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The Dissertation Committee for Urvi Hasmukhlal Gala Certifies that this is the approved version of the following Dissertation:

IMPROVED PROSTATE CANCER THERAPEUTICS THROUGH KINETISOL®

ENABLED AMORPHOUS SOLID DISPERSIONS OF ABIRATERONE

Committee:

Robert O. Williams III, Supervisor

Dave A. Miller

Hugh D. Smyth

Feng Zhang

IMPROVED PROSTATE CANCER THERAPEUTICS THROUGH KINETISOL®

ENABLED AMORPHOUS SOLID DISPERSIONS OF ABIRATERONE

Urvi Hasmukhlal Gala

Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin

in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

The University of Texas at Austin December 2019 Dedication

To my family.

Acknowledgements

I would like to thank my supervisor, Dr. Robert O. Williams III, for his encouragement, support and invaluable guidance during the entire course of the program. I would like to express my earnest gratefulness to Dr. Dave A. Miller, for encouraging and supporting me to pursue doctoral studies. I am also thankful to Dr. Miller for his mentorship and constant guidance throughout the process. I would also like to thank Dr. Ed Rudnic, Dr. Dave Miller, Dr. Chris Brough, Mr. Raj Sheel and Dr. Justin Keen of DisperSol Technologies for financial support for my research and helping guide my research objectives.

I would like to extend my gratitude to my dissertation committee members, Dr. Hugh Smyth, Dr. Feng Zhang and Dr. Dave Miller for their valuable suggestions. I am also thankful to the entire faculty of College of Pharmacy including Dr. Salomon Stavchansky, Dr. Feng Zhang, Dr. Maria Croyle, Dr. Zhengrong Cui, Dr. Janet Walkow and Dr. Kelly Revels for providing enriching courses and encouraging thoughtful discussions. My sincere thanks to College of Pharmacy staff Char Burke, Yolanda Abasta and Jay Hammam for always being helpful. I would also like to thank all my fellow graduate students and lab members at College of Pharmacy for their support and friendship.

My sincere thanks to Mr. Marshall Cisneros and Ms. Vy Nguyen of DisperSol Technologies for their invaluable help. I would also like to thank the entire team of DisperSol Technologies including Devon MacDonald, Marshall Cisneros, Sandra Kucera, Angela Spangenberg and Jackie Jury for supporting and cheering me up during my low times.

I would also like to acknowledge and thank Dr. Harsh Chauhan, my supervisor during Master’s program at Creighton University for his encouragement. I am forever grateful to

v my faculty at Institute of Chemical Technology, Mumbai, who laid the foundation of scientific rationalizing in me.

Most importantly I thank God and my family and I owe all my achievements to them. My father Mr. Hasmukhlal Gala and my mother Mrs. Ranjanben Gala have instilled values and ethics in me, which have helped me reach this point. My sister Dr. Mita Gala and Mrs. Kanan Chheda have been my pillars of strength. I am lucky to have a second set of parents in form of my in-laws Mr. Narendra Savla and Mrs. Nisha Savla who constantly encourage me to achieve the best. I am also thankful to my brother in laws Mr. Dhiren Chheda and Mihir for boosting me up during my hard times. My nieces Prisha and Freya deserve a big thanks for adding that extra happiness in my life. Finally, I thank my husband Romil Savla, the very reason I moved to Austin and all the great things happened in my life. Romil’s love, care, immense sacrifices and unwavering support has helped me sail happily through this process and I will be forever grateful.

vi Abstract

IMPROVED PROSTATE CANCER THERAPEUTICS THROUGH KINETISOL®

ENABLED AMORPHOUS SOLID DISPERSIONS OF ABIRATERONE

Urvi Hasmukhlal Gala, Ph.D.

The University of Texas at Austin, 2019

Supervisor: Robert O. Williams III

A majority of anticancer drugs have inherently poor water solubility, which limits their oral bioavailability and therapeutic potential. In Chapter one, the ability of amorphous solid dispersions (ASDs), to improve the dissolution, pharmacokinetics, efficacy and safety of anticancer drugs is demonstrated. Abiraterone is a poorly water soluble drug used in treatment of prostate cancer. Its commercial formulation Zytiga®, contains slightly more soluble prodrug, abiraterone acetate. Zytiga has poor oral bioavailability, high pharmacokinetic variability and high food effect, which limits its therapeutic potential. Abiraterone has a high melting point and limited solubility in organic solvents, which makes its ASD development difficult with conventional technologies. In Chapter two, KinetiSol® technology, which is a solvent free thermokinetic process, is utilized and an abiraterone ASD is developed for the first time. In addition to contemporary long- chain polymers, a short-chain oligomer is explored for development of binary and ternary KinetiSol processed ASDs (KSD) of abiraterone. Hydroxy propyl beta cyclodextrin (HPBCD) is identified as a suitable carrier for abiraterone KSD. In Chapter three, the impact of drug loading on abiraterone-HPBCD KSD properties is investigated. 10 to 50 % w/w drug loaded abiraterone KSDs are developed. The solid-state interaction studies revealed that abiraterone forms a complex with HPBCD in the KSD. Overall, as drug vii loading increased, the stability, in-vitro and in-vivo performance decreased. Thus, 10% drug loading is found to be optimum. In Chapter four, an optimal KSD of the prodrug abiraterone acetate is developed and compared with abiraterone KSD. A physicochemical stability study revealed, that the abiraterone acetate KSD is chemically unstable, while abiraterone KSD is both physically and chemically stable. Both KSDs have similar in- vitro and in-vivo performance. Thus, it is concluded that the active drug abiraterone’s KSD is more ideal and overcomes the issues associated with the prodrug use. In Chapter five, a preclinical prostate cancer xenograft model is developed to investigate the pharmacokinetic and pharmacodynamic performance of abiraterone KSD. This study demonstrated a statistically significant tumor growth inhibition of 33.1% by the abiraterone KSD. Thus, these studies suggest the potential for the abiraterone KSD formulation to improve therapeutic outcomes for prostate cancer patients.

