg/kg), and saline as a control (q4d x 4). Mice survival and changes in body weight were observed daily over two weeks in all groups. The highest dose that does not cause toxicity (as defined by a median body weight loss of 15 % of the control or abnormal behavior including hunched posture and rough coat) was used to identify

MTD. Changes in histopathology such as inflammation, or presence of necrotic cells would indicate cytotoxicity occurring after treatment.

Toxicology studies

Healthy Balb/c mice were treated with POx alone, POx/PTX and Taxol® at MTD dose. The following day mice were sacrificed and blood were withdrawn and a comprehensive blood chemistry panel were performed. Major organs including heart, liver, kidney, spleen, lung and brain were harvest, fixed in formalin, and subjected to pathological analysis by H&E staining.

In addition, tumor bearing animals two weeks post fourth injection were sacrificed and organs were harvest according to the same procedures as healthy mice.

PK and biodistribution studies

Tumor-free mice. Female Balb/c mice (6-8 weeks of age) were administered a single dose of Taxol® (20 mg/kg) or POx/PTX micelles (20, 75 or 150 mg/kg) containing 3H-labelled PTX (5

Ci/mouse) via tail vein. At various sampling times (0.083, 0.5, 1, 2, 6, 24, 72, and 168 h post) a group of animals (n=3) were euthanized and blood collected from cardiac puncture were analyzed for PTX plasma concentration by measuring radioactivity. The tissues (brain, lung, kidney, spleen, liver, and heart) were also removed, washed in ice-cold saline, weighted and homogenized in a glass tissue homogenizer (TearorTM, BioSpec Products, Inc.), followed by radioactivity level determination using a Tricarb 4000 (Packard, Meriden) to quantify tissue distribution.

Tumor-bearing mice. Female nude mice (6-8 weeks of age) were implanted with 8x106

A2780 OC cells in 50% growth medium and 50% Matrigel (BD Biosciences) by subcutaneous injection. When tumors were about 200 mm3 volume, mice were randomized (n=4 per group) such that the mean and medium tumor weights were similar between groups. Mice were then administered a single dose of above-mentioned formulations. At various time points, blood and tissue samples were obtained accordingly.

Efficacy and POx/PTX tumor accumulation studies

A2780 OC xenograft model. Female athymic nude mice (6-8 weeks) were subcutaneously inoculated in the right flank with 8 x 106 human A2780 OC cells (Sigma Aldrich) resuspended in 50% growth medium and 50% Matrigel. Two sets of experiments were performed: early stage tumor treatment starting after tumor sizes reach ca. 100-200 mm3; or late stage tumor

83 treatment when tumor sizes reach ca. 400mm3. Animals were randomized into groups of seven mice such that the mean tumor volumes were similar between groups and then administered with following formulations: 1) Normal Saline; 2) Taxol® (20 mg/kg PTX at determined MTD dose); 3) Abraxane® (45 and 90 mg/kg PTX at determined ½ MTD and MTD doses); 4) PTX loaded micelles (75 and 150 mg/kg at determined ½ MTD and MTD doses). The formulations were administered via tail vein following q4d x 4 regimen (on the days 0, 4, 8,

12). Tumor growth were monitored twice weekly for 15 weeks or an earlier end-points defined as the tumor reaches a volume >1400 mm3, animal weight loss is > 15%, or animals become moribund. Tumor length (L), width (W) and tumor volume (TV) were calculated as TV =1/2 

L  W2. Survival and body weight were monitored daily. Tumors were removed at the end of the observation and subjected to histopathological examination.

Orthotopic model of LCC6-MDR human TNBC. The LCC6-MDR cells (obtained from Dr. R.

Clarke, Georgetown University Medical School, Washington, DC) expressing high levels of Pgp were originated from LCC6-WT cells stably transfected with a retrovirus vectored mdr1 gene

[239]. The parent LCC6-WT cells were derived from estrogen receptor (ER)-negative, aggressive and metastatic MDA-MB-435 cells. We developed an orthotopic model by directly transplanting LCC6-MDR cells into mice mammary fat pad (5 million cells/nude mouse) and measured the activity of POx/PTX vs other commercial formulations.

T11 orthotopic, syngeneic transplant (OST) cancer model. T11 model mice are assessed using described practices of the Mouse Phase 1 Unit (MP1U) of UNC (e.g., tumor regression, large cohort size, etc.). When tumors were noted to be approximately 10-50 mm3 in size, animals were treated as described and tumor response was assessed by weekly caliper

84 measurements. Data in Fig. 5 are normalized to tumor size at the time of therapy initiation, with volumes calculated using the formula Volume = TV =1/2  L  W2. Tumor-bearing mice were euthanized at the indicated times for morbidity, tumor ulceration, or tumor volume more than 3000 mm3.

PK and data analysis

PK parameters were assessed with Phoenix WinNonlin (version 6.0) using non- compartmental analysis. Statistical comparison of efficacy and tumor accumulation data is one-way ANOVA with Holm-Sidak post-hoc test for multiple comparisons at a significance level of p<0.01 (Graphpad Prism, version 5.1.). If groups fail the normality or equal variance test,

ANOVA on ranks with the Tukey post-hoc test will be used for multiple comparisons.

3.4 Results and Discussion

Characterization of PTX-loaded POx micelles

The POx/PTX formulation was prepared by a very simple and highly reproducible thin film hydration method [38, 50] (Fig. 3.1a and b, Fig. 3.2 and 3.3). POx/PTX micelles spontaneously self-assemble during rehydration of the dry film. The drug loaded micelles appeared to be of spherical morphology[39] and about 30-50 nm effective diameter with a narrow size distribution (Fig. 3.1c). We achieved drug loading of up to 47.3% (Table 3.1), highlighting the high reproducibility of our approach between different labs and polymer batches [38, 39, 50]. These solutions are clear, lyophilizable, and exhibit low viscosity as well as high stability in both saline (Fig. 3.1d) and water (Fig. 3.4a, b; Table 3.2). Due to their low viscosity, they can be readily injected as prepared. There is no significant size change with dilution or dispersing media (Fig. 3.5) and surface charge of micelles as measured by ζ- potential is essentially neutral in 10 mM NaCl solution (Fig. 3.6). Such formulation properties compare very favorably to other PTX formulations in clinics and in late stage clinical trials. We identified, however, a significant burst release at the highest drug loading (POx/PTX 50/45 g/L), which can be somewhat reduced by lowering drug content (POx/PTX 50/40 g/L) (Fig. 3.1e).

The subsequent in vitro and in vivo experiments were therefore performed mostly using

POx/PTX 50/40 formulation unless otherwise specified. A significant difficulty of nanoscopic and particularly self-assembled drug delivery systems is the removal of endotoxins. POx are very thermostable and can be heated to 200 °C for 24 h to efficiently remove endotoxins without compromising polymer properties (Fig. 3.7).

MTD and toxicology profiles of POx/PTX in nude or healthy mice

We investigated MTD in a regimen of every fourth day for a total of 4 injections (q4d x 4), the same schedule was employed in subsequent antitumor efficacy studies. NCI-Nu/Nu mice were injected with POx/PTX micelles (80, 100, 120, 150 and 175 mg PTX/kg body weight) and compared to Taxol® and Abraxane®. MTD was deﬁned as the maximum dose that causes neither toxicity-induced animal death nor greater than 15% of body weight loss, or other noticeable sign of toxicity during the entire experimental period. For the clinically approved formulations our studies confirmed MTDs reported in the literature (20 mg/kg for Taxol® and

90 mg/kg for Abraxane®).

POx/PTX treated mice barely showed weight loss and no noticeable changes in behavior up to 150 mg/kg dose (Fig. 3.8a). We found that mice administered four times with 175 mg/kg micelles, lost over 15% body weight and were sacrificed. However, a single injection of 200 mg/kg of POx/PTX micelles was well tolerated without obvious sign of toxicity. We also tested

POx polymer alone at equivalent dose (187.5 mg/kg and up to 500 mg/kg), and no remarkable changes in general activity and body weight were observed, suggesting that POx has very low toxicity in vivo (Table 3.3).

Based on these data the MTD for POx/PTX micelles under q4d x 4 regimen was determined at 150 mg/kg, more than seven fold of Taxol® (20 mg PTX/kg) and almost twice the value of Abraxane® (90 mg/kg) (Fig. 3.8b). Although it is not straightforward to correlate

MTD in mice or other animals and human, the relative MTDs of Taxol®, Genexol-PM®,

Abraxane® and NK105 correlated reasonably well with human MTD or recommended dose

[102, 240, 241]. Therefore, it is reasonable to hypothesie that the present formulation could present a formulation for which MTD in humans can be extended beyond current possibilities.

We confirmed the low compliment activation previously observed for POx/PTX [38] using an alternative protocol (Nanotechnology Characterization Laboratory protocol NCL ITA-5). The

POx polymer itself and POx/PTX do not activate complement, similar to negative control PBS at a concentration range from 0.006-1.5 mg/mL (1.5 mg/mL corresponds to approx. 10 fold the value we would expect to observe in humans), while Cremophor EL at 1.5 mg/mL (a clinically relevant dose) revealed strong complement activation comparable to positive control cobra venon factor (CVF) (Fig. 3.9a) [38]. Further in vitro results showed that POx and

POx/PTX formulation are not hemolytic and do not induce platelet aggregation (Fig.3. 9b, c, d). In fact, we found evidence that the polymer and the POx/PTX formulation can result in prolonged activated partial thromboplastin time (APTT) without affecting the prothrombin time (PT) (Fig. 3.9e, f). The effect of retarding coagulation can be beneficial to BC patients as it was demonstrated that haemostatic alterations and pro-coagulant systems, especially the formation of venous thromboembolism (VTE), frequently occur following chemotherapy [242,

243].

In addition, in vitro cytotoxicity of PTX/POx in hepatic and kidney cells was similar to

Abraxane® (Fig. 3.10-3.11 and Table 3.4). In vivo, safety of POx/PTX was also evaluated in nude mice by examining clinical chemistry parameters for kidney and liver function, and histopathology of major organs of animals two week after four injections (q4d x 4) at MTD dose (Fig. 3.8c and Table 3.5). There were no significant changes for blood urea nitrogen (BUN), alanine amino transferase (ALT), albumin values and other blood chemistry parameters.

Histology reported no toxicity to lung, and spleen, brain, and heart for all formulations tested.

There are mild toxic degenerative changes of centrilobular hepatocyte atrophy in the

Abraxane and Taxol treated samples (Fig. 3.8c, solid arrow in liver samples). This was also observed in POx and POx/PTX samples, albeit markedly attenuated. In saline control, this change was absent. Also in the kidney, Abraxane and Taxol treated samples exhibit signs of mild toxicity with few scattered atrophied tubules with fluid to proteinaceous accumulations to occasional casts (Fig. 3.8c, dashed arrow in kidney samples). Only very little mild and scattered tubular damage was observed in POx/PTX samples (see supporting information for a more detailed analysis). Therefore, signiﬁcant improvement in safety profile highlights the potential clinical advantages of the presented delivery system over current clinical formulations, allowing high dosage of PTX to be given in order to maximize therapeutic effect.

Antitumor efficacy and tumor accumulation of POx/PTX

After this preliminary experiment using only a singly injection, we evaluated antitumor efficacy of POx/PTX in tumor models including A2780 human OC xenografts, LCC6-MDR multidrug resistant human TNBC orthotopic models and T11 orthotopic syngeneic transplants

(OST) BC models using a q4d x 4 regimen.

A2780 human OC xenografts [155]: At the MTD of 150 mg/kg, POx/PTX 50/40 exhibited very significant tumor inhibition (p<0.001) when treating small tumors (ca. 100-200mm3).

After third dose, tumors were completely eradicated in 100 % of the animals while Taxol® delayed tumor growth until the fourth injection after which the lesions started to grow back rapidly (Fig. 3.12a, c). For later stage tumors (treatment starting from tumor volume ca.

400mm3), Taxol® (Fig. 3.13a) did not control tumor growth requiring animals to be sacrificed, while POx/PTX micelles again resulted in complete remission of tumors in all animals even 120 days post treatment. Survival analysis by Kaplan-Meier estimate demonstrated that POx/PTX was the superior treatment for both small and large tumors (100% survival, p<0.0001 compared to Taxol®, Fig. 3.12d and Fig. 3.13c).

POx/PTX 50/40 formulation at MTD and ½ MTD doses (150 and 75 mg/kg) were also compared with Abraxane® (MTD 90 and ½ MTD 45 mg/kg, Fig. 12b) when treating small tumors (ca. 100-200mm3). We observed that both Abraxane® and POx/PTX can control tumor growth well at MTD doses, but 3 out of 7 mice in Abraxane® group experienced severe peripheral neuropathy as evidenced by paralysis and over 15% weight loss that required sacrificing the animals (Fig. 3.12d). Also at ½ MTD dose, POx/PTX eradicated all tumors while

Abraxane® led to significant tumor shrinkage but eventually 40% of mice relapsed. As a result, survival in the Abraxane® regimen was the same for MTD and ½ MTD group (Fig. 3.12d).

Animal body weight (Fig. 3.12e) and signs of toxicity were monitored for all treatment groups and we found that most of animals did not experience weight loss except above-mentioned

Abraxane® MTD individuals. Efficacy in treating large tumors (ca. 400 mm3) was similar to small tumor treatment (Fig. 3.13a-c).

Orthotopic model of LCC6-MDR human TNBC [239]: In this model, the MTD as established for POx/PTX 50/40 and Abraxane® in tumor free mice proofed slightly too high. Thus, the experiments were conducted at doses of 80 mg/kg (Abraxane®) and 120 mg/kg (POx/PTX).

These two treatment groups successfully reduced tumor growth, POx/PTX more so than

Abraxane® (Fig. 3.14a). However, Abraxane® did not lead to increased survival, while POx/PTX extended survival significantly (Fig. 3.14b).

T11 OST BC model [244-247]: This is a very aggressive tumor model that represents recently identified claudin-low subtype of TNBC with very poor prognosis. This syngeneic transplant model recapitulates well clinical tumor types, in which an adequate genetic engineered mouse model cannot be identified. Tumor cells were transplanted from Balb/c

TP53-/- mice with RAS/RAF/MAPK pathway activation and elevated number of tumor initiating cells. Therefore, it represents a very faithful model that is clinically relevant to specific subtype of TNBC. Several chemotherapeutic agents including carboplatin, paclitaxel, erlotinib and lapatinib were tested in this model as single or two drug combinations but were not efficacious [247]. The remarkable results using the POx/PTX 50/40 formulation in A2780 and

LCC6-MDR prompted us to test in T11 model. We observed clear trend of tumor inhibition in

POx/PTX 50/40 ½ MTD group (75mg/kg) and significant suppression at MTD dose (150 mg/kg, p<0.01) (Fig. 3.14c). Furthermore, POx/PTX 50/40 at ½ MTD dose also exhibited survival benefits over other groups (Fig. 3.14d). This is a remarkable improvement considering that we employed a single agent treatment for very aggressive T11 model. It should be noted that the current formulation we have successfully formulated a number of other drugs (including lapatinib) in conjunction with PTX using the same technology and demonstrated synergism in vitro [50].

Table 3.1 Drug concentration, loading efficiency (LE), loading capacity (LC), drug loaded micelle size and PDI of POx/PTX formulations.

Final Drug Loading Loading Effective POx Conc. (Feeding) Efficiency Capacity Diameter PDI (g/L) (g/L) (LE, %) (LC, %) (nm)

50 45 (50) 91.6 47.8 76.7 0.162

50 40 (45) 88.7 44.4 54.7 0.151 50 20 (20) 100 28.7 23.2 0.155

Table 3.2 Size and PDI of two POx/PTX formulations before and after lyophilization.

Formulations Before Lyophilization Re-dispersed in Water

Effective Effective Polymer/ POx PTX Diameter PDI Diameter PDI drug ratio (g/L) (g/L) (nm) (nm)

1.25/1 50 40 54.7 0.151 52.3 0.194 2.5/1 50 20 23.2 0.160 22.3 0.175

Table 3.3 Monitoring of animal death and weight in the MTD experiments.

Dose Animal Formulations Weight loss (mg/kg) death 20 0/3 3.47% Taxol® 25 1/3 Animal Death 90 0/3 Weight gain Abraxane® 120 1/3 Animal Death 80 0/3 Weight gain POx/PTX 100 0/3 Weight gain 50/40 120 0/3 Weight gain Micelles 150 0/3 Weight gain 175 3/3 >15% 20 2/3 Animal Death Cremophor 25 3/3 Animal Death 187.5 0/3 Weight gain POx 500 0/3 Weight gain 1000 1/3 Animal Death

Table 3.4 Summary of cytotoxicity to liver Hep G2 and kidney LLC-PK1 cells.

LLC-PK1 cells Hep G2 cells Formulations IC50, μM, 48 h IC50, mM, 48 h POx alone > 1000 >5 POx/PTX 50/40 0.00005 >5 Taxol® 0.03 0.005 DMSO-PTX 0.0005 0.03 Abraxane® 0.0007 1

Table 3.5 Clinical chemistry parameters of Balb/c mice receiving POx and POx/PTX.

Formulations Parameters Normal range Saline POx POx/PTX Taxol® ALT 17 – 77 37.0 38.8 36.3 28.7 ALP 35 – 222 27.7 15.3 9.0 22.0 Total bilirubin 0.0 - 0.9 0.3 0.3 0.3 0.3 Glucose 140 – 263 197.0 199.3 305.3 178.3 Total protein 3.9 – 6.4 5.1 5.0 5.0 5.3 Creatinine 0.2 – 0.9 <0.2 <0.2 <0.2 <0.2 BUN 9 - 33 15.7 14.0 16.3 14.0 Albumin 2.5 – 4.6 4.0 3.9 4.0 4.3 Phosphate N/A 9.0 8.9 8.0 8.6 Serum globulin N/A 1.1 1.1 0.9 1.0

Abbreviations used in the table: ALT, alanine amino transferase; ALP, alkaline phosphatase; BUN, blood urea nitrogen. ALT, ALP units in U/L; bilirubin, glucose, creatinine, BUN, phosphate units in mg/dL; total protein, albumin, serum globulin units in g/dL.

Figure 3.1 Preparation and physicochemical properties. a, Chemical and schematic structures of POx and PTX. b, Loading procedure and transmission electron microscopy (TEM) of the resulting formulation. c, Size distribution, d, Stability and e, Drug release profiles of POx/PTX 50/40 (blue) and POx/PTX 50/20 (red) micellar formulations. c, Size distribution measured by DLS, d, size and PDI measurement over time at RT, and e, drug release in phosphate-buffered Saline (PBS) at 37 °C at 50/45, 50/40 and 50/20 polymer/drug ratios.

Figure 3.2 SOP for small scale (1-5 mg) micelle production

Figure 3.3 SOP for large scale (200 mg) micelle production

Figure 3.4 POx/PTX 50/40 formulation. a, The formulation was lyophilized without adding any cryo-protectants. Each vial contains 10mg PTX and 12.5mg POx. b, Stability of POx/PTX 50/40 and 50/20 formulations in water over 13 days by measuring size and PDI in DLS.

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Figure 3.5 Size of POx/PTX 50/40 micellar nanoformulation in water, PBS or 10mM nacl. a, Intensity size and b, Volume size distribution measurements of POx/PTX 50/40 micelles. c, summary table of size measurements from a, and b,. Samples were prepared at 1 or 0.1 mg/ml (polymer concentration) in pure water, PBS or 10 mM nacl. Measurements were performed at samples’ native pH and 25 oC.