viii Table of Contents

List of Tables ...... xvii

List of Figures ...... xix

Chapter One: Harnessing The Therapeutic Potential Of Anticancer Drugs Through Amorphous Solid Dispersions ...... 1

1. Abstract: ...... 1

2. Graphical Abstract: ...... 2

3. Introduction:...... 2

4. Poor Water Solubility Of Anticancer Drugs: ...... 5

5. Application Of ASDs To Anticancer Drugs: ...... 7

6. Benefits Of Oral Administration Of Anticancer Drugs And Current Market Scenario: ...... 12

6.1. Benefits of Oral Administration of Anticancer Drugs: ...... 12

6.2. Current Market Scenario for Oncology Drug Products: ...... 15

7. Challenges And Consequences Associated With Oral Administration Of Poorly Water-Soluble Anticancer Drugs: ...... 17

7.1. Drug-Related Challenges: ...... 18

7.1.1. Solid State: ...... 18

7.1.2. Ionization:...... 20

7.1.3. Stability: ...... 21

7.1.4. Drug–drug interaction: ...... 22

7.2. Physiology-Related Challenges: ...... 25

7.2.1. Transmembrane efflux of drugs: ...... 25

7.2.2. Pre-systemic metabolism: ...... 28

ix 7.2.3. Transporter saturation: ...... 29

7.3. Other Challenges:...... 30

7.3.1. Prandial state: ...... 30

7.3.2. Patient/Oncologist Related Challenges: ...... 33

8. Alleviating The Consequences Through ASDs: ...... 35

8.1. Enhancing Oral Bioavailability: ...... 37

8.2. Reducing Pharmacokinetic Variability: ...... 43

8.3. Enhancing Pharmacokinetic Linearity: ...... 46

8.4. Enhancing Efficacy: ...... 48

8.5. Reducing Toxicity:...... 50

9. Case Studies Of Commercial Oncology Products Based On ASDs: ...... 51

9.1. Vemurafenib (Zelboraf®, Roche/Genentech): ...... 53

9.2. Regorafenib (Stivarga®, Bayer): ...... 54

9.3. Everolimus (Afinitor®, Novartis): ...... 55

9.4. Venetoclax (Venclexta®, AbbVie): ...... 55

9.5. Olaparib (Lynparza®, AstraZeneca): ...... 56

10. Conclusion: ...... 57

11. Acknowledgements And Disclosure: ...... 58

12. References:...... 58

Chapter Two: Improved Dissolution And Pharmacokinetics Of Abiraterone Through KinetiSol® Enabled Amorphous Solid Dispersions ...... 87

1. Abstract: ...... 87

2. Graphical Abstract: ...... 88

3. Introduction:...... 88 x 4. Materials And Methods: ...... 93

4.1. Materials: ...... 93

4.2. Methods: ...... 94

4.2.1. Development of KSDs: ...... 94

4.2.2. Physicochemical characterization of KSDs: ...... 95

5. Results And Discussion: ...... 99

5.1. Development of binary KSDs: ...... 99

5.2. Physicochemical characterization of binary KSDs: ...... 103

5.3. Dissolution of binary KSDs: ...... 107

5.4. Selection of a suitable ternary component for KSDs: ...... 110

5.5. Development of ternary KSDs: ...... 115

5.6. Physicochemical characterization of ternary KSDs: ...... 117

5.7. Dissolution of ternary KSDs: ...... 118

5.8. Pharmacokinetic Study in Beagle Dogs:...... 119

6. Conclusions:...... 123

7. Acknowledgements and Disclosure: ...... 123

8. References:...... 123

Chapter Three: The Effect Of Drug Loading On The Properties Of KinetiSol® Processed Abiraterone–Hydroxypropyl β Cyclodextrin Solid Dispersions ...... 131

1. Abstract: ...... 131

2. Graphical Abstract: ...... 132

3. Introduction:...... 132

4. Materials And Methods: ...... 136

4.1. Materials: ...... 136 xi 4.2. Methods: ...... 137

4.2.1. KinetiSol® Processing: ...... 137

4.2.2. Milling: ...... 137

4.2.3. Melt-quenching Abiraterone: ...... 138

4.2.4. X-Ray Powder Diffraction: ...... 138

4.2.5. Modulated Differential Scanning Calorimetry:...... 139

4.2.6. HPLC Analysis:...... 139

4.2.7. Solid-state Nuclear Magnetic Resonance Spectroscopy: ...... 140

4.2.8. Raman Spectroscopy: ...... 140

4.2.9. Phase Solubility Analysis: ...... 141

4.2.10. Stability Analysis: ...... 141

4.2.11. In Vitro Dissolution Study: ...... 141

4.2.12. Tableting: ...... 142

4.2.13. In Vivo Pharmacokinetic Study in Beagle Dogs:...... 142

4.2.14. Pharmacokinetic Analysis: ...... 143

5. Results:...... 143

5.1. Development of KSDs: ...... 143

5.2. Physicochemical analysis of KSDs:...... 145

5.3. Solid-state interaction between abiraterone and HPBCD in KSDs: ...... 147

5.4. Solution-state phase solubility profile: ...... 154

5.5. Stability of KSDs: ...... 154

5.6. In Vitro and In Vivo Performance of KSDs: ...... 155

6. Discussion: ...... 159 xii 6.1. Development of KSDs: ...... 159

6.2. Physicochemical analysis of KSDs:...... 161

6.3. Solid-state interaction between abiraterone and HPBCD within KSDs: ...... 163

6.4. Solution-state phase solubility profile: ...... 167

6.5. Stability of KSDs: ...... 168

6.6. In Vitro and In Vivo Performance of KSDs: ...... 168

7. Conclusions:...... 170

8. Acknowledgements And Disclosure: ...... 171

9. References:...... 171

Chapter Four: Comparative Evaluation Of KinetiSol® Processed Amorphous Solid Dispersions Of Abiraterone Acetate And Abiraterone: Is The Prodrug Necessary? .178