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Figure 3.6 Zeta-potential of POx/PTX 50/40 micelles in water or 10 mM nacl by zeta sizer. a, zeta potential distribution measurements of POx/PTX 50/40 drug loaded micelles. b, Summary table of zeta-potential measurement from a. Samples were prepared at 1mg/ml (polymer concentration) in pure water or 10 mM nacl. Measurements were performed at samples’ native pH, 25 oC, and applied voltage 150v.

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Figure 3.7 Characterization of POx polymers. a, 1H-NMR spectrum, b, polymer structure with designated NMR peak assignments in a, c, Gel permeation chromatography (GPC) spectrum, d, FT-IR spectrum, and e, UV spectrum of POx polymer before (red) and after (black) heating at 200 oC for 24 h. f, Thermal gravimetric analysis (TGA) spectrum of POx with three stages heating: 25-200 oC (10 oC/min), 200 oC (hold 720 min), and 200-1000 oC (10 oC/min).

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Figure 3.8 MTD in nude mice and toxicology profiles. a, Mice body weight (% of initial) in MTD experiment. b, MTD of POx/PTX 50/40 formulation (150mg/kg), Taxol® (20mg/kg) and Abraxane® (90mg/kg) in a q4d x 4 regimen. c, Histological examination of brain, heart, spleen, liver, lung, and kidney tissues by hemotoxylin & eosin (H&E) staining from animals treated with saline, POx (500mg/kg) or POx/PTX (50/40, 150mg/kg). Tissues were harvested two weeks after the last dose of q4d x 4 regimen.

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Figure 3.9 In vitro toxicity evaluation. a, Compliment activation, b, Hemolysis, c, Platelet aggregation, d, Prothrombin time (PT), e, Thrombin time and f, Activated partial thromboplastin time (APTT) of POx polymer or POx/PTX micelles (concentration range from 0.006-1.52 mg/mL, 1.52 mg/mL corresponds to approx. 10 fold the value we would expect to observe in humans) as compared to Taxol®. Negative control (NC) was PBS.

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Figure 3.10 Cytotoxicity to LLC-PK1 normal pig kidney epithelial cells. Viability and plasma membrane integrity of LLC-PK1 cells exposed to a, POx, b, POx/PTX 50/40, c, Taxol®, d, DMSO-PTX and e, Abraxane® with increasing concentration from 10-10 to 102 μM. Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay and membrane integrity was measured using lactate dehydrogenase (LDH) leakage assay. 106

Figure 3.11 Cytotoxicity to Hep G2 human liver hepatocellular carcinoma cells. Cell viability and membrane integrity of Hep G2 cells exposed to a, POx, b, POx/PTX 50/40, c, Taxol®, d, DMSO-PTX and e, Abraxane® with increasing concentration from 10-13 to 10-1 μM. Cell viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide (MTT) assay and membrane integrity was measured using lactate dehydrogenase (LDH) leakage assay.

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Figure 3.12 Efficacy of POx/PTX 50/40 in A2780 tumors. a, Tumor growth inhibition of POx/PTX 50/40 formulation at MTD dose (150 mg/kg) compared to Taxol® at MTD dose (20 mg/kg), and b, at MTD and ½ MTD dose (150 and 75 mg/kg) compared to Abraxane® MTD and ½ MTD dose (90 ad 45 mg/kg). Each formulation was injected on days 0, 4, 8, 12. Data is expressed as mean ± s.e.m., n=7. ***P<0.001. c. A representative image of mice 2 days after first dose: left, saline treated; middle, Taxol® treated; right, POx/PTX 50/40 treated. d, Kaplan- Meier survival plot for all groups in a. and b. ****p<0.0001. e, Changes of body weight of animals in each group (n = 7). 108

Figure 3.13 Efficacy of POx/PTX 50/40 in A2780 tumors (treatment starting at later stage, tumor volume of ca. 400 mm3). a, Tumor growth inhibition of POx/PTX 50/40 formulation at MTD dose (150 mg/kg) compared to Taxol® at MTD dose (20 mg/kg), and b, at MTD and ½ MTD dose (150 and 75 mg/kg) compared to Abraxane® MTD and ½ MTD dose (90 ad 45 mg/kg). Each formulation was injected on days 0, 4, 8, 12. Data is expressed as mean ± s.e.m., n=7. ***P<0.001. c, Kaplan-Meier survival plot for all groups in a. and b. ****p<0.0001. d, Changes of body weight of animals in each group (n = 7).

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Figure 3.14 Efficacy of POx/PTX 50/40 in LCC6-MDR and T11 tumors. a, Tumor growth inhibition of POx/PTX 50/40 (120mg/kg, adjusted dose from MTD) in LCC6-MDR, and c, POx/PTX 50/40 MTD and ½ MTD dose (150 and 75 mg/kg) in T11 models compared to Taxol® at MTD dose (20 mg/kg). Each formulation was injected on days a, 0, 4, 8, and 12, or c, 2, 6, 10, 14, and 18. Saline and POx polymer alone were used as controls. Data is expressed as mean ± s.e.m., n=7. *p<0.05, **p<0.01. b, Kaplan-Meier survival curve of all groups in a, and d, for all groups in c.

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CHAPTER IV: OTHER APPLICATIONS OF P2 POX POLYMERS (1) PREPARATION, IN VITRO AND IN VIVO EVALUATION OF POX MICELLES WITH HIGH CAPACITY FOR 3RD GENERATION TAXOIDS 1

4.1 Summary

The clinically and commercially successful taxanes, PTX and DTX suffer from two major drawbacks, very low aqueous solubility and the risk of developing resistance. Here, we present a method that overcomes both drawbacks in a very simple manner. We formulated 3rd generation taxoids, able to avoid common drug resistance mechanisms with doubly amphiphilic P2 POx, a safe and highly efficient polymer for formulation of extremely hydrophobic drugs. We found excellent solubilization of different 3rd generation taxoids irrespective of drug structures with essentially quantitative drug loading and final drug to polymer ratios around unity. The hydrodynamic size of highly loaded micelles with less than

100 nm is excellently suited for parenteral administration. Moreover, a selected formulation with the taxoid SB-T-1214 is about one to two orders of magnitude more active in vitro than paclitaxel in the multidrug resistant breast cancer cell line LCC6-MDR. In contrast, in wild-type

LCC6, no difference was observed. Using a q4d x 4 dosing regimen, we also found that POx/SB-

T-1214 significantly inhibits the growth of LCC6-MDR orthotropic tumors, outperforming commercial paclitaxel drug Taxol® and Cremophor formulated SB-T-1214. 1 This chapter previously appeared as an article in press. The original citation is as follows: He, Zhijian et al., “Poly(2-oxazoline) based micelles with high capacity for 3rd generation taxoids: Preparation, in vitro and in vivo evaluation”, Journal of Controlled Release, 2015, doi:10.1016/j.jconrel.2015.02.024.

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4.2 Introduction

Taxanes can arrest cells in the G2/M phase upon binding to the β-tubulin subunits to promote their polymerization and stabilize microtubules, leading to apoptosis through cell- signal cascades. Several commercially successful and clinically significant taxoids have been developed, such as PTX, DTX, and CBZ [9]. They are heavily used in the treatment of breast, lung and OC as well as other malignancies [90, 248]. Unfortunately, these taxoids suffer from two major setbacks.

The first problem is that these compounds are very poorly soluble [16] and require the use of toxic excipients in their clinical formulations, such as Cremophor EL and ethanol in

Taxol® (PTX), or Polysorbate 80 and ethanol in Taxotere® (DTX), or Jevtana® (CBZ). These excipients can cause severe hypersensitivity reactions [18]. Therefore, to reduce this toxicity the patients receiving these medications must be pre-treated with antihistamine, corticosteroid and H2 antagonist and be immediately removed from therapy if the hypersensitivity reactions are observed. Although many, potentially safer formulations have been developed for PTX and other taxanes [18, 38, 39, 50, 192, 249-251], including the protein-drug nanoparticle, Abraxane® and the polymeric micelle drug, Genexol-PM®, the drug payload in these formulations remains relatively low and does not exceed 10% [252] for

Abraxane®, 17% [100] for Genexol-PM® and 23% [36] for NK105. Also, significant improvement of the therapeutic outcome and patient survival, for example for treatment with

Abraxane®, remains to be verified [127].

The second problem is the development of drug resistance in response to the therapy using taxanes. Specifically, cancer cells resistance to PTX, DTX and CBZ can involve 112 overexpression of ABC transporters (i.e. Pgp; MDR-associated protein 1, MRP1), or point mutations in tubulin [253]. This problem was extensively addressed in literature [253, 254].

Thus, the “2nd-generation taxoids” were developed in which the C3-phenyl group in taxoids was replaced with an alkenyl or alkyl group and the C10 position was modified with various acyl groups [255]. Such 2nd-generation taxoids were shown to be 1 to 2 orders of magnitude more potent than the parent drugs against drug-resistant human BC cells [255]. Moreover, further substitution (t-Boc group) at the C3’N position of the 2nd-generation taxoids further enhanced their potency against drug-resistant cancer cell lines (specifically Pgp+ mediated

MDR and tubulin mutations). Examples of such compounds, termed “3rd-generation taxoids” include SB-T-1213, SB-T-121302, SB-T-121303, SB-T-1214, SB-T-121402, SB-T-1216, and SB-T-

121602 (Fig. 4.1a,b) investigated in the present contribution. The new-generation taxoids were shown to be effective in LCC6-MDR (Pgp+ human BC cell line); NCI/ADR-RES (Pgp+ human OC cell line); 1A9PTX10 and 1A9PTX22 (human OC cells originated from A2780 cell line possessing point mutations in class I-tubulin), as well as CFPAC-1, PANC-1, MIA PaCa-2 and

BxPC-3 (human pancreatic cancer cell lines overexpressing MDR genes mdr1, mrp1, mrp2, and lrp). Furthermore, the taxoid SB-T-1214 was evaluated and demonstrated its efficacy against

Pgp+ DLD-1 human colon tumor xenografts in severe combined immune-deficient (SCID) mice

[255].

Unfortunately, the 3rd-generation taxoids remain very poorly water-soluble and require the use of appropriate drug carrier systems. We have recently discovered a novel polymeric drug carrier system, based on block copolymers of hydrophilic PMeOx and mildly hydrophobic

PBuOx. Interestingly, despite the low hydrophobic character of pBuOx, we have found that

113 block copolymers (in particular triblock copolymers) of pMeOx and pBuOx exhibit a surprisingly high efficacy (both relative and absolute) for the solubilization of extremely hydrophobic drugs, including taxanes [38, 39, 50]. The capacity of POx micelles with respect to such taxanes is unprecedented. For example, our POx/PTX micelles have ca. 4 to 5 times higher loading and ca. 10 to 20 times higher drug concentration in injectable formulation than the clinical alternatives of Taxol®, Genexol-PM®, and Abraxane®. POx in general have received increasing attention recently as alternatives to PEG based systems [198, 201, 256, 257] and first-in-man studies are expected to commence in 2015 [258]. Here, we intend to combine the possibilities of our POx-based drug delivery platform for safe and efficient drug formulation and delivery with the advantages of 3rd-generation taxoids, which can overcome MDR mechanisms. Thereby we set out to develop a formulation, which safely addresses the pressing clinical challenge of drug resistance in cancer patients.

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4.3 Materials and Methods

Materials

Reagents and monomers for polymer synthesis as well as (3-[4,5-dimethylthiazol-2-yl]-

2,5-diphenyltetrazolium bromide) (MTT) was obtained from Sigma-Aldrich Inc. (St. Louis, MO or Steinheim, Germany). PTX and DTX were purchased from LC Laboratories (Woburn, MA).

All other materials were from Fisher Scientiﬁc Inc. (Fairlawn, NJ), and all reagents were HPLC grade. MDA435/LCC6 (LCC6-WT) and MDA/LCC6mdr1 (LCC6-MDR) cells were obtained from Dr.

R. Clarke, Georgetown University Medical School, Washington, DC. LCC6-MDR cells, which express high levels of Pgp, were derived from LCC6-WT cells transfected with a retrovirus engineered to constitutively express the mdr1 gene [239]. Cells were cultured in DMEM medium (Gibco 11965-092) supplemented with 10% FBS and 1% penicillin−streptomycin. The

T11 orthotopic syngeneic transplant model is derived from the p53 null strain described by Dr.

Medina [259]. The T11 tumors were originally developed by serial orthotopic transplantation of a murine breast tumor derived from a p53-null mouse into a syngeneic p53 competent recipient, carrying sporadic, somatic K-Ras mutation and exhibiting an RNA expression pattern characteristic of the human claudin-low disease. Tumors growing out of this GEM were evaluated through RNA Microarray analysis as described recently [260]. Most tumors were determined to be of the triple-negative phenotype and T11 was chosen as the most representative Claudin-low. T11 cells were cultured in RPMI medium containing 10% FBS and

1% penicillin/streptomycin. Nude mice were purchased from NCI and housed in UNC DLAM animal facility.

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Preparation and characterization of POx/taxoids micelles

The amphiphilic triblock copolymers [P(MeOx33-b-BuOx26-b-MeOx45), Mn = 10.0 kg/mol, dispersity (Ð = 1.14)], whose synthesis has been described previously [39], was used to prepare formulations of the 3rd-generation taxoids in polymeric micelles. Drug loaded POx micelles were prepared using the thin film hydration method [38]. Briefly, predetermined amounts of POx and drugs (stock solution 10-20 g/L in ethanol) were combined with small amount of ethanol and mixed well. Following a complete removal of ethanol (first, by drying the solution under a stream of air and second, in vacuo), the formed thin film was re-dispersed with appropriate amounts of deionized (DI) water or saline and heated at 50-60 °C for 5-20 min (heating time was dependent on the drug concentration). Samples were allowed to cool to RT and centrifuged at 10,000 rpm for 3 min (Sorvall Legend Micro 21R Centrifuge, Thermo

Scientific) to remove residual solid (if present). Only transparent supernatant was used for the subsequent experiments. The hydrodynamic diameter and PDI of the micelles were determined by DLS using a Malvern Nanosizer and monitored for up to 9 days at r.t. for stability test.

High Performance Liquid Chromatography (HPLC) Analysis of drugs in POx micelles

The amounts of drugs solubilized in POx micelles were quantified via reverse-phase HPLC using an Agilent Technologies 1200 series HPLC system using a Nucleosil C18 5μm column (250 mm × 4.6 mm). The sample was diluted 20 times using mobile phase (specified below) and injected (20 μL) into the HPLC system. A mixture of acetonitrile (ACN)/water (55/45, v/v) was used as mobile phase. The flow rate was 1.0 mL/min, and column temperature 30° C.

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Detection wavelength was 228 nm.

The following equations were used to calculate the drug loading capacity (LC), loading efficiency (LE): m LC drug 100% , (1) mdrug  mexcipient

m LE  drug 100%  mdrug added , (2)

where mdrug and mexcipient are the weight amounts of the solubilized drug and polymer excipient in the solution, while mdrug added is the weight amount of the drug added to the dispersion. Drug concentration (DC) was determined by HPLC and calculated against free PTX standards.

In vitro drug release

The drug release from POx micelles was studied using the membrane dialysis method against PBS, pH 7.4 at 37 °C. Briefly, the drug loaded POx micelle formulations were diluted with PBS to yield solutions of approximately 0.1 mg/mL of each drug. Then the resulting solutions (100 μL) were placed in 100 μL floatable Slide-A-Lyzer MINI dialysis devices with a

MWCO of 3.5 kDa (Thermo Scientific) and suspended in 20 mL of PBS. One device was used for every time point. At each time point the sample was withdrawn from the dialysis device and the remaining drug amount of sample was quantified by HPLC.

In vitro cytotoxicity assay

In vitro cytotoxicity of drug-loaded POx micelles was determined using MTT assay. Four

117 formulations, namely Taxol®, Abraxane®, POx/PTX and POx/SB-T-1214 were compared using each cell line. Briefly, cells were seeded in 96-well plates at a density of 4000 cells/well 48 h prior to drug treatment. Cells were treated for 24 h with respective drug formulations each prepared at series of dilutions in the full medium. After this incubation, medium was removed and cells were further incubated with fresh medium for another 72 h. Subsequently, the medium was again removed and 100 μL of fresh medium with MTT (100 μg/well) reagent was added for additional 4 h incubation at 37 °C. Finally, the medium was discarded, and the formed formazan salt was dissolved in 100 μL of DMSO and absorbance was read at 562 nm using a plate reader (SpectraMax M5, Molecular Devices). Cell survival was calculated as normalized to control untreated wells. Data is presented as means (n = 6) ± standard error means (SEM). The mean drug concentration required for 50% growth inhibition (IC50) was determined using Graphpad Prism 5 software.

In vivo MTD of drug-loaded POx micelles

All animal experiments were carried out with approval of the University of North Carolina

Institutional Animal Care and Use Committee. MTD evaluation for POx/SB-T-1214 micellar formulations was performed in dose escalation study in 6-8 week old female NCI nu/nu mice.

Animals (n = 3 per group) received i.v. injections (tail vein) of 20, 40, 60, 90, and 120 mg/kg of

SB-T-1214 in POx micelles using a q4d x 4 regimen (total 4 times repeated dosing, every 4th day with saline as a control). Mice survival and changes in body weight were observed daily over two weeks in all groups following the last injection. The highest dose that did not cause animal death or noticeable toxicity (as defined by a median body weight loss of 15% of the

118 control or abnormal behavior including hunched posture and rough coat) was used as MTD for efficacy experiment.

In vivo efficacy study

The efficacy of POx/SB-T-1214 polymeric micelles was evaluated in LCC6-MDR orthotopic

BC model. Briefly, 100 μl of cell solution containing 50 % (v/v) 8x106 LCC6-MDR cells suspended in DMEM medium (vide supra) and 50 % (v/v) Matrigel are implanted into mammary fat pad of 8-week-old female nude mice using a 25 G needle. Every 4 days, perpendicular tumor diameters were measured by digital caliper and used to calculate tumor volume according to the formula: volume = Dd2/2, where D equals larger diameter and d equals smaller diameter. When tumor volumes reached about 300 mm3, animals were treated with all formulations by q4d x 4 regimen. Following treatment groups (n = 7) were compared: 1) Saline; 2) POx Polymer; 3) Taxol® (20 mg/kg PTX); 4) Abraxane® (80 mg/kg PTX);

5) Cremorphor (Cre)/SB-T-1214 (20 mg/kg); and 6) POx/SB-T-1214 micelles (20 mg/kg). Tumor volume and survival were monitored 2 times per week. Mice were sacrificed when tumor reached volume of 2000 mm3 or developed ascites metastasis.

The efficacy of POx/SB-T-1214 micelles was also investigated in T11 murine BC orthotopic syngeneic transplant (OST) model (Claudin-low subtype). Tumor volumes reached about 10-

50 mm3 on the 5th day following T11 cell transplant. This was defined as day 0. On day 4, we started to treat animals with all formulations by q4d regimen until tumor remission or experimental endpoints. The following treatment groups (n = 10) were compared: 1) Saline;

2) Taxol® (20 mg/kg PTX); 3) Cremophor (Cre)/SB-T-1214 (20 mg/kg, MTD dose); and 4)

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POx/SB-T-1214 micelles (20 mg/kg). Tumor volume and survival were monitored 3 times per week. Mice were sacrificed when tumor reached a volume of 3500 mm3 or upon signs of ulceration.

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4.4 Results

Formulation of new taxoids in POx micelles

Here, we employed an amphiphilic triblock copolymer [P(MeOx33-b-BuOx26-b-MeOx45),

rd Mn = 10.0 kg/mol, ĐM = Mw/Mn = 1.14)] polymeric micelle formulations of 3 -generation taxoids using a thin-film approach. The chemical structures of investigated taxoids and PTX are depicted in Fig. 4.1a-c. Stock solutions of these drugs and the polymer were prepared and combined in appropriate ratios. The solvent was removed and the resulting polymer film was hydrated using deionized water or USP saline (Fig. 4.1d), resulting in the formation of the drug loaded polymer micelles. The polymer concentration in the final formulation was set to 10 g/L, while the drug concentration was varied from 5 g/L to 15 g/L. The actual maximum loading capacity, LC that was achieved for the different drugs was between 40 and 50 wt.% for 10 g/L or 12 g/L (15 g/L in one case (SB-T-121602)), respectively (Fig. 4.1e). Similar to previous studies [38, 39], the drug loading efficiency, LE were high, until the maximal LC values were achieved, after which LC dropped considerably for all taxoids investigated (Table 4.1). Due to the limited amounts of compounds available, no extensive stability tests were performed.

However, during our experiments, we did not encounter any stability issues and the stability of PTX in POx micelles has been extraordinarily high in previous studies [38, 39].

Physicochemical characterization and drug release of POx/SB-T-1214 micelles

According to previous studies [253, 255], SB-T-1214, a 3rd-generation taxoid, is an excellent candidate to overcome drug resistance. It exhibited high activity in vitro against many drug resistant cancer cell lines including LCC6-MDR, NCI/ADR-RES, 1A9PTX10, 1A9PTX22,

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CFPAC-1, PANC-1, MIA PaCa-2, BxPC-3, and CFPAC-1. Furthermore, its anti-tumor activity was also evaluated in vivo using a Pgp+ human colon cancer DLD-1 xenograft tumor model [255].

In this study Tween 80/ethanol was used as excipients for solubilizing the drug.

Therefore, we solubilized SB-T-1214 in POx polymeric micelles for further in vitro and in vivo activity studies. In order to scale up the formulation for the animal work, we set the polymer concentration at 50 g/L and used 10, 20 and 45 g/L as the initial drug feeding concentrations. Still at such high concentrations the drug incorporation into the micelles was nearly quantitative and the LC values achieved were excellent – 16 wt% (10 g/L), 28 wt.% (19.2 g/L) and 46 wt.% (41.8 g/L), respectively. These formulations will be denoted 50/10, 50/20 and 50/40, respectively. Noteworthy, the highest achieved drug concentration of 41.8 g/L in the POx micelles is ca. 9500 fold of the intrinsic solubility of SB-T-1214 in water at RT (4.4 mg/L, determined by optimized shake-flask method). The size of the drug-loaded POx micelles was determined by DLS. The data suggest that the size (z-average hydrodynamic diameter Dz) of the drug-loaded micelles depends on the loading but remains below 100 nm: 15 nm, 26 nm and 75 nm for 50/10, 50/20 and 50/40 formulations, respectively. Moreover, the drug-loaded micelles were rather well defined displaying a relatively small PDI (<0.2) (Fig. 4.2a) and nearly spherical morphology as was evidenced by TEM micrograph (Fig. 4.2b, c). These results correspond well to our previous results with PTX formulations [39]. We monitored the formulations for 9 days at r.t. and observed no significant changes in the particle size and PDI

(Fig. 4.2d, e), suggesting that the micelles were stable at r.t. for at least 9 days. Also, no drug crystallization was observed by visual inspection. This is important to note, since taxoids typically exhibit a tendency for crystallization and it is often difficult to obtain formulations

122 that are stable in aqueous media.

In contrast, as we applied sink conditions in a dialysis experiment to test the potential release of the drug from the micelles, the drug was continuously released, with > 80% of drug being released after 24 h. Under the present experimental conditions, a burst release was observed. The release was essentially identical for both 50/20 and 50/40 formulations.

However, the release of the drug from the 50/10 formulation was significantly slower with less of a burst release character (Fig. 4.2f). Accordingly, there was little concern that the drug would not be released, despite the excellent stability in the absence of sink conditions.

In vitro cytotoxicity of POx/SB-T-1214 micelles in cancer cells

Since SB-T-1214 was known to be effective against MDR cell lines that overexpress Pgp

[255], we evaluated the in vitro drug efficacy of the 50/40 formulation and compared to

Abraxane®, Taxol® and POx/PTX formulations in wild-type LCC6-WT and multidrug resistant

LCC6-MDR cells using MTT assay.

In multidrug resistant LCC6-MDR cells, the cytotoxicity profile of POx/SB-T-1214 clearly shifted to lower concentrations as compared to the other three formulations of PTX. IC50 value was determined as 34.6 ng/mL for POx/SB-T-1214, which was much lower than 769, 536, and

1385 ng/mL, determined for Abraxane®, Taxol® and POx/PTX, respectively (Fig. 4.3a). We also performed MTT assays in LCC6-WT cells and observed IC50 values of the same order of magnitude for all four formulations, specifically 5.8 for POx/SB-T-1214 and 9.4, 18.4 and 17.8 ng/mL for Abraxane®, Taxol® and POx/PTX, respectively (Fig. 4.3b). When comparing the effectiveness of the drug formulations in resistant vs. wild-type cells using the Resistance

123 factor (R/S) = (IC50 for drug resistant cell line, R)/(IC50 for drug-sensitive cell line, S), we found that POx/SB-T-1214 had R/S value of ca. 6 while for Abraxane®, Taxol® and POx/PTX values of around 82, 29 and 78, respectively were obtained. This result suggests that POx/SB-T-1214 is very potent against both non-resistant wild-type and MDR cells, while the other three formulations containing PTX are only active against wild-type and are less efficient against

MDR cells (Table 4.2).

In addition, we tested the extremely aggressive T11 murine cancer cell line, which is characterized as a claudin-low subtype of TNBC and known for its extremely poor prognosis.

Similar to the result observed using LCC6-MDR cells, the IC50 was about 23 times lower for

POx/SB-T-1214 (43 ng/mL) as compared to POx/PTX (983 ng/mL) (Fig. 4.3c; Table 4.2).

In vivo MTD studies

The MTD evaluation was performed in a dose escalation manner in healthy 6-8 week female nude mice, which received 20, 30, 40, and 60 mg/kg of POx/SB-T-1214 (50/40) micelles using a q4d x 4 regimen. At 30 mg/kg dose, the animal lost more than 15% body weight after the second dosing. Therefore, the MTD was determined as 20 mg/kg at this dosing regimen

(Fig. 4.4a).

We hypothesized that at higher polymer content, the drug release might be slower

(analogous to our in vitro results) and thus, the MTD might be higher. Therefore, we investigated whether changes in the formulation or the dosing regimen would lead to an increase of MTD. However, adjustments of the formulations were not successful in this respect.

Since changes of the polymer/drug ratio in the formulation (50/10 or 50/20 at 40 mg/kg, Fig.

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4.4b) did not improve the MTD in any way, we modified the treatment regimen by increasing dose intervals to weekly injection for 4 weeks (q7d x 4). This improved the situation slightly.

Mice body weight loss remained below 15% until the third injection at day 21. Thus, we concluded that MTD was less than 40 mg/kg under this weekly dosing regimen (Fig. 4.4b).

In vivo efficacy in the LCC6-MDR model

In vivo efficacy of POx/SB-T-1214 was evaluated in the orthotopic LCC6-MDR mouse model using MTD doses for all groups to achieve the best therapeutic effects possible (Fig.

4.5). The Cremophor (CRE)/SB-T-1214 treatment group displayed a similar growth rate as groups treated with saline and POx polymer alone groups - all showing the tumor volume increased from ca. 300 to 2,000 mm3 during 4 weeks. Taxol® slightly decreased the rate of the tumor growth but the difference was not statistically significant as compared to saline,

CRE/SB-T-1214 and POx groups. In contrast, in the POx/SB-T-1214 treatment group, the tumor volume reached only ca. 700 mm3 on the 28 day. (Fig. 4.5a). Abraxane® at 80 mg/kg (MTD dose) also showed significant tumor inhibition compared to CRE/SB-T-1214. However, while the tumor growth curve in the Abraxane®-treated group was similar to that in the POx/SB-T-

1214 group, no statistically significant difference in survival was observed between the

Abraxane® and saline groups (Fig. 4.5b). In contrast, treatment with POx/SB-T-1214 significantly extended the survival time with a median survival of 67 days (p = 0.0003). Median survival in Abraxane®, CRE/SB-T-1214, Taxol® and saline groups were 37, 46, 37 and 33 days, respectively (Fig. 4.5b). Representative images of mice at day 26 with orthotopic tumors clearly show the differences in the tumor burden between these groups with a visibly

125 decreased tumor burden in the POx/SB-T-1214-treated mice (Fig. 4.5c).

Body weight loss over 15% or other signs of severe toxicity were not observed in any treatment group, although the animals in the CRE/SB-T-1214 and POx/SB-T-1214 groups showed about 10% weight loss after four injections, which was regained after 3 weeks (Fig.

4.5d). Injection site inflammation and sometimes prompt shock following injections (animals eventually recovered) were seen in Taxol® and CRE/SB-T-1214 groups, which was probably associated with the excipient Cremophor EL comprising the formulations.

We also tested our POx/SB-T-1214 (50/40) formulation in the very aggressive T11 orthotopic syngeneic transplant (OST) model. At 20 mg/kg, POx/SB-T-1214 was able to suppress the tumor growth to some extent. The treatment outcomes in the CRE/SB-T-1214 and Taxol® groups differed significantly. In the CRE/SB-T-1214 treatment groups the tumors rapidly proliferated to 3,000 mm3 within 20 days and no effect of the treatment was observed compared to the control (Fig. 4.6a). The Kaplan-Meier survival plots (Fig. 4.6b) show that 90% of mice in this treatment group needed to be sacrificed at day 19, which is a worse outcome as compared to saline control. The animals treated with Taxol® fared only little better, if at all.

In contrast, POx/SB-T-1214 significantly improved survival time such that, at day 20, only one mouse needed to be sacrificed. The survival curve declined gradually and 30% of mice survived until day 27.

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4.5 Discussion

New and clinically relevant formulations of taxanes and taxoids remain a matter of considerable interest. For instance, an amphiphilic block copolymer consisting of PEG and 4- phenyl-1-butanol modified polyaspartate was designed to physically entrap PTX. During the self-assembly process, the polymer forms micelles, which incorporate PTX into their core through hydrophobic interactions between the drug and modified polyaspartate (hydrophobic segment). This formulation, designated NK105, can incorporate 23% (w/w) PTX and has shown less toxicity and enhanced efficacy compared to free drug in the preclinical and clinical development [37, 240]. Another example is the targeted PEG-PLGA polymer formulation of

DTX using an RNA aptamer A10 as targeting moiety that binds to the extracellular domain of the prostate-specific membrane antigen on the surface of prostate cancer cells [261]. It has been shown that the targeted nanoparticles enhance cellular uptake compared to their non- targeted counterparts in vitro and in vivo. However, this delivery system may be limited by its rather large particle size (160-290 nm) and its very low drug loading (<1%).

Similar to our previous work on POx polymeric micelles for PTX [38, 39, 50], we used the triblock copolymer with a central hydrophobic BuOx block and two flanking hydrophilic MeOx blocks in this study. The molar mass of the polymer is approximately 10 kg/mol. Therefore, while the polymer micelles (> 10 nm) are likely to be well above the renal excretion threshold, the unimers are well below this threshold and thus expected to be rapidly excreted by renal filtration. Since the synthesis of POx is based on a living cationic ring-opening polymerization, the polymers are well defined and accessible in a reproducible manner. In addition, POx

(co)polymers alone displayed very little, if any, toxicity up to concentrations of 10 g/L in 127 various cell lines [38, 199, 202, 214]. We have previously demonstrated the extremely high loading capacity of the POx micelles for several hydrophobic drugs [38, 39, 50]. In the present work, for all taxoids studies LC values between 45 and 50% were achieved while SB-T-121303 could be loaded at over 50%.

Similar to previous studies [39, 50] the drug loaded spherical micelles were found to be well defined (PDI <0.2) and relatively small with Dz of 15 to 75 nm, which did not change in size over 9 days at r.t.. The morphology and size of the loaded micelles during drug release has not been determined so far. In a recent study [39], we investigated the morphology of the micelles in dependence of different PTX concentrations. Based on these results, it can be expected that for the low drug concentrated formulation (50/20 25 and 50/10) no change in micelle size should occur during drug release. Comparing the results of Schulz et al. and present study, we deduce that the micelle size and likely the morphology are very similar, whether PTX or other taxoids are loaded. On the other hand, for the high loaded micelles a decrease in size might be likely with decreasing drug concentration. For the formulation 50/20 and 50/40 there might be also a change in morphology upon complete release of the drug. In the above mentioned study a change of morphology of the micellar core towards a raspberry- like shape was observed via small angle neutron scattering (SANS) at 9wt% PTX and higher.

There is also the possibility of formation of wormlike micelles at very low PTX concentration.

However, we would also like to note that such studies of size and morphology were obviously not performed in vivo. Such an endeavor would be virtually impossible at the current state of art. Relevance of in vitro release and size and morphology for in vivo performance is in any way questionable.

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In the present study, the POx micelle formulation of taxoid SB-T-1214 improved the drug efficacy in the LCC6-MDR model as compared to PTX formulation or SB-T-1214 formulated with Cremophor EL and ethanol, the vehicle used in the commercial Taxol® formulation. The reason for this increased efficacy remains unknown to date. A detailed pharmacokinetic study may help to elucidate this matter, but this was outside the scope of the present study. We also found that Abraxane® effectively reduced the tumor growth, but did not prolong the survival.

It is noteworthy that Abraxane® was much less effective than SB-T-1214 in vitro but at MTD exhibited similar tumor growth inhibition as SB-T- 1214 in vivo. In this regard we would like to point out that a correlation between in vitro tumor growth inhibition and in vivo efficacy is not straightforward and generally should not be expected. In this regard in vitro experiments performed on cell monolayers may only reflect that a researched compound is pharmacologically active. In contrast, the in vivo efficacy accounts for a much more complex set of factors including a drug distribution to the tumor, cancer cells heterogeneity, interactions with the tumor microenvironment and contribution of the off-target side effects at the level of the whole organism. The “mismatch” between in vitro and in vivo activities is well documented in the literature for many drugs. For example, two highly selective progesterone receptor modulators showed 4-fold potency difference in vitro, while exhibiting similar efficacy in rats against mutagen 7,12-dimethylbenz[a]anthracene induced breast tumor [262].

Another example involves the derivatives of statin-class drugs. One such derivative cerivastatin, is 6 to 7 times more potent than another derivative, pitavastatin in vitro in U87 glioma and MDA-432 BC cell lines. However, cerivastatin demonstrated similar, if not worse,

129 tumor inhibition compared pitavastatin in vivo [263]. Moreover, a mismatch between in vitro and in vivo efficacy was also reported for different formulations of paclitaxel, specifically,

Taxol® and Abraxane®. Thus, Demeure et al. reported that Abraxane® has similar in vitro inhibition (IC50 = 0.33 μM = 282 ng/mL) in H295R cells, as Taxol® (IC50 = 0.35 μM = 299 ng/mL).

However, Abraxane® showed a significantly better efficacy than Taxol® in vivo in H295R xenograft adrenocortical cancer model. Another example also suggests that Abraxane® has similar in vitro activity as Taxol® but outperforms the latter in tumor inhibition in vivo in pediatric solid tumors [264]. Overall, the improved efficacy and higher response rate to

Abraxane® in preclinical and clinical studies compared to other drug formulations [252] may be attributed to the gp60 mediated transport of paclitaxel-loaded albumin into tumor cells or its binding to an extracellular matrix protein, SPARC (secreted protein acidic rich in cysteine), which increases Abraxane® accumulation in the tumor. We also would like to point out that in our work Abraxane® is used in vivo at its MTD dose (80 mg/kg), and is much more efficient in tumor inhibition than Taxol® at 20 mg/kg while having comparable efficacy to POx/SB-T-

1214 micelles at 20 mg/kg. At the same time the effect of Abraxane® on the animal’s lifespan non-significant compared to Taxol® and both agents are much less effective in this regard than POx/SB-T-1214 micelles.

In addition to LCC6-MDR tumors, we also evaluated our POx/SB-T-1214 formulation using the T11 murine cancer model. This is an extremely aggressive model that faithfully recapitulates Claudin-low BC, a subtype of TNBC recently classified via gene expression profiling, exhibiting particularly poor prognosis [202, 260]. It is an OST model, which was established via isolating cells from the mammary gland of genetically engineered balb/c mice

130 which were null for p53, and genetically engineering them to tumor cells carrying claudin-low subtype and subsequently transplanting the cells orthotopically. Abraxane® was excluded in this set of studies due to expected immunogenicity upon injecting human albumin to immuno- competent mice. It should be noted that most previously tested chemotherapeutic drugs were non-effective in this model and typically tumor growth curves of groups treated with a single drug typically show no difference to control [246].

The in vitro MTT results suggested that SB-T-1214 is more active than PTX in T11 cells.

The T11 cells might be intrinsically resistant to chemotherapy with agents such as PTX. Despite the inability to produce long-term survivors, the performance of our formulation in vivo is very promising when one takes into account the inability for other single drug chemotherapeutic regimens to achieve any efficacy in this model [246]. Although these aggressive tumors will ultimately continue to grow, combination therapies [49] that involve our POx micelle delivery system and new generation taxoids along with other anticancer drugs are worth exploring in the future. The present platform readily allows for combination therapy [50] and is therefore very well suited for exploring new treatments of such challenging cancer models.

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Table 4.1 Data of solubilization experiments. Polymer concentration was set to 10 g/L. Data is presented in means ± standard deviation (n = 3).

Note: DC, final loaded drug concentration; LE, loading efficiency (loaded drug concentration/initial drug feeding concentration*100%); LC, loading capacity (final drug wt./total micelles wt.*100%).

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Table 4.2 IC50 values of POx/SB-T-1214 micelles vs. other PTX formulations

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Figure 4.1 Preparation of POx/taxoid micellar nanoformulations. a, b, Chemical structures of 3rd-generation taxoids. c, Chemical structure of PTX. d, Schematic showing of formation of POx/taxoid micelles formed through self-assembly. e, Drug loading of taxoids in POx micelles. POx concentration was fixed at 10 g/L while taxoid feeds were 5, 10, 12 and 15 g/L, respectively.

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Figure 4.2 Physicochemical properties of various POx/SB-T-1214 polymeric micelle formulations. a, Size (z-average, Dz) and size distribution of POx/SB-T-1214 at 50/40 and 50/20 ratios measured by DLS. b,c, TEM micrograph of POx/SB-T-1214 at 50/40 and 50/20 ratios. Scale bar = 100 nm. Stability of the POx/SB-T-1214 micelles at r.t. as determined by the size d, and PDI e, measurements over time. f, Drug release profiles of SB-T-1214 from POx micelles at different polymer/drug ratios of 50/40, 50/20 and 50/10. The drug release study was performed at 37 °C in PBS buffer at pH 7.4.