1. Abstract: ...... 178

2. Graphical Abstract: ...... 179

3. Introduction:...... 179

4. Materials And Methods: ...... 183

4.1. Materials: ...... 183

4.2. Methods: ...... 184

4.2.1. KinetiSol® Processing: ...... 184

4.2.2. Milling: ...... 185

4.2.3. X-Ray Powder Diffraction: ...... 186

4.2.4. HPLC Analysis:...... 186

4.2.5. Physicochemical Stability Analysis: ...... 187

4.2.6. In Vitro Dissolution Study: ...... 188

xiii 4.2.7. Supersaturation Study: ...... 188

4.2.8. Tableting: ...... 189

4.2.9. In Vivo Pharmacokinetic Study in Beagle Dogs:...... 189

4.2.10. Pharmacokinetic Analysis: ...... 190

5. Results & Discussion: ...... 190

5.1. Development of abiraterone acetate KSDs: ...... 190

5.1.1. Identification of a suitable carrier for abiraterone acetate KSDs: ...... 190

5.1.2. Identification of an optimal drug loading for the abiraterone acetate KSD: ...... 199

5.2. Development of abiraterone KSDs: ...... 202

5.3. Comparison of the physicochemical stability of the abiraterone acetate KSD and abiraterone KSD: ...... 202

5.4. Comparison of the in vitro performance and supersaturation ability of abiraterone acetate KSD and abiraterone KSD: ...... 208

5.5. Comparison of the in vivo performance of the abiraterone acetate KSD and the abiraterone KSD:...... 211

5.6. Consideration of other implications of abiraterone acetate use: ..213

6. Conclusion: ...... 214

7. Acknowledgements And Disclosure: ...... 214

8. References:...... 214

Chapter Five: Improved Apparent Solubility Of Abiraterone Leads To Enhanced Therapeutics In A Preclinical Model ...... 220

1. Abstract: ...... 220

2. Graphical Abstract: ...... 221

3. Introduction:...... 221

xiv 4. Material And Methods: ...... 226

4.1. Materials: ...... 226

4.2. Methods: ...... 226

4.2.1. Abiraterone KSD Preparation: ...... 226

4.2.2. Solubility Study: ...... 227

4.2.3. Pharmacokinetic Study:...... 227

4.2.4. Pharmacokinetic Analysis: ...... 228

4.2.5. Xenograft Model Development:...... 228

4.2.6. Tumor Growth Inhibition Study:...... 229

4.2.7. Tumor Growth Inhibition Analysis: ...... 229

5. Results And Discussion: ...... 230

5.1. Apparent solubility of neat abiraterone API and abiraterone KSD: ...... 230

5.2. Pharmacokinetic performance of neat abiraterone acetate API and abiraterone KSD:...... 231

5.3. Pharmacodynamic performance of neat abiraterone acetate API and abiraterone KSD: ...... 237

6. Conclusion: ...... 241

7. Acknowledgements And Disclosure: ...... 241

8. References:...... 242

Chapter Six: Concluding Remarks And Future Direction ...... 247

1. Dissertation Conclusion: ...... 247

2. Future Direction: ...... 248

3. References:...... 250

xv Appendix ...... 251

Bibliography ...... 268

Vita ...... 319

xvi List of Tables

Table 1.1 Benefits of administering anticancer drugs orally...... 13 Table 1.2. List of poorly water-soluble anticancer drugs that are substrates for major

efflux pumps located on the intestinal membrane. [167, 172-174]: ...... 27 Table 1.3. List of poorly water-soluble anticancer drugs demonstrating prominent

food effects on their pharmacokinetics...... 31

Table 1.4. Commercial Oncology Products Based on Amorphous Solid Dispersions. ....52 Table 2.1. Primary polymers/Oligomers selected for development of binary KSDs of

abiraterone [35, 42-44]...... 101 Table 2.2. Binary KSD compositions, processing parameters, and their corresponding

appearance...... 102 Table 2.3. Relative area under the drug dissolution curve for the selection of suitable

ternary components (i.e., secondary polymers)...... 114

Table 2.4. Ternary KSD composition, processing parameters, and appearance...... 116

Table 2.5. Results from the in vivo pharmacokinetic (PK) study in male beagle dogs. .122

Table 3.1. KSD composition and processing parameters...... 144 Table 3.2. Raman peak positions for neat abiraterone API, melt-quenched abiraterone

API, Lot 1 PM, and Lots 1 to 5 KSDs...... 152 Table 3.3. Raman peak shifts for melt-quenched abiraterone API, Lot 1 PM, and Lots

1 to 5 KSDs...... 153

Table 3.4. Results from in vivo pharmacokinetic (PK) study in male beagle dogs...... 158 Table 4.1. Abiraterone acetate KSD compositions, processing parameters and their

corresponding appearance...... 193 Table 4.2. Abiraterone acetate KSD compositions with higher drug loading and their

processing parameters...... 199 xvii Table 4.3. Abiraterone KSD composition and its processing parameters...... 202

Table 4.4. Results from in vivo pharmacokinetic (PK) study in male beagle dogs...... 212

Table 5.1. Pharmacokinetic study design...... 231

Table 5.2. Tumor growth inhibition study design...... 239

Table 5.3. Results of tumor growth inhibition study...... 241

xviii List of Figures

Figure 1.1. Schematic representation of the process of solubilization of anticancer drugs in their crystalline state, neat amorphous state, and in an ASD. Reproduced with permission. The schematic is modified with respect to

anticancer drug states and solubilization steps [38]...... 11 Figure 1.2. Oncology drug products approved by the US FDA between 2000 and

2018, categorized based on route of administration...... 16 Figure 1.3. Oral anticancer drugs, categorized based on type of formulation.