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Fig. 4.3 In vitro cytotoxicity of various PTX and SB-T-1214 formulation in a, LCC6-MDR cells, b, LCC6-WT, and c, T11 cells (mean ± SEM, n = 6).

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Fig. 4.4 Establishment of the safe dose of SB-T-1214 in nude mice. a, MTD of POx/SB-T-1214 = 50/40 formulation using a q4d x 4 treatment regimen in escalating doses from 20-60mg/kg. b, MTD of POx/SB-T-1214 = 50/20 and 50/10 using a q4d x 4 regimen or 50/40, 40 mg/kg using a q7d x 4 regimen.

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Fig. 4.5 Efficacy of various drug formulations in LCC6-MDR tumors. a, Tumor growth inhibition of POx/SB-T-1214=50/40 formulation (20 mg/kg) compared to Taxol® (20 mg/kg), Abraxane® (80 mg/kg) and CRE/SB-T-1214 (20 mg/kg), saline as well as POx polymer alone (equivalent polymer amount as POx/SB-T-1214 micelle formulation). Each formulation was injected on days 0, 4, 8, 12. Data is expressed as mean ± SEM, n = 7. *** p < 0.001 (vs. saline group). b, Kaplan-Meier survival plot for all groups. c, A representative image of treated mice. Left, saline group; middle, CRE/SB-T-1214 group; right, POx/SB-T-1214 group. d, Body weight loss for each treatment group.

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Fig. 4.6 Efficacy of various drug formulations in T11 OST tumors. a, Tumor growth inhibition of POx/SB-T-1214 = 50/40 formulation compared to Taxol®, CRE/SB-T-1214 as well as saline. Each formulation was injected on days 2, 6, 10, 14 and 18. Data is expressed as mean ± SEM, n = 10, ** p < 0.01. b, Kaplan-Meier survival plot for all groups.

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CHAPTER V: OTHER APPLICATIONS OF P2 POX POLYMERS (2) SYNERGISTIC COMBINATIONS OF MULTIPLE CHEMOTHERAPEUTIC AGENTS IN HIGH CAPACITY POX MICELLES 1

5.1 Summary

Many effective drugs for cancer treatment are poorly water-soluble. In combination chemotherapy, needed excipients in additive formulations are often toxic and restrict their applications in clinical intervention. Here, we report on amphiphilic POx micelles as a promising high capacity delivery platform for multi-drug cancer chemotherapy. A variety of binary and ternary drugs combinations of PTX, DTX, 17-allylamino-17- demethoxygeldanamycin (17-AAG), etoposide (ETO) and bortezomib (BTZ) were solubilized in defined polymeric micelles achieving unprecedented high total loading capacities of up to 50 wt.% drug per final formulation. Multi-drug loaded POx micelles showed enhanced stability in comparison to single-drug loaded micelles. Drug ratio dependent synergistic cytotoxicity of micellar ETO/17-AAG was observed in MCF-7 cancer cells and of micellar BTZ/17-AAG in MCF-

7, PC3, MDA-MB-231 and HepG2 cells.

1 This chapter previously appeared as an article in Molecular Pharmaceutics. The original citation is as follows: Han, Yingchao*, He, Zhijian* et al., “Synergistic combinations of multiple chemotherapeutic agents in high capacity poly(2-oxazoline) micelles”, Molecular Pharmaceutics, 2012, 9(8):2302-13. * Authors contributed equally.

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5.2 Introduction

Single drug therapy of cancer is rarely successful due to inherent and developing drug resistance of tumors. Even those tumors that initially respond to such therapy subsequently nearly inevitably adopt resistance by generating inhibitors against apoptotic stimuli or activating multi-drug resistant genes (e.g.: MDR1) [265]. Furthermore, most tumors comprise heterogeneous cell populations and are sustained in growth by small populations of “tumor initiating cells” (TICs), which have high proliferation potential and are inherently resistant

[266-269]. Survival of TICs during chemotherapy can result in tumor progression and recurrence. Thus clinical use of combination chemotherapies became a standard of treatment for most types of cancers [270-273]. Such therapy regimens commonly involve sequential administration of multiple drugs that can act along different and synergistic pathways and kill cancer cells better. Combining such drugs in one vehicle could simplify treatments and make it less hazardous to patients. Yet, many drugs are incorporated in different vehicles that are often incompatible with each other. Therefore, common vehicles that include multiple drugs are needed for next generation therapies.

Some researchers have sought such multi-drug delivery systems. For instance, micelles of stearate-grafted chitosan oligosaccharide (CSO-SA) were used for co-delivery of PTX and doxorubicin [274]. Another study employed nanoparticles of PLGA for simultaneous delivery of vincristine (VCR) and verapamil (VRP) [275]. A liposomal delivery formulation for quercetin and VCR was also developed [276]. Additionally, Kwon et al. reported that PEG-b-PLA micelles can deliver multiple drugs including combinations of PTX/17-allylamino-17- demethoxygeldanamycin (17-AAG), etoposide (ETO)/17-AAG, DTX/17-AAG and PTX/ETO/17-

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AAG [277].

To make this approach practical and translate it to clinic the multi-drug vehicles must be safe to patients and have high loading capacity with respect to multiple drugs. The latter remains a principal limitation for most drug delivery systems carrying water-insoluble drugs.

For example, most polymeric micelle systems have a loading capacity for water-insoluble drugs not exceeding 10 to 15 % by weight of the dispersed phase [38]. Such mediocre loading can be an impediment for even a single drug formulation, while for a multi-drug vehicle it becomes a principal roadblock to pharmaceutical development. Since treatment doses in chemotherapeutic regimens cannot be significantly altered, the low loading of multiple drugs would require using prohibitively high doses of the vehicle proper, which could result in the vehicle-derived toxicity. A well-known example of dose limiting vehicle toxicity is Taxol®, a clinical formulation of PTX, which contains as excipient Cremophor EL (PEGylated castor oil) that can induce hypersentitive reactions as well as neuro- and nephrotoxicities during IV infusions [18, 19, 21].

Therefore, in an attempt to develop a multi-drug vehicle this work focused on polymeric micelles of amphiphilic POx block copolymers that were recently shown to have an unprecedented high capacity for PTX of up to 45 % wt.. [38] POx has attracted increasing interest for biomedical applications due to their tunable properties and structure, which compare favorably to polyethers, such as PEG [278]. The POx backbone with pending amide moieties is highly hydrated while the alkyl side chains of varying length can provide for amphiphilic character of the monomer units (non-ionic polysoap) and determine the water solubility of the polymer. Variation of the length of the alkyl side chain in POx allows fine-

142 tuning the hydrophilic-lipophilic balance of each monomer unit, as well as of the entire polymer chain. For example, a short side chain POx, pMeOx is highly hydrophilic while pEtOx shows some amphiphilicity, comparable to PEG [279] and displays a temperature-dependent water solubility (lower critical solution temperature). Both, pMeOx and pEtOx exhibit stealth properties and biodistribution similar to PEG when administered alone or grafted onto liposomes [214, 279-281]. Poly(2-propyl-2-oxazoline) displays lower critical solution temperature around 25°C (n-propyl) and 47°C (i-propyl) [219]. PBuOx is water-insoluble at RT and suitable to build a hydrophobic block in POx block copolymers, in which PMeOx or PEtOx are used as hydrophilic blocks. Such block copolymers form stable polymeric micelles and their interaction with biological entities (e.g. rate of endocytosis) can be fine-tuned through the polymer structure [199]. We have recently shown that the PBuOx core of such micelles can efficiently incorporate PTX. As a result drug-containing aggregates of defined size are produced, in which the drug can comprise nearly half of the aggregate mass [38]. These PTX- loaded POx micelles remained stable throughout lyophilization/redispersion cycles and exhibited low toxicity and complement activation.

This chapter addresses two principal new questions (Fig. 5.1). First, whether different multiple water-insoluble drugs can be simultaneously incorporated into such POx micelles with high loading capacity preserved. Second, whether these drugs can be combined in one

POx micelle formulation to form pharmacologically synergistic chemotherapeutic combinations of high potency to kill cancer cells. The results suggest that POx micelles are a promising high-capacity multi-drug delivery platform for combination cancer chemotherapy.

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5.3 Materials and methods

Materials

The amphiphilic triblock copolymers (1.batch: P(MeOx40-b-BuOx21-b-MeOx34), Mn = 9.1 kg/mol, ĐM (Mw/Mn) = 1.14; 2. batch: P(MeOx33-b-BuOx26-b-MeOx45), Mn = 10.0 kg/mol, ĐM

(Mw/Mn) = 1.14) were synthesized as described in the previous study [38]. PTX, DTX, 17-AAG and bortezomib (BTZ) were purchased from LC Laboratories (Woburn, MA). ETO was obtained from Sigma-Aldrich Inc. (St. Louis, MO). Alexa Fluor 647 (AF 647)-N-hydroxysuccinimide (NHS) ester labeling kits (A20173) and BODIPY® FL PTX (P7500) were purchased from Invitrogen. All other materials were from Fisher Scientific Inc. (Fairlawn, NJ) and all reagents were HPLC grade

(see all drug structures in Fig. 5.2). The MCF-7 (HTB-22TM), PC3 (CRL-1435TM), HepG2 cells (HB-

8065TM) and MDA-MB-231 (HTB-26TM) were originally obtained from ATCC.

Preparation of drug loaded POx micelles

Drug loaded POx micelles were prepared using the film hydration method [38]. Pre- determined amounts of POx and drugs (stock solution 10 - 20 g/L in ethanol) were combined with small amount of ethanol and mixed well. Following removal of ethanol (40 °C, air flow), the formed thin film was further dried in vacuo to remove residual solvent. The dried film was subsequently re-dispersed with appropriate amounts of deionized (DI) water and heated at

50 - 60 °C for 5 - 20 min (heating time dependent on the drug concentration; for example,

5 min for low concentration and up to 20 min for high concentration) to procure drug loaded polymeric micelles. POx micelles co-loaded with multiple drugs were prepared accordingly with final polymer concentration of 10 g/L and each drug concentration of 4 g/L.

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HPLC analysis of drugs in POx micelles

The amounts of drugs solubilized in POx micelles were quantified via reverse-phase HPLC using an Agilent Technologies 1200 Series HPLC system using a Nucleosil C18−5μ column 5 μm column (250 mm × 4.6 mm). The product was centrifuged at 10,000 rpm (9630×g) for 3 min

(Sorvall Legend Micro 21R Centrifuge, Thermo Scientific) to remove residual solid and the supernatant was diluted 20 times using mobile phase (specified below) and injected (20 µL) into the HPLC system. For PTX, 17-AAG and the combination of PTX/17-AAG, a mixture of acetonitrile (ACN)/methanol (MeOH)/water (39/38/23, v/v/v) was used as mobile phase. The retention times of PTX and 17-AAG were 5.0 and 7.1 min respectively. Similarly, ACN/water

(55/45, v/v) was applied for DTX, 17-AAG and DTX/17-AAG. The flow rate was 1.0 mL/min and column temperature was 30 °C. Detection wavelength was 227 nm for PTX and DTX, and

333 nm for 17-AAG. The retention times of DTX and 17-AAG were 8.1 and 13.3 min respectively. For all ETO-containing samples, a step-wise gradient was used. First, the analyte was eluted for 10 min with ACN/MeOH/water (0.1 % phosphoric acid, 1% MeOH) (5/5/90, v/v/v) followed by second 10 min elution of ACN/water (0.1 % phosphoric acid, 1% MeOH) at a ratio of 60/40 (1 min transition). Column temperature was 40°C and detection wavelength for ETO was 227 nm. Under these conditions, the retention times of ETO, PTX and 17-AAG were 9.1, 16.9 and 18.7 min, respectively. Accordingly, for samples containing BTZ, another two-step gradient was applied: ACN/water (0.1 % phosphoric acid) 90/10 (v/v) for the first minute; and ACN/MeOH/water (0.1 % phosphoric acid) 35/35/30 (v/v/v) for the following

10 min. The flow rate was 2.0 mL/min and column temperature was 55°C. Detection was performed at 270 nm for BTZ. The retention times of PTX, 17-AAG and BTZ were 1.5, 1.7 and

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3.6 min respectively.

Drug loading calculations

The following equations were used to determine drug loading capacity (LC), loading efficiency (LE) and drug loading (DL):

m LC drug 100%, (1) mdrug  mexcipient

m LE  drug 100%, (2)  mdrug added

m DL  drug 100%, (3)  mexcipient

where mdrug and mexcipient are the weight amounts of the solubilized drug and polymer  excipient in the dispersion, while mdrug added is the weight amount of the drug added to the dispersion.

DLS

The size distribution of micelles was investigated with DLS using a Nano-ZS (Malvern

Instruments Inc., UK). The product was centrifuged at 10,000 rpm for 3 min and the resulting polymeric micelles were diluted 10 times with DI H2O to yield 1 g/L final polymer concentration prior to the measurement. The intensity-mean z-averaged particle size

(effective diameter) and the PDI obtained from cumulant analysis were given to reflect hydrodynamic diameters of drug loaded POx micelles. Measurements were repeated over prolonged time periods in order to evaluate stability of drug loaded micelles.

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Confocal laser scanning microscopy (CLSM)

POx was labeled with AF647 according to the manufacturer's protocol. Briefly, POx (10 mg/mL, 0.5 mL in PBS) was treated with AF647-NHS ester and stirred for 2 h at RT in the dark.

Labeled POx was separated from free dye by gel filtration (First Bio-Rad BioGel P-30 resin in

PBS, then Sephadex LH20 in methanol). The calculated labeling degree was about 25 % according to the method in protocol. Next PTX (BODIPY® FL PTX: PTX=1:40) and 17-AAG were co-loaded into AF647 labeled POx micelles (no dilution with unlabeled POx) according to the method of 2.2.1. CLSM was carried out using LSM710 (Carl Zeiss MicroImaging GmbH,

Germany). MCF-7 cells were seeded into 4-well plates (Lab-Tek®II Chambered #1.5 German

Coverglass System) at a density of 40,000 cells per well for 24 h, and then incubated with fluorescently labeled free PTX, polymer alone or POx micelles co-loaded with PTX and 17-AAG for 1 h and 4 h. Cell nuclei were stained with Hoechst 33342 before CLSM.

Cytotoxicity assay

MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazoliumbromide) assay was conducted to evaluate in vitro cytotoxicity of drug loaded POx micelles. Briefly, cells were seeded in 96-well plates at a density of 4000 cells/well 24 hrs prior to drug treatment.

Subsequently, cells were treated with micelle-formulated drugs at series of dilutions in full medium. Following 24 hrs treatment the incubation medium was removed and cells incubated with fresh medium for another 72 hrs. The medium was removed and 100 L fresh media with

MTT (100 μg/well) reagent was added for an additional 3 h incubation at 37 °C. The media was discarded and the formed formazan salt was dissolved in 100 L DMSO and absorbance

147 was read at 562 nm using a plate reader (SpectraMax M5, Molecular Devices). Cell survival rates were calculated as normalized to control untreated wells. Each concentration was tested in six wells and data presented in means ± standard error means (SEM). The mean drug concentration required for 50% growth inhibition (IC50) was determined using CompuSyn software (Version 1.0, ComboSyn Inc., U.S.) using the median effect equation: Fa =

m -1 [1+(IC50/D) ] , where Fa is the fraction of affected cells, D is drug concentration, and m is the

Hill slope.

Combination index (CI) analysis

CI analysis based on Chou and Talalay method was performed using CompuSyn software

[282]. Briefly, for each level of Fa the CI values for binary drug combinations were calculated according to the following equation: CI = (D)1 / (Dx)1 + (D)2 / (Dx)2, where (D)1 and (D)2 are the concentrations of each drug in the combination resulting in Fax100% growth inhibition, and

(Dx)1 and (Dx)2 are the concentrations of the drugs alone resulting in Fax100% growth inhibition

[282, 283]. CI values for drug combinations were plotted as a function of Fa. Generally, the CI values between Fa = 0.2 and Fa = 0.8 are considered valid. The best-fit CI value at IC50 was used to show and compare the synergistic effects of drug combinations with different drug ratios or for different cell lines. CI values less than 1 or more than 1 demonstrate synergism or antagonism of drug combinations, respectively.

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5.4 Results Preparation and characterization of drug loaded POx micelles

A film hydration method (from ethanol) was used to prepare drug loaded polymeric micelles of POx triblock copolymers, P(MeOx40-b-BuOx21-b-MeOx34 (batch 1, Mn = 9.1 kg/mol,

ĐM = 1.14) and P(MeOx33-b-BuOx26-b-MeOx45 (batch 2, Mn = 10.0 kg/mol, ĐM = 1.14).

According to this method first the drug(s) were blended with the block copolymer, dried, and then the blend was re-dispersed in water. The incorporation of drugs in micelle dispersions was quantified by HPLC (Table 5.1). First, in line with our previous account we were able to dissolve 3.9 g/L of PTX and DTX in 10 g/L copolymer (Table 5.1). Furthermore, we were able to obtain stable micellar solutions of nearly 40 g/L DTX and PTX in 50 g/L copolymer in both distilled water and PBS, which is approx. 100,000 and 7,000 times more than the solubilities of these drugs alone (0.0005 g/L [16] and 0.0055 g/L [277] for PTX and DTX, respectively)

(Table 5.1). In subsequent experiments comparing different drugs and drug mixtures we kept the POx and drug concentrations constant (10 g/L and 4 g/L, respectively) while preparing the solutions. Under these conditions 3.45 ± 0.21 g/L 17-AAG, 3.62 ± 0.18 g/L ETO, and 3.12 ± 0.12 g/L BTZ were solubilized as single drugs (Table 5.1). These apparent solubility values again greatly exceed the inherent solubility of the drugs alone (0.01 g/L 17-AAG, 0.058 g/L ETO, and

0.002-0.004 g/L BTZ).

From the standpoint of the loading efficiency (LE), i.e. the fraction of the drug incorporated into micelles vs. total drug added to the system, the PTX and DTX appeared to be favorable having LE values of about 97 %. The ETO and 17-AAG displayed LE values of about

91 % and 86 %, respectively, while BTZ had lower but very good LE of 78 %. Another key measure of drug solubilization is the loading capacity (LC), i.e. the fraction by weight of the 149 solubilized drug vs. the weight of the entire dispersed phase. In agreement with our previous finding, LC for PTX alone could reach as much as 45 %. Moreover, the same high LC value of

45 % was achieved for DTX. Thus a very small amount of polymer excipient was needed to solubilize these drugs, which are normally very challenging from the standpoint of formulation.

Remarkably, very high LC values were also observed for drug combinations (Table 5.1). Thus,

PTX or DTX in combination with 17-AAG displayed the highest total LC values for binary drug combinations of about 43 % (and over 90 % LE for each drug). ETO combinations with PTX or

17-AAG also showed over 41 % LC and nearly 90 % LE. The LC values of the binary combinations comprising BTZ (PTX/BTZ and 17-AAG/BTZ) were slightly lower but still approached 40 %. Finally, the ternary drug combinations PTX/17-AAG/ETO and PTX/17-

AAG/BTZ displayed very high total LC of about 48 to 49 % and LE of ca. 80 % for each drug

(Table 5.1). That means that only about 1 g of POx copolymer is needed to solubilize 1 g of such drug mixtures.

Size distribution and stability of drug-loaded POx micelles

The size distribution and dispersion stability of the drug-loaded POx micelles are crucial factors for their successful parenteral application. We measured the particle sizes in the dispersions of the drug loaded micelles using DLS and examined the size alterations over various periods of time. The micelles loaded with the single drugs, PTX, DTX or 17-AAG, had small hydrodynamic diameters, 36 nm, 18 nm and 33 nm, and moderate size distributions

(PDI), 0.26, 0.30 (bimodal), and 0.18, respectively. The DLS profiles of the micelles loaded with

ETO or BTZ were bi- or multimodal with broad size distributions suggesting formation of heterogeneous particle populations (Fig. 5.3a). By analyzing these data along with the 150 dispersion stability profiles, we noticed that the larger sizes and broader size distribution patterns of the ETO- and BTZ-loaded micelles were accompanied with their lower stability compared to the micelles loaded with any of the three other drugs. To be more specific, micelles loaded with ETO or BTZ started precipitating after 2 days. In contrast the more uniform and smaller micelles loaded with PTX or 17-AAG remained stable for at least 14 days

(Fig. 5.3b). This excellent stability combined with relatively small PDI values suggested that the PTX or 17-AAG loaded micelles were thermodynamically stable.

Interestingly, the micelles with binary drug combinations containing either ETO or BTZ co-loaded with PTX exhibited more uniform size distribution compared to micelles loaded with ETO or BTZ alone. Their hydrodynamic diameters were 42 nm and 36 nm and PDI values

0.25 (bimodal) and 0.20, respectively. A similar trend was observed for the micelles containing combinations of either ETO or BTZ with 17-AAG, which also were more homogeneous than the micelles with ETO or BTZ alone. The hydrodynamic diameters of these micelles were 79 nm and 57 nm and PDI values 0.20 and 0.13, respectively (Fig. 5.4a). Furthermore, micelles with binary drug combinations containing either PTX or 17-AAG exhibited high stability similar to stability of single-drug micelles loaded with PTX or 17-AAG. For example, the ETO/PTX,

BTZ/PTX and ETO/17-AAG micelles did not change size for 14 days and BTZ/17-AAG micelles had constant size for 7 days (Fig. 5.4b). Finally, micelles loaded with ternary drug combinations

PTX/17-AAG/ETO and PTX/17-AAG/BTZ had hydrodynamic diameters of ca. 53 nm and 99 nm and relatively small PDI values (0.14 and 0.19, respectively) (Fig. 5.5a). Both dispersions were stable for at least 14 days (Fig. 5.5b).

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Internalization of drug-loaded in POx micelles

The BODIPY® FL PTX and AF647-labeled POx block copolymer were used to examine drug and block copolymer internalization and co-localization in MCF-7 cells by CSLM. As shown in

Fig. 5.6a, after 1h incubation the free PTX was distributed in cytoplasm. In contrast the labeled copolymer appeared to be primarily localized in the perinuclear region and mainly, but not exclusively localized in vesicular bodies (Fig. 5.6b). This distribution pattern did not change significantly for the drug-loaded micelles (Fig. 5.6c). There was little co-localization of drug and polymer albeit the drug appeared to be concentrated around the vesicles and/or some other subcellular compartments, in which the polymer was also present. This may indicate that the drug-loaded micelles serve as intracellular drug depots, from which the drugs are released over time. However, the rate of the release is probably faster than the observation time since the overall distribution pattern did not change significantly after 4 h exposure of the same formulation to the cells (Fig. 5.7). Comparing the time scale of this experiment with the time scales observed in the release studies one may conclude that the intracellular release of the drug is faster than its release in the surrounding solution.

In vitro cytotoxicity and analysis of synergistic effects of drug combinations

The toxicity of POx block copolymer alone was determined in MCF-7, PC-3, HepG2 and

MDA-MB-231 cells using MTT assay. In all cell lines, the copolymer displayed little if any toxicity up to the concentration of 200 g/mL (data not shown). To exclude any cytotoxic action of the copolymer on cells the POx concentration used in subsequent experiments did not exceed 40 g/mL. The cytotoxicity of drug combinations solubilized in POx micelles was

152 determined using MCF-7 breast adenocarcinoma cells. Previous reports have demonstrated synergistic cytotoxicity of PTX and 17-AAG in this as well as several other cancer models.37

Therefore, we analyzed the effects of the other binary drug combinations ETO/17-AAG and

BTZ/17-AAG at different drug weight ratios. The first combination, ETO/17-AAG, showed clear synergy at 1:1 ratio, limited synergy (up to Fa = 0.6) at 1:2 ratio, but little synergy or antagonism at 1:0.5 and 1:0.7 ratios (Fig. 5.8). The second combination, BTZ/17-AAG, displayed strong synergy at 1:0.6 and 1:1 ratios and antagonism at 0.5:1 and 0.3:1 ratios (Fig. 5.9).

Since the BTZ/17-AAG combination displayed very pronounced synergistic cytotoxicity in the MCF-7 cancer cell line, we further evaluated this combination in the human prostate cancer, PC3, triple negative human breast carcinoma, MDA-MB-231, and human hepatocellular carcinoma, HepG2 (Fig. 5.10). While this combination (at 1.1:1 ratio) displayed little synergy in HepG2 cells, in both PC3 and MDA-MB-231 cells the synergy was very pronounced (Fig. 5.10). Specifically, the IC50 values of micellar BTZ, 17-AAG and BTZ/17-AAG formulations were 0.062, 4.6 and 0.017 g/mL for PC3 cells, 0.083, 0.67 and 0.006 g/mL for

MDA-MB-231 cells, and 0.095, 0.012, and 0.018 g/mL for HepG2 cells. The corresponding CI values of micellar BTZ/17-AAG combination at IC50 were 0.14, 0.045 and 0.84 for PC3, MDA-

MB-231 cells HepG2 cells, respectively.

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5.5 Discussion

Early publications using polymeric micelles with non-covalently incorporated

(“solubilisized”) drugs go back more than two decades [284], and this technology has undergone clinical evaluation [285]. Separately, combinations of different anticancer drugs in one polymeric vehicle, such as liposomes, nanoparticles, or polymer conjugates have been explored for some time [275, 276, 286]. However, multiple drug combinations co-solublized in polymeric micelles were reported relatively recently by the group of Kwon [49, 277].

Although polymeric micelles are extremely attractive vehicles due to simplicity of preparation and no need of chemical modification of the drug molecules, achieving high loading capacities have been challenging especially for very poorly soluble drugs. This also was a serious obstacle for developing multi-drug combinations in single polymeric micelles. Therefore, recent discovery of high capacity POx micelles that can solubilize such poorly soluble drugs as PTX using nearly 100 times less excipient than the commercial Taxol® and 10 times less than

Abraxane® is a major breakthrough [38]. For comparison, using PEG-b-PLA micelles Kwon et al. [277] reported PTX solubility of 3.54 g/L with LC of ca. 9.3 % (note that these authors present data for DL, which are recalculated in LC in Table 5.2). In contrast, using POx micelles we report therein PTX solubility of 38.7 g/L with LC of 43.6 %. Thus, over ten times more drug was solubilized with only 50 g/L POx) block copolymer compared to 30 g/L PEG-b-PLA.

Furthermore, in the present study extending our previous results POx micelles were shown to have excellent capacity for solubilization of several other poorly soluble drugs, such as DTX,

17-AAG, ETO and BTZ, and their combinations.

The advantages of the POx micelles compared to other polymeric micelle drug delivery

154 systems are clearly seen with several single drugs reported here for the first time. Thus, 10 g/L

POx) block copolymer was sufficient to solubilize 3.45 g/L 17-AAG with LC of 25.6 %. Nearly same amount of this drug (3.9 g/L) was solubilized with as much as 30 g/L PEG-b-PLA (LC 10.2%)

[277]. For ETO these values were 3.62 g/L ETO in 10 g/L POx) (LC 26.6%) vs. 3.31 g/L ETO in 30 g/L PEG-b-PLA (LC 8.7%). For DTX - 3.87 g/L DTX in 10 g/L POx) (LC 27.9%) or 40.6 g/L DTX in

50 g/L POx) (LC 44.8%) vs. 3.31 g/L DTX in 30 g/L PEG-b-PLA (LC 10.3%) [277].

Furthermore, the efficacies of solubilization of the drug blends in POx micelles were similar or better than those observed for single drugs. For example, as the initial drug feed increased from 4 g/L (single drug), to 8 g/L (binary drug combinations), and then to 12 g/L

(ternary drug combinations) the total LC was also increased from 23.8 - 27.9 % to 39.5 - 43.3 % and up to 48.4 - 48.7 % (POx concentration 10 g/L). This compares favorably to other micellar or liposomal multi-drug formulations described in literature. For example, Kwon et al. used 30 g/L PEG-b-PLA copolymer to solubilize two-drug mixture of 3.9 g/L PTX and 3.9 g/L 17-AAG at total LC of 20.6 %. Again, essentially the same concentrations of these drugs were dissolved with 3-times less (10 g/L) POx) block copolymer (total LC 43.1 %). The same amount of this block copolymer solubilized 3.7 g/L ETO and 3.4 g/L 17-AAG (total LC 41.1 %) vs. 3.5 g/L ETO and 4.2 g/L 17-AAG solubilized in 30 g/L PEG-b-PLA copolymer (total LC 20 %). Another direct comparison is a ternary drug combination of 3.5 g/L PTX, 3.6 g/L 17-AAG and 3.2 g/L ETO solubilized using 30 g/L PEG-b-PLA block copolymer at total LC of 26.2 % [277]. Nearly same concentrations of these drugs are solubilized with only 10 g/L POx) block copolymer (total LC of 48.7 %).

Needless to say the drug solublization capacities of POx micelles are superior not only to

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PEG-b-PLA micelles but also to other multi-drug-loaded delivery systems. For example, CSO-

SA micelles incorporated PTX and DOX with LC of only about 5.72 % wt. (PTX: 4.20 %; DOX:

1.52 %) [274]. Hou et al. [275] prepared VCR and VRP co-loaded in PLGA nanoparticles at a total LC of 7.9 %. Chiu et al. [276] developed an original multi-drug liposomal formulation comprising egg sphingomyelin/cholesterol/PEG2000 ceramide/quercetin co-encapsulating quercetin in lipid bilayer and vincristine in aqueous core. The optimal formulation contained about mol. 5 % quercetin and the molar ratio of vincristine to quercetin was 2:1, suggesting that total LC was about 13 - 14 %.

In addition to the high loading capacity the POx micelle formulations displayed high stability [277]. In many cases reported herein, no drug precipitation or changes in the particles sizes for as long as two weeks were observed. For comparison, PTX solubilized in PEG-b-PLA block copolymer micelles under similar conditions precipitated in only 24 hrs [277]. The single drug POx micelle formulations containing ETO or BTZ were more disperse and least stable compared to other drug formulations as they exhibited particle size increase and aggregation started after two to four days. However, when these drugs were blended in binary or ternary drug combinations in POx micelles the stability of the formulations was markedly improved and they displayed more uniform particle sizes compared to single drug formulations.

Noteworthy, Kwon et al also demonstrated a similar synergistic improvement in stabilities of multi-drug loaded PEG-b-PLA micelles [49, 277]. These authors speculated that such increased stability is due to favorable drug-drug molecular interactions in the multiple drug-loaded micelles. Albeit hypothetical, such assumption also seems to be most reasonable explanation for the observed phenomena in our case. On the other hand, in such case, one might expect

156 a significant effect of drug release upon co-solubilization which we did not observe. We also hypothesize that high capacity and stability of the drug-loaded POx micelles can be attributed in part to H-bonding between amide bonds of the BuOx block and the H-bond donors in the drug molecules incorporated in the micelle cores. However, using the described experimental setup, no conclusive evidence was obtained at this time by ATR-IR.

Altogether, POx micelles exhibited greater capacity for drug loading and stability compared to the PEG-b-PLA micelles for every drug studied. A marked increase in the drug loading and decrease in the amount of polymeric excipient needed to solublize chemotherapeutic drugs in POx micelles is likely to translate to lower excipient-related side effects [38]. In this account POx micelles appears to be an extremely attractive drug delivery system and may have great advantage over current methods potentially increasing the safety of clinical interventions. For instance, the current clinical formulation of PTX, Taxol®, contains only 1 % wt. of active drug, the remainder of the formulation being Cremophor EL and ethanol, which is known to induce hypersentitive reactions as well as neuro- and nephrotoxicities during IV infusions unless countermeasures are taken [18, 19, 21, 38]. In a more current PTX formulation, Abraxane®, the drug loading was increased to about 10 % wt., but this technique appears to be challenging from a technological point of view for solubilization of multiple drugs. Dimethyl sulfoxide/lipid or Cremophor EL were employed to solubilize 17-AAG, and

PEG-sorbitan monooleate (Tween 80) was used in ETO formulations, which resulted in many incidents in clinical trials such as patients incompliance (early withdrawal) and serious side effects [287-290]. As consequence combination therapy involving such hydrophobic drugs has been restricted so far due to the lack of a versatile and non-toxic delivery system for combined

157 delivery of hydrophobic drugs. In this regard low toxicity of POx may be an additional advantage. Early studies on the toxicity of PEtOx reported a very high acute oral LD50 of >4 g/kg

(rats) and acute percutaneous absorption LD50 of > 4 g/kg (rabbit) [291]. Viegas et al. [279] recently showed that PEtOx is essentially non-toxic when administered i.v. into rats at amounts of 2 g/kg. Also, repeated administration of 50 mg/kg did not reveal any adverse effects. Our own studies on the biodistribution and excretion using radiolabled PMeOx and PEtOx homopolymers showed a fast distribution of the hydrophilic homopolymers throughout the entire organism (mice) as well as a very rapid renal excretion [214]. Taken together the safety, biodistribution and excretion data for POx polymers one might hope that they can be advanced to the pharmaceutical sector to develop a versatile drug delivery platform.

To further support our rationale for development of multi-drug loaded POx micelles, we studied the tumor cell growth inhibition using selected drug combination and various drug ratios. The studies revealed that synergistic effects can indeed be achieved using ETO/17-AAG and BTZ/17-AAG binary drug formulations and that this effect is strongly influenced by the drug ratio and cell lines used. As discussed previously [271], any synergistic effect greatly depends on drug dosages, combination ratios, cell lines and intervention schedules. For example, 17-AAG is effective in treating solid malignancies and leukemia through inhibiting heat shock protein 90 (HSP90), a chaperone, which interferes with misfolding of various oncogenic proteins [292]. Using a H358 human non-small-cell lung cancer xenograft model,

Nguyen et al. [293] found that 17-AAG conferred 5 to 22 fold increase in antitumor efficacy when combined with PTX, a drug that inhibits tumor cell division by stabilizing microtubuli.

17-AAG was also able to suppress FMS-like tyrosine kinase 3 (FLT3), a client of HSP90 whose

158 mutations as internal tandem duplications (ITD) often correlates with poor prognosis [294]. In

IDT mutated-FLT3 leukemia cells, DNA-repairing related proteins such as Rad51 and checkpoint kinase 1 (Chk1) were under-expressed, making them sensitive to ETO, a topoisomerase II antagonist. Therefore, co-administration of 17-AAG and ETO can be synergistic for suppressing leukemia cells specifically with ITD mutated-FLT3. Indeed, we observed a strong synergy of the binary drug combination ETO/17-AAG for selected drug ratios.

Another synergistic binary combination reported in our study is BTZ/17-AAG. BTZ has been approved to treat multiple myeloma. It inhibits the proteasome machinery undermining the cancer cell ability to eliminate aberrant proteins and thus evade apoptotic checkpoints

[295]. Combination of BTZ with PTX delayed the tumor growth and achieved high tumor- inhibitory effects on Lewis lung carcinoma [296]. Mimnaugh et al. [297] combined 17-AAG with BTZ and found significant inhibition of BC cell proliferation that was superior to either drug used alone. Apart from the mechanism basis, such synergistic effects depend strongly on optimized ratios of combined drugs, so that synergism may be observed at molar drug ratios that differ significantly from unity [271]. For example, co-delivery of camptothecin and doxorubicin to glioma cells at a molar ratio of 1.5:1 resulted in synergistic activity, whereas strong antagonism was observed at a ratio of 5:1 [271]. Similarly, in our study both binary drug combinations ETO/17-AAG and BTZ/17-AAG were synergistic at some drug ratios but were additive or antagonistic at the other ratios. Therefore, optimization of the drug dosages and ratios in POx micelles will be further considered for combination therapy studies in appropriate disease models using the presented POx co-delivery platform. It is worth pointing

159 out that in view of considerable differences between the cell culture and animal model experiments the optimization of the drug ratios may not be a trivial task and would require understanding of the pharmacokinetics and pharmacodynamics of each individual drug in the multi-drug composition. Furthermore, translating the results of such laboratory studies to the clinical treatment regimens is always challenging and will be greatly helped by thorough understanding of the absorption, distribution, metabolism, and excretion (ADME) of the multi-drug containing polymeric micelles.

Finally, the chemical versatility [298] of POx block copolymers can be exploited for the preparation of targeted micelles in the future, potentially increasing the therapeutic opportunities with this high-capacity micelle multi-drug delivery platform. The fine-tuning of the amphiphilic contrast of POx monomer units, blocks and the entire polymer should also have direct implementations in the morphology of the micellar aggregates and allow to design the shape and size of these aggregates to match the specific requirements to accommodate a variety of drugs as well as optimize size dependent pharmacokinetics and biodistribution behavior of the carrier [298, 299].

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Table 5.1. Solution concentration, LE and LC of drugs in POx aqueous dispersions (n = 3 ± SD)a

Solution drug Drugs LE (%) LC (%) Total LC (%) concentration (g/L) PTX 3.88±0.20 97.0±5.1 27.9±1.0 27.9±1.0 DTX 3.87±0.14 96.8±3.5 27.9±0.7 27.9±0.7 17-AAG 3.45±0.21 86.2±5.3 25.6±1.2 25.6±1.2 ETO 3.62±0.18 90.6±4.6 26.6±1.0 26.6±1.0 BTZ 3.12±0.12 77.9±3.1 23.8±0.7 23.8±0.7 PTX 3.66±0.15 91.6±3.7 20.8±0.5 43.1±1.2 17-AAG 3.93±0.23 98.2±5.6 22.3±0.9 DTX 3.92±0.10 98.1±2.6 22.3±0.4 43.3±0.6 17-AAG 3.70±0.10 92.6±2.5 21.0±0.4 PTX 3.59±0.33 89.8±8.3 21.0±1.6 41.6±1.6 ETO 3.54±0.36 88.6±9.0 20.7±1.7 ETO 3.69±0.17 92.2±4.2 21.6±0.7 41.1±0.9 17-AAG 3.38±0.09 84.4±2.2 19.8±0.2 PTX 3.27±0.33 81.6±8.3 19.4±1.2 40.3±2.4 BTZ 3.52±0.38 87.9±9.5 20.9±1.4 BTZ 3.27±0.13 81.8±3.2 19.8±0.7 39.5±0.7 17-AAG 3.25±0.17 81.3±4.3 19.7±0.9 PTX 3.01±0.12 75.4±3.0 15.5±03 17-AAG 3.19±0.16 79.9±4.1 16.4±06 48.7±1.1 ETO 3.27±0.16 81.8±3.9 16.8±06 PTX 3.18±0.14 79.5±3.5 16.4±0.5 17-AAG 3.03±0.11 75.7±2.7 15.6±0.3 48.4±0.7 BTZ 3.17±0.03 79.4±0.7 16.4±0.2 DTX 40.60±3.61 81.2±7.2 44.8±2.2 44.8±2.2b PTX 38.71±2.62 77.4±5.24 43.6±1.6 43.6±1.6c

a Unless stated otherwise the POx copolymer [P(MeOx40-b-BuOx21-b-MeOx34)] was used and its concentration in the dispersion was 10 g/L. b 50 g/L POx and DTX was used in this experiment. c 50 g/L POx and PTX was used along with a different batch of POx copolymer

[P(MeOx33-b-BuOx26-b-MeOx45)].