“Reproduced from [13]. Reproduced with permission”...... 17 Figure 1.4. Challenges and consequences associated with the oral administration of

poorly water-soluble anticancer drugs...... 34 Figure 1.5. Role of ASDs in alleviating the challenges and consequences associated with the oral administration of poorly water-soluble anticancer drugs, thereby leading to improved oncological therapeutic outcomes. [ASD–

amorphous solid dispersions, MTC-minimum toxic concentration, MEC-

minimum effective concentration, Cmax = maximum plasma drug

concentration, Cmin = minimum plasma drug concentration, AUC = area under the plasma drug concentration time curve, t = duration of drug

exposure] ...... 36 Figure 1.6. (a) pH-solubility profile of GT0918 (proxalutamide) at 37 °C and (b) plasma concentration–time profiles of GT0918 (proxalutamide) in

beagle dogs after a single-dose oral administration of pH-modified solid dispersion tablets (□) and conventional tablets (Δ). Reproduced with

permission [269]...... 44

xix Figure 1.7. (a) Kaplan–Meier plots of overall survival data from trial 25026 showing a trend for exposure response. Low and high vemurafenib exposure were

defined by patients with Cmin,tn values < or ≥ 39.0 μg/mL [Figure taken from [297]]. (b) Comparison of dose-normalized exposure data among three capsule formulations: Phase I crystalline vemurafenib and two ASD vemurafenib formulations (MBP) [Reproduced with permission

[39]]...... 54

Figure 2.1. Structure of Abiraterone Acetate and Abiraterone...... 88 Figure 2.2. Activation energy diagram for the solubilization of a drug from a

crystalline form (right) and from an ASD (left)...... 91 Figure 2.3. X-Ray diffractograms of abiraterone API (top) and abiraterone binary

PMs, KSDs (bottom)...... 104 Figure 2.4. mDSC thermograms of abiraterone API (top) and abiraterone binary

KSDs (bottom)...... 106 Figure 2.5. In vitro, non-sink, gastric transfer dissolution profiles of neat abiraterone API, generic abiraterone acetate tablets, and binary KSDs. The red region: 0.01N HCl. The blue region: FaSSIF. The inset represents the

enlargement of the dissolution profile in FaSSIF...... 110 Figure 2.6. In vitro, non-sink, gastric transfer dissolution profile of abiraterone API, generic abiraterone acetate tablets, Lot 5 KSD and Lot 5 KSD with different secondary polymer candidates. Red region: 0.01N HCl. Blue

region: FaSSIF. The inset represents the enlargement of the dissolution

profile in FaSSIF...... 113

Figure 2.7. X-ray diffractograms of abiraterone ternary KSDs...... 117

xx Figure 2.8. In vitro, non-sink, gastric transfer dissolution profiles of the Lots 5, 6, and 7 KSDs. Red region: 0.01N HCl. Blue region: FaSSIF. The inset

represents the enlargement of the dissolution profile in FaSSIF...... 119 Figure 2.9. In vivo plasma concentration versus time profiles from oral dosing of the generic abiraterone acetate tablet, the Lot 2 Tablet, the Lot 5 Tablet, and

the Lot 6 Tablet in fasted non-naïve male beagle dogs...... 121

Figure 3.1. Structure of abiraterone and hydroxypropyl β cyclodextrin...... 134 Figure 3.2. X-ray diffractograms of (Top) neat abiraterone API, melt-quenched

abiraterone API and HPBCD; (Bottom) for the Lot 1 PM and the Lots 1 to 5 KSDs. The dotted circles in the top figure indicate the characteristic peaks of abiraterone API. The dotted lines in the bottom figure indicate

the peak position regions of these characteristic peaks...... 146

Figure 3.3. mDSC thermogram of the Lot 1 PM and the Lots 1 to 5 KSDs...... 147 Figure 3.4. 13C ssNMR spectra of neat abiraterone API, HPBCD, the Lot 1 PM, and

the Lots 1 to 5 KSDs. The dotted rectangles indicate the regions of sp3 hybridized carbon atoms, the C3 carbon atom, and sp2 hybridized carbon

atoms of neat abiraterone API...... 149 Figure 3.5. 2D 13C–1H HETCOR spectra of neat abiraterone API (black), HPBCD

(red), and the Lot 3 KSD (blue). 1H cross sections at 103.9 ppm in the

13C dimension are shown in the 2D spectra...... 150 Figure 3.6. Phase solubility profiles for abiraterone–HPBCD in 0.01N HCl and

FaSSIF...... 154 Figure 3.7. X-ray diffractograms of Lots 1 to 3 KSDs at 90 °C and 150 °C. The dotted lines indicate the peak position region of abiraterone

characteristic peaks...... 155 xxi Figure 3.8. In vitro, non-sink, gastric transfer dissolution profiles of neat abiraterone API and Lots 1 to 5 KSDs. The red region indicates 0.01N HCl, and the

blue region indicates FaSSIF...... 156 Figure 3.9. In vivo average plasma concentration vs. time profiles from oral dosing of Zytiga® and the Lots 1 to 3 Tablets in fasted, non-naïve, male beagle

dogs...... 157 Figure 3.10. In vitro and in vivo relative performance percentages of KSDs with

various drug loadings...... 159

Figure 4.1. The presystemic conversion of abiraterone acetate to abiraterone...... 180 Figure 4.2. Polymer/oligomer space mapped for abiraterone acetate KSD