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Table 5.2 Comparison of our results with others for drug formulation in LC and DL valuesa

Our results G.S. Kwon group’s results Drugs Solution drug Solution drug LC (%) DL (%) LCd (%) DLe (%) concentration (g/L) concentration (g/L) PTX 3.88 27.9 38.8 3.54 9.3 10.3 DTX 3.87 27.9 38.7 4.27 10.3 11.5 17-AAG 3.45 25.6 34.5 3.90 10.2 11.3 ETO 3.62 26.6 36.2 3.31 8.7 9.6 BTZ 3.12 23.8 31.2 / / / PTX 3.66 3.92 43.1 75.9 20.6 25.9 17-AAG 3.93 3.88 DTX 3.92 4.62 43.3 76.2 20.5 25.8 17-AAG 3.70 4.01 PTX 3.59 41.6 71.3 / / / ETO 3.54 ETO 3.69 3.49 41.1 70.7 20.0 25.0 17-AAG 3.38 4.21 PTX 3.27 40.3 67.9 / / / BTZ 3.52 BTZ 3.27 39.5 65.2 / / / 17-AAG 3.25 PTX 3.01 3.50 17-AAG 3.19 48.7 94.7 3.61 26.2 35.6 ETO 3.27 3.17 PTX 3.18 17-AAG 3.03 48.4 93.8 / / / BTZ 3.17 DTXb 40.6 44.8 81.2 / / / PTXc 38.71 43.6 77.4 / / /

a Unless stated otherwise the POx copolymer P(MeOx40-b-BuOx21-b-MeOx34) was used and its concentration in the dispersion was 10 g/L. b 50 g/L POx and DTX was used in this experiment. c 50 g/L POx and PTX was used d along with a different batch of POx copolymer P(MeOx33-b-BuOx26-b-MeOx45). Recalculated value according to LC equation. e Original reported data. (H.C. Shin, A.W.G. Alani, D.A. Rao, N.C. Rockich, G.S. Kwon, Multi-drug loaded polymeric micelles for simultaneous delivery of poorly soluble anticancer drugs, J. Control. Release 140 (2009) 294-300) m LC  dr ug 100% m  m dr ug excipient (1)

m DL  drug 100%  m excipient (2)

162 

Figure 5.1 Chemical structure of amphiphilic POx copolymer, P(MeOx40-b-BuOx21-b-MeOx34), and schematic representation of multiple drug loaded micelles.

163

H3C O O O O OH N O O O H OH H HO HO PTX O O O O H O O CH HO O 3 H O O H O OH CH O O O 3 OH H3C O OH H3C O N O O O H OH HO ETO O O O O DTX O CH3

O H OH N O H C O H 2 N N B CH3 N OH N H H O O H3C H N H CO O 3 BTZ H3CO CH3

H3C 17-AAG OCONH2

Figure 5.2 Chemical structures of anti-cancer drugs.

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Figure 5.3 a, Size distribution (as determined by DLS) of POx micelles loaded with single drugs: PTX (), 17-AAG (), DTX (), ETO (), and BTZ ( ). b, Stability studies of POx micelles loaded with single drugs as in (a) by plotting average size (nm) and PDI over consecutive time points (days). b, Measurement ended at 14 days. Lines between data points are for illustration purpose only.

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Figure 5.4 a, Size distribution of POx micelles co-loaded with two drugs: PTX/17-AAG (), PTX/ETO (), PTX/BTZ (), ETO/17-AAG (), BTZ/17-AAG ( ) and DTX/17-AAG () by DLS. b, Stability studies of POx micelles co-loaded with two drugs as in a, by plotting average size (nm) and PDI over consecutive time points (days). Lines between data points are for illustration purpose only.

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Figure 5.5 a, Size distribution of POx micelles co-loaded with three drugs: PTX/17-AAG/ETO () and PTX/17-AAG/BTZ (). b, Stability studies of POx micelles co-loaded with three drugs as in (a) by plotting average size (nm) and PDI over consecutive time points (days). Lines between data points are for illustration purpose only.

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Figure 5.6 CLSM images of MCF-7 cells incubated for 1 h with free a, PTX, b, POx copolymer alone and c, POx micelles co-loaded with PTX and 17-AAG. Blue: Cell nuclei staining by Hoechst 33342; Green: BODIPY® FL PTX; Red: AF647 labeled POx. Scale bars are 20 m.

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Fig. 5.7 CLSM images of MCF-7 cells incubated with a, free PTX, b, POx block copolymer alone and c, POx micelles co-loaded with PTX and 17-AAG for 4 h. Blue: Cell nuclei staining by Hoechst 33342; Green: BODIPY® FL PTX; Red: AF647 labeled POx. Scale bars are 20 μm.

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Figure 5.8 a, Cytotoxicity of single drugs and binary drug combination, ETO/17-AAG solubilized with POx micelles in MCF-7 cells (mean ± SEM, n = 6) and b, the corresponding CI vs. Fa plot (right). Please note, the total drug concentration is presented. The IC50 values for single micellar ETO and 17-AAG were 7.8 and 0.35 g/mL, respectively. At the optimum ratio 1:1 the ETO/17-AAG combination displays IC50 = 0.29 g/mL (total drug, 0.143 g/mL ETO and 0.147 g/mL 17-AAG) and CI = 0.45. All drug formulations contain 10 g/L POx stock solutions; ETO and 17-AAG concentrations are 3.50 and 1.87 g/L (1:0.5); 3.83 and 2.75 g/L (1:0.7); 3.72 and 3.82 g/L (1:1); 1.85 and 3.66 g/L (1:2).

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Figure 5.9 a, Cytotoxicity of single drugs and binary drug combinations, BTZ/17-AAG solublilzed in POx micelles in MCF-7 cells (left, mean ± SEM, n = 6) and b, the corresponding log CI vs. Fa plot (right). Note that the total drug concentrations are presented. The IC50 values for single micellar BTZ and 17-AAG were 0.04 and 0.6 g/mL, respectively. At the optimum ratio (1:0.6) the BTZ/17-AAG combination displays IC50 = 0.006 g/mL (total drug, 0.00365 g/mL BTZ and 0.00235 g/mL 17-AAG) and CI = 0.099. All drug formulations contain 10 g/L POx in stock solutions; BTZ and 17-AAG concentrations are: 1.04 and 3.58 g/L (0.3:1); 1.80 and 3.27 g/L (0.5:1); 3.27 and 3.25 g/L (1:1); 3.02 and 1.95 g/L (1:0.6).

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Figure 5.10 a, IC50 values of micellar 17-AAG, BTZ and BTZ/17-AAG combination in PC3, MDA- MB-231, and HepG2 cancer cells and b, the CI vs. Fa plot. The micellar BTZ/17-AAG combination was examined at 1:1.05 ratio (POx: 10 g/L, BTZ: 4.13 g/L, 17-AAG: 3.78 g/L).

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CHAPTER VI: A LOW PROTEIN BINDING CATIONIC POLY(2-OXAZOLINE) AS A NON-VIRAL VECTOR 1

6.1 Summary

Developing safe and efficient non-viral gene delivery systems remains a major challenge.

We present a new cationic poly(2-oxazoline) (CPOx) block copolymer for gene therapy that was synthesized by sequential polymerization of non-ionic 2-methyl-2-oxazoline (MeOx) and a new 2-oxazoline monomer, 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline, followed by deprotection of the pendant secondary amine groups. Upon mixing with plasmid DNA (pDNA)

CPOx forms small (diameter ≈ 80 nm) and narrowly dispersed polyplexes (PDI < 0.2), which are stable upon dilution in saline and against thermal challenge. These polyplexes exhibited low plasma protein binding and very low cytotoxicity in vitro compared to the polyplexes of pDNA and poly(ethylene glycol)-b-poly(L-lysine) (PEG-b-PLL). The transfection efficiency of the polyplexes in B16 murine melanoma cells was low after 4 h but increased significantly for 10 h exposure time, indicative of slow internalization of polyplexes. Addition of Pluronic P85 boosted the transfection using CPOx/pDNA polyplexes considerably. The low protein binding of CPOx/pDNA polyplexes is particularly interesting for the future development of targeted gene delivery.

1 This chapter previously appeared as a manuscript accepted by Macromolecular Bioscience. The original citation is as follows: He, Zhijian et al., “A Low Protein Binding Cationic Poly(2- oxazoline) as Non-Viral Vector”, Macromolecular Bioscience, 2015, doi: 10.1002/mabi.201500021.

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6.2 Introduction Gene therapy has been explored to treat devastating genetic disorders such as Leber's congenital amaurosis [300, 301], X-linked adrenoleukodystrophy (ALD) [302], β-thalassemia

[303], severe combined immune deficiency (ADA-SCID) [304], hemophilia [305, 306] as well as acquired diseases such as cancer [307-309] or neurodegenerative disorders [310]. The non- viral gene delivery vectors are intensively pursued as an alternative strategy to the viral-based vectors that exhibit high transfection efficiency but also often have high cost and safety issues

[311, 312]. In contrast non-viral vectors are less active but relatively safe, cost-efficient and easy-to-manipulate systems, which are flexible to formulation design, amenable to modifications using target ligands, and capable to condense large plasmid DNAs [313-317].

One major category of non-viral vectors is polycations – the positively charged macromolecules that are typically exemplified by poly(L-lysine) (PLL) [318, 319], polyethylenimine (PEI) [320-325] or chitosan [325-329]. Upon mixing with the negatively charged DNA the polycations produce electrostatic complexes called “polyplexes”, which have been studies extensively as gene delivery vehicles [330]. Unfortunately, most polycations and polyplexes display high cytotoxicity [318, 331-334]. Grafting of PEG to polycations is known to increase polyplexes stability. Thus, excess of polycations is avoided and toxicity may be reduced [323, 335-340].

Although the use of PEG in drug and gene delivery systems has been well established, this polymer is known to have several shortcomings. In particular, it is prone to oxidative degradation, which is typical of polyethers [195-197]. After administration to the body low molar mass PEG is predominantly cleared via kidney and considered safe [341], yet several studies found evidence of persisting PEG in vivo [342-344]. Moreover, approximately 25% of 174 patients have pre-existed anti-PEG antibodies, which may fail those treatments employing

PEGylated therapeutics in clinic [193, 194].

Poly(2-oxazolines) (POx) have been suggested as alternatives to PEG due to their highly tunable structure, versatile properties and favorable biological safety profiles [198, 201]. POx are accessible via living cationic ring opening polymerization (LCROP) of 2-oxazolines, a robust and manageable synthetic approach, which allows incorporating various functionalities in the polymer using different initiation and termination reagents, or 2-substituted monomer side chains. LCROP can result in excellently controlled POx length and dispersity (Mw/Mn = Đ < 1.2).

The pending amide moieties of POx are highly hydrated while the alkyl side chains can be fine- tuned by adjusting chain length to achieve amphiphilicity. Both poly(2-methyl-2-oxazoline)

(PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) exhibit “stealth” properties and long circulation time comparable or superior to PEG when grafted onto liposomes or surfaces [38,

198, 199, 214, 256, 280, 281, 345]. Viegas et al. have reported that a conjugated of PEtOx and bovine serum albumin was less immunogenic as compared to the PEGylated protein in rats

[279].

There have been attempts to develop gene delivery systems via partially hydrolyzing

PEtOx into linear PEI-POx diblock polymers [220, 221]. One major drawback of this approach is its complexity - the polymer first, has to be hydrolyzed, and second, coupled to unhydrolyzed chain to obtain desired block copolymers. Moreover, this approach does not allow introducing other functional side chains. The direct synthesis of cationic POx by LCROP has been challenging because the growing polymer chains are very sensitive to and could be terminated by nucleophiles [198]. Cesana et al. [222] previously synthesized the first cationic

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2-oxazoline monomers bearing tert-butyloxycarbonyl (Boc) protected primary amines.

Polymerization of this monomer followed by deprotection of Boc groups generated POx with pendant cationic groups. However, Boc-protected pendant primary amine monomers provided difficulties for direct polymerization due to the interference from protected-primary amine (nucleophile) to the ring opening process. Hartlieb et al. [223] applied a very similar synthetic approach to Cesana’s and obtained hydrogel scaffolds to capture or enrich DNAs intended to be applied in bioanalytical systems such as gene chips. More recently, Rinkenauer et al. [224] has explored a number of cationic 2-oxazoline polymers by polymer-analogue modification of POx side chains with primary or tertiary amines. They found that 1) long hydrophobic side chain induced high cytotoxicity of the resulting polymer and 2) primary amines with amine content at least 40% were required for efficient transfection.

Here, we developed a novel cationic POx copolymer (CPOx) comprising a non-ionic hydrophilic PMeOx block and a cationic poly[2-(N-methyl)aminomethyl-2-oxazoline]

(PMAMeOx) block. We hypothesized that pendant secondary amine groups tethered by a short alkyl spacer, methylene, will reduce hydrophobicity and thus, toxicity while still retaining the ability to form polyplexes. The polymer was synthesized by LCROP of 2-methyl-

2-oxazoline (MeOx) and a new 2-(N-methyl, N-Boc-amino)-methyl-2-oxazoline (Boc-

MeAmMeOx) monomer. After deprotection, a polycationic polymer CPOx was obtained. We explored the potential of this block copolymer for gene delivery for macrophage transfection with potential applications in tumor immunotherapy.

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6.3 Materials and methods

Materials

3-(4,5-dimethylthiazol-2-yl)-2,5-diph3enyltetrazolium bromide (MTT), Boc-sarcosin, 2- methyl-2-oxazoline (MeOx) and branched PEI (25,000 Da) were purchased from Sigma-Aldrich

(Steinheim, Germany and St. Louis, US, respectively). Isobutylchloroformiate was purchased from Alfa Aesar (Karlsruhe, Germany), N-Boc-sarcosine from Bachem (Bubendorf,

Switzerland). Dry solvents were stored under argon and over mol sieve. PEG105-PLL51 was from

Alamanda Polymers™ (Huntsville, AL) with Mn=13 kg/mol and dispersity Ð = Mw/Mn = 1.09.

Pluronic P85 (lot # WPYE537B) was kindly provided by BASF Corporation (North Mount Olive,

NJ).

NMR spectra were recorded on an Inova 400 (1H: 400 MHz) at RT. The CMC of the polymers were determined by Wilhelmy plate method using Sigma 703D Tensiometer. The molar mass (Mn, GPC) and dispersity Ð (Mw/Mn) were measured by gel permeation chromatography (GPC) performed on a Polymer Laboratories GPC-120 (column setup: 1 x PSS

GRAM analytical 1000 and 1 x PSS GRAM analytical 100 obtained from Polymer Standards

Services, Mainz, Germany) using solvent of N,N-dimethyl acetamide (DMAc) (5 mmol/L LiBr,

1 wt% H2O, 70 °C, 1 mL/min) as eluent and poly(methylmethacrylate) standards. Pre-cast Tris-

HCl gels and Precision Plus Protein™ All Blue Standards were from Bio-Rad (Hercules, CA).

SYPRO® Ruby protein gel staining and cell culture reagents including Dulbecco’s Modified

Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin (P/S) were purchased from Invitrogen (Carlsbad, CA). NAP™ desalting columns and LH-20 were from GE

Healthcare (Piscataway, NJ). Luciferase (Luc) assay kit was from Promega (Madison, WI). gWIZ-

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Luc plasmid DNA (pDNA) was from Genlantis (CA, USA). Carboxyrhodamine-Azide (AZ105) was purchased from Click Chemistry Tools (Scottsdale, AZ 85260). All other reagents and supplies were from Fisher Scientific (Pittsburgh, PA) unless noted otherwise. The monomers and solvents used for the polymerization of POx were purified by distillation under reduced pressure. Other commercially available chemicals (Sigma-Aldrich) were used without further purification. RAW264.7 murine macrophage and B16 human melanoma cell lines were from

American Type Culture Collection (ATCC, Manassas, VA) and cultured according to ATCC protocol.

Polymer synthesis

Synthesis of Boc-MeAmMeOx

Boc-MeAmMeOx monomer was synthesized as presented in Figure 6.1 by ring-closing reaction following the general procedure of Levy and Litt and a newer version of Weberskirch et al. [225, 226]. Using a triple-necked flask with a thermometer and dropping funnel, 21.06 g (111.7 mmol) N-Boc-sarcosine were dissolved in 350 mL tetrahydrofuran (THF) and 15.45 mL (111.7 mmol) triethylamine were added, the mixture was cooled to 3 °C.

Isobutylchloroformiate (14.5 mL, 111.7 mmol) were added dropwise under vigorous stirring.

Subsequently, 12.95 g (111.7 mmol) chloroethylamine hydrochloride were dissolved in 50 mL dimethylformamide (DMF) and added dropwise followed by 15.45 mL triethylamine. The yellowish solution was stirred at RT for 1 h. The reaction mixture was filtered and concentrated.

After dilution with dichloromethane, the solution was extracted thrice each with 10% aqueous soda and brine. The combined aqueous phases were extracted with dichloromethane. From

178 the combined organic phases, all volatiles were removed the residue dissolved in 150 mL methanol (MeOH) and 22.505 g (162.8 mmol) of K2CO3 were added. The mixture was stirred under inert atmosphere overnight at RT and then refluxed for 5 h. Volatiles were removed at reduced pressure and the residue was dried at 0.002 mbar. From the yellowish solid the product was obtained by distillation at 3.6 x 10-3 mbar. At 76-83 °C, the product was fractionated and the product was obtained as a colorless liquid (13.683 g, 28.8%).

1 H-NMR (d3-MeOD, 300 MHz, 295 K): 4.256 (t, 2H, N-CH2-CH2-O), 3.954 (s, 2H, C-CH2-

N(Me)(Boc)), 3.735 (t, 2H, N-CH2-CH2-O), 2.818 (s, 3H, N-CH3), 1,35 (d, 9H, O-C(CH3)3) ppm.

13 C-NMR (d3-MeOD, 75 MHz, 295 K): 166.2/4 (C-CH2-N(Me)(Boc)), 156.1/155.7 (N-C(O)-

O-C(CH3)3), 80.1 (N-C(O)-O-C(CH3)3), 68.0 (N-CH2-CH2-O), 53.2 (N-CH2-CH2-O), 45.7/44.9 (C-

CH2-N(Me)(Boc)), 34.1 (N-CH3), 27.2 (N-C(O)-O-C(CH3)3) ppm.

Synthesis of Propargyl-P(MeOx50-b-MeAmMeOx16)

The polymer was synthesized following published methods [38, 198, 227] and its reaction strategy is depicted in Figure 6.3. Briefly, under dry and inert condition 63.1 mg (0.3 mmol,

1eq) of propargyl toluenesulfonate (initiator, 1) and 1276.5mg (15 mmol, 50eq) of 2-methyl-

2-oxazoline (MeOx, 2) were dissolved in 3 mL dry acetonitrile at RT. The mixture was heated

(microwave-assisted synthesizer, 150W maximum, and 130 °C) for 5 min. After cooling to RT, the cationic monomer for the second block, Boc-MeAmMeOx (1011.4 mg, 4.7 mmol, 16 eq,

179 and the polymer (4) was obtained as colorless powder after lyophilization of an aqueous solution in water. De-protection of Boc group was performed in trifluoroacetic acid

1 H-NMR (CDCl3, 298K): δ=4.1-3.9 (br, 37H, -N-CO-CH2-N-Boc, a); 3.47(br, 249H, N-CH2-

CH2, b); 2.87 (br, 63H, boc-N-CH3, c); 2.05 (m, 147, -N-CO-CH3, d); 1.4 (s, 180, tertbutyl, e).

Synthesis of carboxyrhodamine 110 fluorescence labeled polymers (F-CPOx)

RT for 1 h. Finally the reaction mixture was purified with NAP10 size exclusion column, dialyzed against deionized water for 2 days with MW cutoff of 3500, (GE healthcare,

Piscataway, NJ) and freeze-dried to give Carboxyrhodamine 110-labeled polymers F-CPOx

180

(Scheme 2, compound 6).[228] Column purification was repeated twice to completely remove free fluorescence dye. The conversion rate (fluorescence labeling efficiency) was about 72% determined from the calibration of free fluorescence dye standard.

Acid-Base Titration

The buffering capacity of the synthesized block copolymer was determined by acid-base titration. Briefly, 2 mg of polymer was dissolved in 10 mL of 0.1 M NaCl to give a final concentration of 0.2 g/L, the pH of the polymer solution was brought to 10 with NaOH, and the solution was subsequently titrated with 0.1 M HCl. 0.1 M NaCl or 0.2 g/L PEG-PLL were also titrated as references.

Formation and characterization of CPOx/pDNA polyplexes

For preparation of polyplex, stock solution of pDNA (1 g/L) and polymer (1 g/L) in 10 mM

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.4) were mixed at various N/P charge ratios (N: amine groups on polymer, 1g polymer ≈ 2.6 mmol amine groups, positive charge; P: phosphate group on pDNA, 1g DNA ≈ 3 mmol phosphate groups, negative charge) and vortexed immediately for 30 sec, then incubated for 30 min at RT before further characterization. The final concentration of pDNA in the polyplexes was 20 µg/ml.

Hydrodynamic diameter, size distribution and zeta-potential of polyplexes were measured by

DLS in triplicate using a Malvern Zetasizer Nano (Malvern Instruments Ltd., Malvern, UK).

Results represent the average from three independent manufactured polyplexes.

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Ethidium bromide exclusion assay, agarose gel electrophoresis, and stability of polyplexes during storage, dilution and heating

The ability of CPOx and PEG-PLL to condense pDNA was confirmed by a standard ethidium bromide (EtBr) exclusion assay via measuring the changes in EtBr/pDNA fluorescence. pDNA (gWIZ-Luc) solutions in 10 mM sodium acetate buffer (pH 5.2) and 10 mM

HEPES, pH 7.4 at concentration of 20 μg/mL were mixed with EtBr (1 μg/mL) and fluorescence measured using 545 nm excitation and 595 nm emission and set to 100 %. Background fluorescence was set to 0 % using EtBr (1 μg/mL) solution alone. Fluorescence readings were taken following a stepwise addition of polymer solution, and condensation curves for each polymer was constructed accordingly.

For agarose gel electrophoresis, 20 μL of each polyplex solution was mixed with 4 μL 6x

DNA gel loading dye and loaded on 1 % agarose gel containing EtBr (0.2 μg/mL). The samples were electrophoresed in 1x Tris-Acetate-EDTA (TAE) running buffer for 1 h at 100 V, and pDNA bands were visualized under UV light (FluorChem E, ProteinSimple, Santa Clara, CA).

In order to analyze the stability upon storage at 4 °C, size and PDI of polyplexes were measured up to 7 days using DLS. Size and PDI of polyplexes were also monitored upon heating, where the temperature was step-wise increased from 25 °C to 40 °C, 55 °C and finally 70 °C.

To ensure proper equilibration, the polyplexes were maintained for 5 min at the desired temperature before the measurement was started.[346]

Transmission electron microscopy (TEM)

The morphology of polyplexes was studied using a LEO EM910 TEM operating at 80KV

182

(Carl Zeiss SMT Inc., Peabody, MA) and digital images acquired using a Gatan Orius SC1000

CCD Digital Camera with Digital Micrograph 3.11.0 (Gatan Inc., Pleasanton, CA). Briefly, a drop of the polyplex solution was deposited on a copper grid/carbon film for 5 min while the excess solution was wicked off with filter paper, and a drop of staining solution (1% uranyl acetate) was allowed to contact the sample for 10 seconds prior to the TEM imaging.

Plasma protein binding assay

150 μL Pooled rat plasma was incubated with 50 μL PEG-PLL/pDNA, PLL/pDNA, and

CPOx/pDNA polyplexes (the final concentration of pDNA was 200 μg/mL; N/P ratios at 5, 10 and 20) for 30 min at 37 °C. Mixtures were centrifuged at 14000 rpm for 40 min to precipitate serum proteins-bound polyplexes from those serum proteins that were not adsorbed.

Unbound components were removed by 3 cycles of washing/centrifugation in 10 mM HEPES buffer (pH 7.4). The serum protein-bound polyplexes pellets were then washed with cold acetone to remove any lipids that were co-precipitated. Serum proteins bound to polyplexes were ultimately desorbed from the pellets using 50 μL 2% SDS (in 0.5 M Tris-HCl, 10% glycerol, pH 6.8) at 100 °C for 5 min. For comparison, the rat plasma proteins were collected from pooled rat plasma by direct precipitation with cold acetone. The amount of protein bound to polyplexes was determined using MicroBCA Assay™. The equal volume of samples (8 μL each) were loaded on SDS-PAGE gel and stained using SYPRO Ruby. The molecular identity of selected protein bands was identified using Liquid chromatography–mass spectrometry (LC-

MS).

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Cell culture and in vitro cell viability assay

RAW264.7 murine macrophages and human melanoma B16 cell lines were cultured in

DMEM supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin/100 μg/mL streptomycin (complete media). All media was obtained from Gibco Life Technologies, Inc.

(Grand Island, NY). For cytotoxicity assay, cells were seeded at a density of 2 x 104 cells per well in 96-well plates and allowed to adhere for 24 h prior to adding various concentrations of polymer ranging from 1 μg/mL up to 1000 μg/mL. PEG-PLL was used as a positive control.

Cells were treated with polymers for 48 h and cell viability was determined by MTT assay (100

μg/well, 3h). Data represents average of six separate wells for each polymer concentration

[227].

Cellular internalization of polyplexes and in vitro transfection

Cellular internalization of polyplexes was studied using confocal microscopy. Briefly, cells were seeded onto sterile Lab-Tek II chambered coverglass. After overnight attachment, the cells were treated with polyplexes [fluorescently labeled polymer (115mg/L)/ TOTO-3 labeled pDNA (20mg/L)] for various time points from 2, 4, 6, 8, 10, 12 to 24h. pDNA labeling with

TOTO-3 was performed according to manufacturer’s protocol (Life Technologies, Grand Island,

NY). At the end of each time point, cells were washed with 1xPBS for 3 times and fixed with

4% paraformaldehyde for 10 min followed by imaging using confocal microscopy (Zeiss 510 microscope). All imaging conditions, including laser power, photomultiplier tube, and offset settings, remained constant for each comparison set. For in vitro transfection study, the

RAW264.7 macrophage or B16 melanoma cells were split one day prior to transfection and

184 plated in 48-well plates at cell density of 3x104 per well for RAW264.7 cells and 1x104 for B16 cells. Before transfection, the cell culture medium was replaced with fresh serum-free DMEM or complete DMEM media. Transfection solutions were prepared in 1.5 mL eppendorf tube by mixing (i) 4 μg gWIZluc plasmid DNA, (ii) 150 μL HEPES, and (iii) cationic polymer at predetermined volume to give desired N/P ratio. The cells in each well were transfected with polyplexes containing 0.5 μg of plasmid DNA at 37oC for 4 h or 10 h (n=3). Then the complexes were removed and cells were incubated for additional 24 h in fresh complete DMEM media or with Pluronic P85 excipient (in complete media) for extra 3 h before changing to complete medium. Cells were washed with 1xPBS and then lysed with 120 μL 1x cell culture lysis buffer

(Promega). The luciferase activity in each sample was measured by Promega Luminometer after adding luciferin substrate.

Statistical Analysis

Unless otherwise specified, all statistical comparisons are made using Student’s t test. A result with p < 0.05 was considered statistically significant.

185

6.4 Results

Synthesis and characterization of the new monomer Boc-MeAmMeOx

A new monomer was designed to yield polymers containing pendant secondary amine groups after polymerization and deprotection. The synthesis was performed by a simple two- step reaction following a standard route of 2-oxazoline synthesis (Figure 6.1). Boc-Sarcosine

(1) was coupled with 2-chloroethylammonium chloride in THF. The 1H-NMR spectrum of the purified Boc-protected monomer is shown in Figure 6.2. All of the signals could be unambiguously assigned.

Synthesis and characterization of the “clickable” Propargyl-P(MeOx50-b-MAMeOx16)

The polymerization was initiated using propargyl toluenesulfonate to incorporate a functional alkyne group at the nonionic PMeOx block end for future “click” chemistry application (Figure 6.3). The molecular mass of Propargyl-P(MeOx50-b-MAMeOx16) determined using GPC (Mn, GPC) was close to the desired value (Mcalc), the dispersity was reasonably low. Since the Boc-protected PMAMeOx block is hydrophobic the copolymer is amphiphilic and formed micelles in an aqueous solution with a CMC value of about 19 mg/L

(2.1 µM) at 20°C as determined using tensiometer and Wilhelmy plate. Boc-CPOx exhibited a characteristic signal of Boc-protection group (tert.-butyl protons) at 1.4 ppm (Figure 6.4a).

After deprotection under standard acidic condition, these protons were largely absent as indicated in Figure 6.4b. However, it is notable that no complete removal of the Boc- protection groups was achieved using a standard protocol. Approx. 10 % of the protection groups remained on the polymer. Even after a second deprotection attempt, approx. 5% of

186 the Boc groups remained visible in the NMR spectrum (Figure 6.4c). For further experiments, we hypothesized that the remaining hydrophobic groups may actually stabilize the complexes.

Thus, we did not conduct further attempts for complete removal of the Boc groups.

Formation and physicochemical characterization of CPOx/pDNA polyplexes

This study investigated the potential use of a POx block copolymer with pending hydrophilic cationic moieties for non-viral gene delivery. In contrast to a recently published polymer analogue addition approach report by Schubert and co-workers [224], we introduced the positive charges via monomer-deprotection route. The buffer capacity of the pending amino groups at low pH may be important for transfection efficiency of CPOx/pDNA polyplexes since buffering effects are believed to facilitate endosomal escape of polyplexes and thereby protect DNA from nuclease degradation in endosomal compartment (proton sponge effect) [347-349]. Therefore, we first measured the buffering capacity of the block copolymer by acid-base titration in 0.1 M NaCl aqueous solution (Figure 6.5a). At the same mass concentration, both CPOx and PEG-PLL showed similar titration curves, however, CPOx appears to buffer slightly better between pH 7.4 and 5.2, relevant for endosomal escape

(Figure 6.5b) [350].

The ability of CPOx to condense pDNA and form polyplexes was then evaluated by ethidium bromide exclusion (Figure 6.6a) and gel retardation (Figure 6.6b) assays. CPOx was mixed with gWIZ-Luc pDNA at various N/P ratios. Condensation curves for polyplexes at pH

5.2 or pH 7.4 both showed typical transition between N/P ratios of 1 and 2.

Gel retardation assay suggested that CPOx is able to condense DNAs and produce

187 polyplexes. When N/P ratio exceeded 2, CPOx/pDNA polyplexes were unable to migrate on the gel and remained immobilized in the vicinity of the gel loading sites.

The further study of polyplexes by DLS (Figure 6.7a) revealed that CPOx indeed effectively condensed DNA into nanoscale-sized particles. Polyplexes at N/P ratio of 2 exhibited large size

(about 132 nm) and high polydispersity (PDI close to 0.3). Interestingly, the measured ζ- potential was slightly negative but essentially neutral (Figure 6.7b). To obtain more uniform

(PDI < 0.2) and smaller polyplexes (< 100 nm), a higher excess of polymer was needed. For example, further increase in N/P ratios to N/P = 5 or N/P = 10 produced smaller complexes of approx. 83 and 78 nm, respectively, with PDIs close to 0.17 displaying slight positive but essentially neutral surface charge (Figure 6.7b and c). The DLS data were in good agreement with what we observed based on the agarose gel electrophoresis.

Following standard deprotection procedure, we were unable to completely remove Boc protection group and about 10% of Boc groups remained on the polymer. However, this particular polymer formed small and defined (relatively uniform) complexes with pDNA. In contrast, the polymer produced after repeated deprotection that still contained a trace amount of Boc groups, <5%, formed less defined complexes with pDNA (Figure 6.7d and e).

This is very interesting and we hypothesize that CPOx containing some Boc groups bearing hydrophobic properties may promote the core formation of CPOx/pDNA complexes.

Examples for such hydrophobic stabilization of polyplexes can be indeed found in the literature [351-354].

We also investigated morphology of polyplexes formed by CPOx and pDNA via TEM

(Figure 6.8). A representative TEM image of the polyplexes at N/P ratio 10 indicated an

188 essentially spherical particle morphology and we estimated a diameter of 50 nm for these particles. This study corroborates the data obtained by DLS (as the TEM sizes are usually less that hydrodynamic diameters obtained by DLS).

Stability of CPOx/pDNA polyplexes

Stability of CPOx/pDNA polyplexes is a critical factor to facilitate the translation of this gene delivery platform for in vivo applications. We observed nearly no changes in the particle size and PDI for the polyplex at N/P ratio 2 (Figure 6.9a and b, black line; Size~130 nm and

PDI~0.25) and 5 (blue line; Size~83 nm and PDI~0.16) after their storage at 4 °C for up to one week in 10 mM HEPES buffer. The polyplex at N/P = 10 were also stable during storage for up to 5 days, while at day 7 their particle size increased from 78 nm to almost 100 nm and their

PDI increased from 0.17 to about 0.3 (Figure 6.9a and b, red line).

We also found that the polyplex at N/P = 1 disintegrated upon 10-fold dilution in 10 mM

HEPES buffer (pH 7.4) as we were unable to detect any nanoparticles after such dilution. In contrast, polyplex at N/P = 5 were stable and exhibited minimal changes in size (~82 nm) and

PDI (0.16) during 10-fold dilution in 10 mM HEPES buffer (Figure 6.10a).

When the CPOx/pDNA polyplex at N/P = 5 was diluted 10-fold in isotonic saline solution

(0.9% NaCl), both the size and PDI increased almost two fold to about 150 nm and 0.4, respectively. This result is a first indication that such polyplexes may be applied in vivo as they were sufficiently stable as nano-sized complexes and did not dissociate immediately in isotonic solutions. We further prepared polyplexes at N/P = 5 directly in 0.9% NaCl instead of

10 mM HEPES buffer, and using this procedure successfully obtained polyplexes with the

189 particle size of 135 ± 1 nm and PDI of 0.275 ± 0.008 (n = 3) (Figure 6.10a). Thermal stability of

CPOx/pDNA polyplexes at N/P = 5 and N/P = 10 was also investigated. The size and PDI were determined as a function of increasing temperature from 25 °C up to 70 °C (Figure 6.10b).

Interestingly, the particle hydrodynamic diameter exhibited a small but steady increase while an increase and strong variation in the PDI at 70 °C may indicate an onset of instability for both

N/P ratios 5 and 10.

Plasma protein binding with polyplexes

Plasma proteins bound to nanoparticle are directly linked to the uptake and further clearance of these nanoparticles by the RES in vivo [334, 355-357]. Hydrophilic POx, i.e.

PMeOx and PEtOx homopolymers, have been long known to exhibit anti-fouling or so called

“stealth” properties, similar to or potentially superior to these of PEG [214, 256, 281, 358].

This prompted us to evaluate the plasma protein binding with our polyplexes. The polyplexes were prepared at various N/P ratios (5, 10, and 20) and incubated with pooled rat plasma for

30 min, then isolated and analyzed by SDS-PAGE (Figure 6.11a). We found very little protein binding to CPOx/pDNA polyplexes at any N/P ratio compared to the PEG-PLL/pDNA or

PLL/pDNA polyplexes, which exhibited strong protein biding in this assay. We further quantified the protein binding to polyplexes using microBCA assay. This assay confirmed very low serum protein binding to CPOx/pDNA polyplexes compared to the two other control polyplexes (Figure 6.11b). Using LC-MS analysis we determined that the major proteins bound to PLL/pDNA polyplexes included IgG heavy and light chains, C3, C4, and C5 proteins of the complement system, and β and γ chain of fibrinogen isoforms (Figure 6.11a, small red box

190 from bottom to top respectively).

Determination of in vitro cytotoxicity of CPOx polymer

Many cationic polymer-based delivery systems have cellular toxicity issues. In order to form stable pDNA complexes, large excess of cationic polymers are often necessary [313, 332,

337], which may induce cytotoxicity due to the direct interaction of these polymers with the cell membranes. In this respect, we studied in vitro cytotoxicity of the CPOx copolymer in the cell lines we were interested to transfect, RAW264.7 murine macrophage and B16 melanoma cells. The cytotoxicity of PEG-PLL was tested in parallel as a comparison. Interestingly, CPOx showed little if any toxic effect on RAW264.7 cells at concentrations up to 500 μg/mL (IC50 > 1 g/L) (Figure 6.12a, black line) while PEG-PLL induced cytotoxicity with an IC50 value close to 41 mg/L (red line). Similar results were observed in B16 melanoma cells where the IC 50 value of

CPOx was about 1 g/L while that of PEG-PLL was close to 32 mg/L (Figure 6.12b). Moreover, the CPOx/pDNA polyplexes were significantly less toxic in RAW264.7 cells than the PEG-

PLL/pDNA polyplexes at the same N/P ratios of 5 or 10 following 4 h or 10 h exposures mimicking the transfection procedure (Figure 6.12c).

Analysis of cellular uptake of CPOx/pDNA polyplex in the macrophage cells

Cellular uptake of CPOx/pDNA polyplex (gWIZ-Luc pDNA, N/P 5) was studied in

RAW264.7 cells using confocal microscopy. For this experiment, the CPOx polymer was fluorescently labeled with carboxyrhodamine 110 (Figure 6.13, green) using the click chemistry method, while pDNA was visualized using TOTO-3 fluorescence dye (Figure 6.13,

191 red). Cells were imaged after incubation with polyplexes for 0.5, 2, 4, 6, 8, 10 and 24 h, respectively. We found that the uptake process in RAW264.7 cells was rather slow. Up to 4 h exposure there was no detectable amount of polyplexes taken up in the cells. After 6 and 8 h exposures the green fluorescence of CPOx and red fluorescence of pDNA was detectable in the cells, while after 10 h exposure the majority of cells were positive for both CPOx and pDNA fluorescence. Furthermore, after 24 h incubation both the green and red label fluorescence concentrated in the perinuclear region of the cells (Figure 6.13). However, significant loss of cell viability was also observed after 24 h of continuous exposure of the cells to the

CPOx/pDNA polyplex. Similar results were obtained for the polyplex at N/P 10 (Figure 6.14).