development...... 191 Figure 4.3. X-ray diffractograms of neat abiraterone acetate API (top) and the Lots 1–8 KSDs (bottom). The dotted circle in the top figure indicates the characteristic peaks of abiraterone acetate API. The dotted lines in the

bottom figure indicate the peak position region of these characteristic peaks, and the dotted rectangle in the bottom figure indicates the sharp

diffraction peak seen in the Lot 1 KSD...... 196 Figure 4.4. In vitro, non-sink gastric transfer dissolution profiles of neat abiraterone

acetate API and abiraterone acetate KSD. The red region represents 0.01N HCl, and the blue region represents FaSSIF. The inset represents

the enlargement of the dissolution profile in FaSSIF...... 198

Figure 4.5. X-ray diffractograms of the Lots 9 and 10 KSDs...... 200 Figure 4.6. In vitro, non-sink gastric transfer dissolution profiles of the Lots 9 and 10 KSDs. The red region represents 0.01N HCl, and the blue region

indicates FaSSIF...... 201 xxii Figure 4.7. Heat–cool–heat DSC thermograms for neat abiraterone acetate API (top)

and neat abiraterone API (bottom)...... 204 Figure 4.8. X-ray diffractograms of the abiraterone acetate KSD (top) and the abiraterone KSD (bottom) at room temperature and under accelerated

conditions...... 206 Figure 4.9. Total impurity profiles for abiraterone acetate KSD and abiraterone KSD

at room temperature and under accelerated conditions...... 207 Figure 4.10. In vitro, non-sink gastric transfer dissolution profiles of neat abiraterone

acetate API, neat abiraterone API, abiraterone acetate KSD, and abiraterone KSD. The red region indicates 0.01N HCl, and the blue region indicates FaSSIF. The inset represents the enlargement of the

dissolution profile in FaSSIF...... 209 Figure 4.11. In vitro supersaturation ability of the abiraterone acetate KSD and

abiraterone KSD...... 210 Figure 5.1. Effect of increased abiraterone exposure on its pharmacodynamics [22, 24-26]. [Blue - action of abiraterone at its standard exposure, Red and Green- action of abiraterone and its potent metabolite D4A at elevated exposure]; [CYP17A1 - 17α-hydroxylase/C17, 20-lyase; 3βHSD - 3β-

hydroxysteroid dehydrogenase; AKR31C - Aldo-keto reductase family

1 member C3; PSA - Prostate specific antigen]...... 224 Figure 5.2. Apparent solubility of neat abiraterone API and abiraterone KSD across

different pH...... 230 Figure 5.3. Pharmacokinetic performance of neat abiraterone acetate API and

abiraterone KSD in male SCID mice...... 235

xxiii Figure 5.4. Dose- exposure relationship of neat abiraterone acetate API and

abiraterone KSD in male SCID mice...... 236 Figure 5.5. Pharmacodynamic performance of neat abiraterone acetate API and

abiraterone KSD in 22Rv1 xenograft model...... 240

Figure 6.1. Illustrative summary of the dissertation...... 249

xxiv

Chapter One: Harnessing The Therapeutic Potential Of Anticancer Drugs Through Amorphous Solid Dispersions

1. ABSTRACT: 1The treatment of cancer is still a major challenge. But tremendous progress in anticancer drug discovery and development has occurred in the last few decades. However, this progress has resulted in few effective oncology products due to challenges associated with anticancer drug delivery. Oral administration is the most preferred route for anticancer drug delivery, but the majority of anticancer drugs currently in product pipelines and the majority of those that have been commercially approved have inherently poor water solubility, and this cannot be mitigated without compromising their potency and stability. The poor water solubility of anticancer drugs, in conjunction with other factors, leads to suboptimal pharmacokinetic performance. Thus, these drugs have limited efficacy and safety when administered orally. The amorphous solid dispersion (ASD) is a promising formulation technology that primarily enhances the aqueous solubility of poorly water-soluble drugs. In this review, we discuss the challenges associated with the oral administration of anticancer drugs and the use of ASD technology in alleviating these challenges. We emphasize the ability of ASDs to improve not only the pharmacokinetics of poorly water-soluble anticancer drugs, but also their efficacy and safety. The goal of this paper is to rationalize the application of ASD technology in the formulation of anticancer drugs, thereby creating superior oncology products that lead to improved therapeutic outcomes.

1 Gala, U.H., D.A. Miller, and R.O. Williams, Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2020. 1873(1): p. 188319. The dissertator has primarily conceptualized and written this review manuscript.

2. GRAPHICAL ABSTRACT:

3. INTRODUCTION: Cancer is a major global health problem. It is one of the leading causes of death worldwide [1]. According to the American Cancer Society’s 2019 statistics, about 1,762,450 new cancer cases and 606,880 cancer-related deaths are projected to occur in the United States alone [2]. Thus, the ever-increasing burden of cancer treatment not only necessitates the discovery and development of new anticancer drugs, but it also calls for the improvement of existing anticancer drugs.

Despite the enormity of the research in the anticancer drug discovery and development space, the success rate for anticancer drugs has remained consistently poor for years [3]. It has been estimated that, only 1 of every 5,000–10,000 prospective anticancer agents receives FDA approval, and only 5% of anticancer drugs entering Phase I clinical trials are ultimately approved [4]. In a recent study, the overall probability of new anticancer drugs successful passing from Phase I to approval was found to be unacceptably low, at 2

3.7% [5]. One of the causes of such a high attrition rate for new anticancer drugs is their poor pharmacokinetics, which largely stems from their poor water solubility [6-9]. It has been estimated that about 75% of new drug development candidates have poor water solubility, and many of these are anticancer drugs [7].

Another approach being explored for anticancer drug development is repurposing, which involves exploring approved non-anticancer drugs for anticancer activity. This approach also faces challenges due to a lack of optimal physicochemical properties of these drugs, such as poor water solubility, which limits their application in oncology [10]. For instance, Rapamycin, an immuno-suppressant, has shown promising anticancer activity, but has poor water solubility and thus attempts are being made to derive its more soluble analogs, in order to facilitate its use in oncology [11, 12]. Among the existing approved oral anticancer drugs, about 65% have poor water solubility and thus they do not achieve their potential therapeutic outcomes with maximum efficacy and minimum toxicity [13]. Oral administration is the most preferred route of drug delivery for anticancer drugs [14]. It offers several advantages, such as ease of administration and reduced therapy cost. Oral administration allows for feasible continuous drug administration [14-16]. One of the prerequisites for successful oral chemotherapy is achieving a reliable and consistent pharmacokinetic profile for the drugs, which enables maximum drug efficacy and minimal toxicity. However, several challenges are associated with the physicochemical properties of drugs and the physiology of the gastrointestinal tract. These challenges limit the achievement of desirable pharmacokinetics, thus they also limit the pharmacodynamics of orally administered, poorly water-soluble anticancer drugs.

The poor water solubility of anticancer drugs leads to suboptimum formulations or requires the use of excipients that have toxic side effects. For example, Nexavar® (sorafenib tosylate), an orally administered kinase inhibitor is used in the treatment of hepatocellular and renal cell carcinoma [17, 18]. According to the biopharmaceutical classification system (BCS), sorafenib belongs to BCS Class II, which is characterized by low solubility and high permeability. Thus, sorafenib has unacceptably low and slow 3

dissolution in the gastrointestinal tract, which is a rate-limiting step in its absorption and, along with its first-pass metabolism, results in low oral bioavailability and wide intersubject variability [19]. Thus, the poor water solubility of sorafenib leads to either sub-therapeutic outcomes or acute toxicity [20, 21]. Paclitaxel, a well-known anticancer drug, has poor water solubility (< 0.03 mg·mL−1) [22]. Thus, the intravenous formulation Taxol® was developed using Cremophor EL (polyethoxylated castor oil) and ethanol to solubilize paclitaxel [23]. The use of Cremophor EL led to acute hypersensitivity reactions in patients. Despite the use of premedication, reactions still occurred in ~44% of patients, and potentially life-threatening reactions occurred in ~3% of patients [24, 25]. Hence, the poor water solubility of existing anticancer drugs poses challenges not only for oral formulations but also for intravenous formulations.

Several attempts to address the poor water solubility of anticancer drugs have been reported, such as the use of prodrugs, polymeric nanoparticles, lipoidal microspheres, solubilizers, and nanocolloids [26-34]. However, these attempts are limited by several challenges, such as low drug loading capacity, complex physical structures, instability, potential material toxicity, altered drug distribution, and clearance [35, 36].

The amorphous solid dispersion (ASD) is a formulation technology in which the drug, also known as the active pharmaceutical ingredient (API), is dispersed in an amorphous carrier [37]. ASDs aid the dissolution of poorly water-soluble drugs primarily by presenting the drug in an amorphous form, thereby lowering the total energy required for the solubilization of the crystalline drug [38]. ASD technology has generated tremendous benefits for therapeutically potent, poorly water-soluble anticancer drugs such as vemurafenib, regorafenib, everolimus, venetoclax, and olaparib [13, 39, 40].

In this review, we discuss the causes of the poor water solubility of anticancer drugs, the application of ASDs in formulating anticancer drugs, and the benefits they offer for delivering anticancer drugs orally. We focus on anticancer drugs because their poor water solubility cannot be mitigated without compromising their potency and stability. We

address the issues encountered during the oral delivery of poorly water-soluble anticancer drugs, and we discuss the role of ASDs in resolving such issues. We focus on using ASD technology not only to enhance the oral bioavailability of poorly water-soluble drugs, but also to improve the pharmacokinetic properties of such drugs and thereby improve their pharmacodynamics. The goal of this paper is to focus our literature analysis on the ability of ASDs to harness the maximum therapeutic potential of anticancer drugs.

4. POOR WATER SOLUBILITY OF ANTICANCER DRUGS: Poor water solubility is an inherent property of many anticancer drugs for two main reasons. First, based on our literature review, there is seldom emphasis given on the physicochemical properties of drug candidates during anticancer drug discovery. Second, certain indispensable hydrophobic structural features are required for anticancer drug permeability, activity, and stability, which impart poor water solubility to the drug.

Historically, one of the major pitfalls in the anticancer drug development pipeline is its negligible focus on drug disposition and pharmacodynamics [41]. The “nanomolar rule” was widely adopted in cancer drug discovery. This rule involves selecting compounds that have nanomolar potency for development [42]. This rule was based on the assumption that such compounds are required at low doses and thus would be safe and efficacious. This rule ignored a critical parameter: the physicochemical properties of these compounds, which affect their pharmacokinetics and thus their safety and efficacy [42]. Thus, compounds such as combretastatins A-4, having excellent cytotoxicity in nanomolar concentrations, were selected for development, but they eventually failed due to their poor water solubility [43, 44].

Recently, a target-based drug discovery and development approach has been adopted for new anticancer drugs. This involves target identification and validation, followed by the development and screening of agents against their targets, followed by lead identification and optimization [45]. The protocols adopted for the screening of agents against their

targets are largely unaffected by the physicochemical properties of the drug—more specifically, their water solubility. For example, in most in vitro experiments, the drugs are dissolved in organic solvents (instead of physiologically relevant aqueous solvents) and their anticancer activity on cell lines is evaluated [42, 46]. Thus, there is no selection bias against poorly water-soluble anticancer drugs in the preclinical stage. As a result, several promising anticancer drug candidates that emerged out of the drug discovery process failed during clinical development due to their poor water solubility (e.g., ABT- 737, wortamanin, fenretinide, sapacitabine) [31, 47-50].