In vitro transfection using CPOx/DNA polyplexes

To evaluate the potency of the CPOx polymer in gene delivery we prepared CPOx/DNA polyplexes at N/P ratios of 5 and 10 and determined their transfection efficiency in B16 human melanoma cells and RAW264.7 murine macrophages, the latter cell line being extremely hard- to-transfect. The PEG-PLL/pDNA polyplexes were used as a comparison reference in the transfection experiments. All transfections were performed in complete DMEM medium containing 10% FBS for 4 h and 10 h. The 10 h time point was determined given the fact that the CPOx/pDNA polyplexes internalized in RAW264.7 cells relatively slowly, and that this time point allowed for considerable uptake of the polyplex in the cells without showing much cytotoxicity (Figure 6.12-6.14).

In the B16 cells the transfection observed using the control PEG-PLL/pDNA polyplexes was significant although relatively low. The transfection observed after exposure of these cells

192 to CPOx/pDNA polyplexes at N/P ratios of 5 or 10 for 4 h was not significantly different from the baseline (Figure 6.15a). When the exposure time was increased to 10 h the CPOx/pDNA polyplexes at N/P = 5 produced significant transgene expression in these cells, albeit several fold lower that that observed using PEG-PLL/pDNA polyplexes. Since the transfection levels were low we explored the effect of a gene expression adjuvant, an amphiphilic block copolymer, Pluronic P85 that was previously shown to increase transgene expression both in vitro and in vivo [359, 360]. In particular, in vitro using different polyplexes this block copolymer was shown to increase cellular uptake of the pDNA, nuclear transport of the pDNA and the transcription of the pDNA residing in the nucleus [359, 361]. Indeed, after adding this copolymer to CPOx/pDNA polyplexes at N/P 10 we observed a considerable increase of the cell transfection although the gene expression levels in this treatment group were still less than those in the PEG-PLL/pDNA polyplexes groups.

In RAW264.7 macrophage cells, PEG-PLL/pDNA polyplexes produced significant although low level of transfection (Figure 6.15b). In contrast, we saw no transfection after either 4 or

10 h incubation of the cells with CPOx/pDNA polyplexes. However, significant levels of the transfection were observed when these cells were exposed to the CPOx/pDNA polyplexes along with either 0.1 % or 0.5 % Pluronic P85. In these cases the gene expression reached nearly same levels as observed with the PEG-PLL/pDNA polyplexes groups.

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6.5 Discussion

Various cationic polymers and polyplexes on their base have been extensively explored as carriers for non-viral gene delivery although their applications in vivo have been hindered by low transfection efficiency, toxicity as well as rapid clearance from the plasma circulation.

Here we designed a new secondary amine-containing oxazoline monomer, and synthesized a new cationic POx block copolymer, CPOx containing pendant secondary amine groups by copolymerization of this new monomer with 2-methyl-2-oxazoline. This new cationic block copolymer exhibited low cytotoxicity, and formed polyion complexes with pDNA, which revealed low serum protein binding compared to the polyplexes based on a conventional cationic copolymer, PEG-PLL. Decreased protein binding may be due to good protective properties of PMeOx, which forms a hydrophilic shell at the surface of the CPOx/pDNA polyplexes. This shell may be more hydrophilic that the PEG shell that stabilizes the PEG-

PLL/pDNA polyplexes as PMeOx is reported to be more hydrophilic than the PEG [279, 362].

We also found that the CPOx/pDNA polyplexes are relatively stable even at elevated temperature. These polyplexes were also stable upon dilution in low ionic strength buffers and saline as well as upon storage at 4°C for at least 1 week. The stability of polyplexes is of particular interest for their applications. While studying interactions of the CPOx/pDNA polyplexes with the cells we found that these polyplexes slowly internalized in RAW264.7 macrophages and accumulated in these cells over 10 h. The slow internalization of the

CPOx/pDNA polyplexes may also be due to good protective properties of the PMeOx shell that diminishes interactions of these polyplexes with the cells. In contrast, amphiphilic POx, which also exhibit very low protein binding,[363] were shown to be internalized very rapidly in a

194 variety of cells [199]. As a general rule, internalization and toxicity of polyplexes formed by

PEG-modified polycations (e.g. PEG-PLL) are less than internalization and toxicity of polyplexes formed by the same polycations that do not carry PEG chains (e.g. PLL) [364, 365].

The newly designed CPOx/pDNA polyplexes appear to be even less toxic than the PEG- containing ones, which may underscore an important difference between the PMeOx and

PEG, both of which are hydrophilic polymers.

To optimize the in vitro transfection using CPOx/pDNA polyplexes the time of the exposure of the cells to these polyplexes was increased to 10 h compared to conventional 4 h.[366] Even at increased exposure the CPOx/pDNA polyplexes revealed very low transfection activity that was same of less than the transfection activity of the PEG-PLL/pDNA polyplexes.

We performed multiple transfection experiments and found that in very difficult to transfect

RAW264.7 macrophages the transfection efficiency of the CPOx/pDNA polyplexes was negligible, however, it was considerably increased when the polyplexes were co-administered to cells with poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblock copolymer,

Pluronic P85. We also noted considerable increase in transfection of B16 mouse melanoma cells with CPOx/pDNA polyplexes and Pluronic P85 systems, compared to the polyplexes alone.

This is a very promising observation especially in view of our prior findings that Pluronic block copolymers, and in particular, Pluronic P85 were shown to greatly improve non-viral gene transfer in vivo, including increasing gene expression in macrophages and other immune response cells [367].

Although the transfection levels observed with the CPOx/pDNA polyplexes without the block copolymer adjuvant were lower compared to PEG-PLL pDNA polyplexes, we view the

195 presented results as an interesting starting point due to the excellent variability of the POx polymer structure, which we will employ in future studies. The slow uptake in macrophage cells also offers an opportunity to minimize non-specific uptake in vivo but also provides a time window to target specific cell populations of cells via attaching affinity ligands on the alkyne terminal group through click chemistry tool. The ‘clickable’ property of the developed

CPOx polymer was evidenced by successfully attaching a fluorescence dye and could be further extensively developed for the targeting purpose. In future studies, with this new type of monomer we will be able to vary the sequence, length and charge density via LCROP approach and obtain optimized structure for gene delivery based on this new platform.

It is well known that macrophages play a key role in immune responses through direct and indirect mechanisms [368, 369]. Very abundant population of macrophage cells derived from monocytic precursors in the blood is believed to be associated with malignant tissues in tumor microenvironment. These special phenotypes of macrophages, often termed tumor- associated macrophages (TAMs), function as pro-tumoral components. Previous reports demonstrated a number of chemoattractants such as CCL2, CCL5 and CXCL1 secreted by tumor or stromal cells are responsible for the accumulation of TAMs to tumor sites. Similar roles may also be played by vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), transforming growth factor beta (TGF 훽 ) and macrophage colony stimulating factor (M-CSF), of which the latter may promote macrophage survival and differentiation. TAMs also facilitate tumor cells producing matrix proteases to degrade extracellular matrix (ECM); therefore promote tumor invasion and metastasis [370]. Gene delivery that targets macrophages; either by re-educating macrophages towards tumoricidal

196 phenotype or directly wiping out these populations offers great opportunities for long-term cancer immunotherapy. Interestingly, our group recently discovered that macrophages can transfect neighboring cells simply by transporting DNAs using excreted lipid vesicles termed

“exosomes” [371, 372]. In this regard, we proposed that transfecting TAMs can provide for possibilities to infiltrate and impact deep tissues of tumors. The transfection efficiency in macrophages has proven to be extremely low and although a number of non-viral gene transfer methods attempted to improve macrophage transfection [373-375] all these methods have drawback of low transfection efficiency and/or high cellular toxicity. This study offers a great opportunity to develop new platform to deliver genes to TAMs with low toxicity.

197

Figure 6.1 Synthesis of the new monomer, Boc-MeAmMeOx (3). i) Triethylamine, isobutylchloroformate, 3 °C, THF, ii) Triethylamine, 2-chloroethylammonium chloride, DMF, iii) MeOH, K2CO3, reflux.

198

1 Figure 6.2 H-NMR spectra (d3-MeOD, 300 K) of the novel monomer 2-(N-methyl,N-Boc- amino)-methyl-2-oxazoline with the assignment of all observed signals.

199

Figure 6.3 Block copolymerization of MeOx (2) and Boc-MeAmMeOx (3) using propargyl tosylate (1) as the initiator and 5% K2CO3 as the terminating agent. The deprotection of the pendant amino group of (4) was performed with TFA to obtain Propargyl-p(MeOx50-b- MAMeOx16) or CPOx, (5). Fluorescence labeled CPOx (F-CPOx), (6) was produced by click reaction of carboxyrhodamine 110-Azide with CPOx.

200

1 Figure 6.4 H-NMR spectra (400 MHz, d3-MeOD) of a, Boc-CPOx and the deprotected CPOx b, after single and c, repeated deprotection procedures, along with the respective structures and the peak assignments. The peak of Boc-protection group (peak e in a,) was greatly attenuated but remained in the deprotected CPOx spectrum (estimated 10% Boc residual in b,). This peak nearly disappeared (estimated <5% Boc residual in c,) after repeated deprotection procedure to further remove residual Boc groups in CPOx.

201

Figure 6.5 Titration curves for CPOx copolymer (black squares) and PEG-PLL (red squares) in 0.1M aqueous NaCl (pH 10, adjusted with NaOH) using 0.1M HCl. As reference, the titration curve of NaCl (open circles) is also presented. The concentration of polymers was 0.2 g/L.

202

Figure 6.6 a, pDNA condensation by CPOx assay by ethidium bromide exclusion. Change in fluorescence intensity afyer addition of the polymer to pDNA (20 mg/L)/ethidium bromide (1 mg/L) solution at various N/P ratios in HEPES (▪, pH 7.4) or sodium acetate buffer (▫, pH 5.2). A relative fluorescence of 100 RFU represents the fluorescence of pDNA/ethidium bromide solution in the absence of CPOx while 0 RFU indicates background fluorescence of ethidium bromide not intercalated into DNA. b, Gel retardation assay of pDNA and polyplexes (prepared in pH 7.4 of HEPES) at various N/P ratios of 1/3, 2, 5, 10 and 20, loaded on 1% agarose gel, stained with ethidium bromide, and visualized under UV light.

203

Figure 6.7 DLS measurements of the a, d, size distributions, b, e, particle size and PDI, and c, ζ-potential of CPOx/pDNA polyplexes as a function of N/P ratio of 2 (black), 5 (blue), and 10 (red). a-c, Polyplexes were prepared using the CPOx samples obtained after single deprtotection of the Boc-protected polymer precursor (4, Scheme 2), d, e, CPOx samples were subjected to the repeated deptorection to decreased Boc content.

204

Figure 6.8 Representative TEM image. CPOx/pDNA polyplex at N/P ratio of 10 indicated a spherical morphology. 10 µL of polyplex solution (10 µg/mL pDNA) were deposited onto copper grid with carbon film for 5 min and then negatively stained with 1% uranyl acetate. (Scale bar, 100 nm).

205

Figure 6.9 a, Size and b, PDI of CPOx/pDNA polyplexes at various N/P ratios: 2 (black), 5 (blue), and 10 (red) as a function of storage time. Each data point represents the average of independently prepared samples (n = 3).

206

Figure 6.10 Stability of CPOx/pDNA polyplexes upon dilution and heating. a, Size (solid square) and PDI (open reverse triangle) of polyplex at N/P=5 upon 10-fold dilutions in 10 mM HEPES buffer, pH 7.4, or 150 mM NaCl or the same polyplex produced directly in 150 mM NaCl. b, Stability of polypexes in 10mM HEPES buffer at N/P = 5 (▪) and N/P = 10 (▫) upon step-wise increased of the temperature from 25 °C to 40 °C, 55 °C and finally 70 °C.

207

Figure 6.11 a, SDS-PAGE of adsorbed plasma proteins on PEG-PLL/pDNA, PLL/pDNA, and CPOx/pDNA polyplexes at N/P ratio of 5, 10 and 20 (8 μL each sample). Plasma proteins were separated on the gel as a standard shown in the first lane left. Protein bands on the SDS-PAGE gel were visualized by SYPRO Ruby staining. b, Total protein adsorption in separated polyplexes as determined by micro BCA assay. Data is presented as protein concentration. ** Comparison with PEG-PLL/pDNA polyplexes, p<0.05.

208

Figure 6.12 In vitro cytotoxicity of a, b, the polymers and c, the polyplexes in a, c, RAW264.7 murine macrophages and b, B16 murine melanoma cells. a, b, Cell monolayers were incubated with respective polymers for 48 h followed by cell viability measurement by MTT assay. c, CPOx/pDNA or PEG-PLL/pDNA polyplexes prepared at N/P 5 or 10 were incubated with RAW264.7 murine macrophages under the same conditions as in the transfection experiments (0.1 microgram pDNA per well of 96-well cell culture plate) for 4 h or 10 h, and then incubated with complete medium for another 24 h before measuring the cell viability using MTT assay. Data represent average ± S.D. (n = 6).

209

Figure 6.13 Cellular uptake of CPOx/pDNA polyplex with N/P 5 in RAW264.7 macrophage cells. Blue, nuclear staining (Hoechst); Green, F-CPOx; Red, TOTO-3 labeled pDNA. Cells were incubated with polyplex for 0, 0.5, 2, 4, 6, 8, 10 and 24 h, then washed with PBS and fixed in 4 % paraformaldehyde before confocal imaging.

210

Figure 6.14 Cellular uptake of CPOx/pDNA polyplex with N/P 10 in RAW264.7 macrophage cells. Blue, nuclear staining (Hoechst); Green, F-CPOx; Red, TOTO-3 labeled pDNA. Cells were incubated with polyplex for 0, 0.5, 2, 4, 6, 8, 10 and 24 h, then washed with PBS and fixed in 4 % paraformaldehyde before confocal imaging.

211

Figure 6.15 In vitro transfection of a, B16 murine melanoma and b, RAW264.7 murine macrophage cells. Cells were transfected with PEG-PLL/pDNA and CPOx/pDNA at N/P ratios 5 or 10 in the complete DMEM medium containing 10% FBS for 4 h or 10 h. The last two bars on the right correspond to cells transfected with CPOx/pDNA polyplex supplemented with 0.1% or 0.5% Pluronic P85. After transfection, cells were incubated with fresh complete medium for another 24 h and then harvested for luciferase measurement. Grey bar, N/P = 5; Black bar: N/P =10. Comparison with cells only, * p<0.05, ** p < 0.01. n.s., non significant.

212

CHAPTER VII: SUMMARY AND FUTURE EXPERIMENTS

In this dissertion, I reviewed the clinical formulation and MOA of taxane drugs, the PK and dose regimens of clinical taxane formulation, the limitations of taxanes, and the new nanoformulations for taxane delivery. A number of new monomers and novel functionalized

POx polymers were subsequently discussed. Their potential to be novel drug delivery systems are partially explored. For example, the clickable handles on the propargyl-P4 polymer has potential to be modified with targeting groups and the resulting targeted P4 can be further applied to deliver proteins, small molecules and siRNAs. In addition, the gradient polymerization using MeOz monomers with MeOx and BuOx will be an interesting future experiment to investigate whether the one-pot synthesis can be realized to reduce time and cost for the P2 polymer. Other than these potential applications of newly synthesized monomers or polymers, the rest of my dissertation focused on the application of P2 polymers for anticancer drug delivery and the CPOx for gene delivery.

P2 represents a novel drug delivery platform capable of incorporating very large amounts of insoluble anticancer drugs PTX in nano-sized formulations which are very simple to prepare, stable on the shelf yet very efficacious in vivo. The nanoformulation features a defined size

(30-50 nm, PDI ~0.1) which is particularly desirable for drug delivery. Its unparalleled high drug loading allows for significantly decrease of the amount of excipient needed which may

213 be connected with the extraordinary high MTD as compared to clinically approved formulations, such as Taxol® and Abraxane®. Consequently, the therapeutic index of P2/PTX micellar formulations is greatly increased. PK data showed a strongly elevated drug exposure to tumor tissues and consequently a significantly enhanced antitumor activity with significantly prolonged survival was observed. These promising preclinical data on P2/PTX nanoformulation provide a robust rational for further development and might indicate that ultimately increased patient survival could be achieved, which is not provided by Abraxane® and Taxol®.

Furthermore, the first example of using P2 block copolymer to deliver 3rd generation taxoids was shown in Chapter IV. All taxoids studied could be incorporated at a nearly

1/1 ratio (wtaxoid/wpolymer) resulting in stable formulation with 50 %wt. active component.

The size of the formulations remained around or below 100 nm as evidenced by DLS and electron microscopy. The efficacy of the selected 3rd-generation taxoid SB-T-1214 in vitro against MDR cancer cell lines was higher than that of PTX, while no difference was observed in the non-resistant cell line. Although the MTD of SB-T-1214 formulated with

Cremophor EL and the POx block copolymer were identical, the tumor inhibition using the POx/SB-T-1214 polymeric micelles was enhanced in two orthotopic multidrug resistant tumor models, LCC6-MDR and T11. The latter model is characterized as a particularly faithful and clinically relevant model TNBC. Survival in this model may be further improved by using drug combinations, for which our POx polymeric micelle platform is well suited.

In addition to formulation of PTX or 3rd generation of taxoids, P2 micelles are able to

214 simuteneously incorporate large amounts of different hydrophobic anti-cancer agents. This carrier system also provides means for the realization of highly stable and well defined binary and ternary drug formulations with unprecedented high drug loading. Interestingly, binary and ternary drug formulations display improved stability and reduced dispersity compared to the single drug containing micelles. The multi-drug loaded micelles can deliver drugs to cancer cells and display synergistic effects against several tumor models. These synergistic effects were dependent on drug ratios, which require further optimization of the corresponding multi-drug formulations. The multi-drug loaded POx micelles represent an attractive platform attractive for further development of combination anti-cancer therapeutics. Future experiments include the determination of synergistic effects of multi-drug loaded P2 micelles in animal models.

Last but not least, I have synthesized the first CPOx with pending secondary amines. The prepared polyplexes via complexing CPOx with pDNA are characterized by low cytotoxicity, low serum protein binding compared to PEG-PLL-based polyplexes. The new polyplexes exhibited excellent stability upon storage and dilution in physiological saline as well as upon thermal challenge. However, the transfection efficiency of the new polyplexes in the macrophage and melanoma cells was not satisfactory, although can be improved using

Pluronic P85 as a gene transfer adjuvant. The CPOx polymer was functionalized with a

“clickable” terminus allowing for attaching the targeting groups, fluorescent dyes and other functional molecules to the resulting polyplexes. In future studies stability and targeting of the new polyplexes in complex biological media can be further explored both in vitro and in vivo. We observed that 10% BOC can help in stabilizing the polyplexes compared with 5% BOC,

215 and we hypothesize that hydrophobicity provided by BOC groups can benefit the electrostatic polyplexes and certainly transfection efficiency. However, a systematic study of structure- property relationship to identify the optimum content of BOC/hydrophobicity requires more quantity of polymer being synthesized which is not available currently. Future experiments can be performed to synthesize more of CPOx polymers with different hydrophobic content to study structure-property relationship. These novel CPOx vectors can be developed as new generation of non-viral gene delivery platform as compared to PEGylated cationic polymer systems.

216

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