A certain degree of hydrophobicity or lipophilicity is necessary for anticancer drugs to cross cell membranes and reach their target site of action [51]. According to Veber et al., compounds that have a topological polar surface area (TPSA) ≤ 140 have acceptable permeability [52]. However, the lower the polar surface area, the lower the water solubility. Thus, in practice, it is difficult to balance the two opposing factors of high solubility with low polarity. ABT-737, which has a lower TPSA of 131, was designed against Bcl-2 and Bcl-xL proteins, which are involved in the regulation of apoptosis. ABT-737 exhibited high potency, but it also exhibited low water solubility and therefore poor bioavailability [47, 53]. ABT-737 was then chemically modified by replacing its nitro group, which reduced its acylsulfonamide acidity. This change increased the drug’s oral bioavailability, but it also caused a decrease in potency [53]. Later, the compound ABT-263 (i.e., navitoclax), which has a TPSA of 128, was developed. This compound exhibited dissolution-limited poor oral bioavailability [53]. Eventually, compound ABT- 199 (i.e., venetoclax), with a TPSA of 172, was developed. It too possessed dissolution- limited poor oral bioavailability without an enabling formulation technology [53]. Thus, in order to achieve good permeability and therapeutic activity, all the compounds designed as Bcl inhibitors were burdened with markedly poor water solubility. Many anticancer drugs require bulky hydrophobic structures such as polycyclics to exert their anticancer effects by binding to their target receptors [54, 55]. For instance, paclitaxel is a widely used anticancer drug. Paclitaxel has a bulky, fused taxane ring in its center,

surrounded by several hydrophobic functional groups, which impart high lipophilicity and low water solubility to the drug [56]. Several attempts have been made to modify the taxane ring and to substitute hydrophilic functional groups. While these analogs imparted slight increases in water solubility, the modified drugs had less potency than paclitaxel, thereby confirming that certain hydrophobic structures were essential for paclitaxel’s antineoplastic activity [57, 58].

Another contributor to the poor water solubility of anticancer drugs is the crystalline form of the drug. The crystalline form has advantages such as high purity and stability. However, to solubilize the crystalline form, its lattice energy barrier must be overcome, which is challenging and leads to slower drug dissolution [59]. On the other hand, the amorphous form of an anticancer drug is more water soluble, but it is also typically physically unstable, hence almost all anticancer drugs are synthesized in crystalline form. For example, bicalutamide is an anticancer drug used in the treatment of prostate cancer, and it has poor water solubility [60]. Several studies have shown that the amorphous form of bicalutamide is more water soluble than its crystalline form. However, the amorphous form of bicalutamide is highly unstable, which leads to recrystallization of the drug [60, 61]. Hence, bicalutamide is commercially available in the poorly water-soluble crystalline form. Thus, poor water solubility is an intrinsic property of several anticancer drugs.

5. APPLICATION OF ASDS TO ANTICANCER DRUGS: According to modified Noyes–Whitney equation [40, 62], 푑퐶 (퐶푠 − 퐶) = 퐷푆 푑푡 ℎ where dC/dt is the rate of dissolution, S is the surface area available for dissolution, D is the diffusion coefficient of the drug, Cs is the saturation solubility of the drug in the dissolution medium, C is the instantaneous concentration of the drug in the medium at

time t, and h is the thickness of the diffusion boundary layer adjacent to the surface of the dissolving drug.

According to this equation, the rate of dissolution of the drug can be increased by increasing the surface area for dissolution, which can be achieved by reducing the particle size. However, there are limitations to the minimum particle size that can be attained without encountering issues such as particle segregation and agglomeration. Other possibilities to increase the dissolution rate include modifying the surface of particles to enhance their wettability, reducing the thickness of the diffusion layer, and maintaining a high concentration gradient. Among these possible methods, the hydrodynamic changes are difficult to invoke in vivo, and sink condition maintenance also depends on the intrinsic properties of the drug [63]. One possibility for increasing the dissolution rate— i.e., increasing the saturation solubility of the drug or its apparent aqueous solubility— appears to be most promising method, and it is attainable by reducing particle size and by adopting an ASD technology [63, 64].

The term solid dispersion was first defined as “… the dispersion of one or more active ingredients in an inert carrier or matrix at solid state prepared by the melting (fusion), solvent, or melting-solvent method” [65]. ASDs are essentially solid glass solutions in which the API is dissolved in an amorphous carrier [37]. The amorphous form of the drug exhibits a disordered structure compared to its crystalline form, and it possesses higher free energy, which leads to higher apparent aqueous solubility and faster dissolution of the drug [66]. However, the neat amorphous form of the drug itself tends to be highly unstable and shows a greater tendency to recrystallize [67]. Thus, neat amorphous forms of drugs are rarely used.

ASDs kinetically stabilize the amorphous form of the drug by dispersing the drug in an amorphous matrix composed of polymers or oligomers. Figure 1.1 illustrates the process of solubilization of anticancer drugs in their crystalline state, their neat amorphous state, and in ASD form. This process involves a pre-step to convert the crystalline drug to a

non-crystalline form of the drug and three basic steps: (1) solvent cavitation, (2) solvation, and (3) maintenance of solvation.

In the pre-step, the crystal lattice of a poorly water-soluble anticancer drug in its crystalline state must be disrupted to convert the compound to a non-crystalline drug. This is an endothermic step that requires high energy to disrupt the crystalline lattice. The non-crystalline state of the drug is highly physically unstable and tends to revert to its crystalline state. There is no significant pre-step for neat amorphous anticancer drugs, since these drugs are already in an amorphous form. However, neat amorphous drugs are also highly unstable and have a greater tendency to recrystallize. Similarly, no significant pre-step is involved for the solubilization of anticancer drugs in an ASD. Moreover, the physical form of a drug in an ASD is stabilized by polymers/oligomers, which prevent recrystallization of the drug.

After the pre-step, the first step of solvent cavitation involves the creation of space in the solvent to host the drug molecules. This is also an endothermic step. The rate and extent of solvent cavitation is higher for an ASD compared to a crystalline or neat amorphous drug. This is because the hydrophilic polymers/oligomers aid in the reduction of surface tension between the drug and solvent, thereby making larger cavities in the solvent more quickly.

After solvent cavitation, the next step involves the solvation of the drug molecules into the solvent, thereby leading to drug solubilization. This is an exothermic step. For crystalline anticancer drugs, only limited amounts of the drug dissolve, corresponding to or slightly greater than its equilibrium solubility. For neat amorphous anticancer drugs and for anticancer drugs in an ASD, more of the drug dissolves, leading to a supersaturated solution.

The final step involves maintenance of the solvated state of the drug. For crystalline drugs, precipitation of the dissolved drug above its equilibrium solubility occurs. For neat amorphous anticancer drugs, the maintenance of their supersaturated solvated state is 9

momentary, and the drug undergoes rapid precipitation. On the other hand, for anticancer drugs in an ASD, the polymers/oligomers prevent drug precipitation and thereby maintain the supersaturation of the solvated drug.

There are several methods for manufacturing ASDs based on heating (e.g., hot melt extrusion) or based on solvents (e.g., spray-drying, freeze drying, thin-film freezing, coprecipitation, electro-spinning) [39, 68-73]. The heating-based methods may not be suitable for thermolabile drugs. The solvent-based methods are limited by the solubility of the drug in organic solvents. In addition, solvent-based methods are often associated with economic, environmental, and safety concerns. We have previously reported that KinetiSol® is a solvent-free, thermokinetic method for manufacturing ASDs, and this method is suitable for several drugs, including thermolabile drugs and drugs that have limited solubility in organic solvents [74-77].

6. BENEFITS OF ORAL ADMINISTRATION OF ANTICANCER DRUGS AND CURRENT MARKET SCENARIO: One might argue that the parenteral administration of anticancer drugs should be the preferred route, since parenteral administration circumvents several challenges associated with the oral administration of poorly water-soluble drugs. However, the abundant benefits of oral administration tend to refute this argument.

6.1. Benefits of Oral Administration of Anticancer Drugs:

Administering anticancer drugs orally addresses unmet personnel needs, unmet pharmacoeconomic needs, as well as unmet pharmacological needs. Table 1.1 illustrates the benefits of administering anticancer drugs orally.

Table 1.1 Benefits of administering anticancer drugs orally.

Benefits References

Personnel Benefits

Patient Perspective Oncologist Perspective

• Independence, home- • Patient compliance [15, 78-80] based therapy • Scheduling ease • Better tolerability [14, 15]

• No needles, pain-free • Ease of dosing, staff savings [81, 82] administration, reduced risk of infections • No hospitalization needed, • Ease of withdrawal, in the [15, 16, 83] reduced travel time and event of toxicity cost • Minimal monitoring/ • Ease of dose modification [82] laboratory testing

In several surveys, cancer One survey found that more than [15, 78, 84- patients clearly preferred oral 80% of oncologists in the US had 87] chemotherapy over increased their prescription of oral intravenous chemotherapy anticancer drugs over the period of (e.g., > 89%) analysis (two years).

Table 1.1 Continued Pharmacoeconomic Benefits

Medication Costs Hospitalization Costs

• Comparatively cheaper • Reduced visits [15] medication • Resources savings [15, 88]

• Staff savings-oncologist, [15] nurses time and availability

One study found that oral An Italian analysis demonstrated [89, 90] chemotherapy was associated that oral capecitabine was the with a 36% reduction in cost for “dominant strategy” in breast cancer patients and a 43% pharmacoeconomic reduction in cost for colon cancer terms, providing a savings from the patients compared to intravenous Italian hospital perspective of chemotherapy. €2,234 per patient compared to intravenous treatment with fluorouracil/leucovorin.

Table 1.1 Continued Pharmacological Benefits

Continuous/Chronic Therapy Co-Therapy

• Opportunity to suppress • Reduces the severity of [14, 91] the target continuously toxicity associated with through chronic dosing combination or co-therapy with oral chemotherapy compared to intravenous chemotherapy • Makes metronomic (low- [92, 93] dose continuous) dosing feasible

One example is the continuous [94] administration of tyrosine kinase inhibitors for better therapeutic outcomes.

6.2. Current Market Scenario for Oncology Drug Products:

We performed a survey (See Appendix for details) of the oncology drug products approved by the US FDA between January 2000 and September 2018. We then categorized these drugs based on their route of administration [95, 96]. Figure 1.2 illustrates the results of the survey. We find that among the 167 oncology drug products surveyed, about 42.8% were intravenously administered. Thus, there are enormous opportunities for developing oral counterparts for currently intravenously administered anticancer drugs, thereby making their therapy more convenient and

economic. Anticancer drugs such as vinorelbine, idarubicin, etoposide, and paclitaxel have undergone the beneficial ‘intravenous to oral switch’ [82, 97]. Yet, there are some shortcomings with their therapies, such as high intersubject pharmacokinetic variability and toxicity, which could be resolved by ASDs.

Figure 1.2. Oncology drug products approved by the US FDA between 2000 and 2018, categorized based on route of administration.

Sawicki et al. surveyed 72 oral anticancer drugs and categorized them based on their formulation approach and BCS class. Figure 1.3 illustrates the types of formulation approach adopted for the surveyed 72 oral anticancer drugs. They found that 65% of the drugs surveyed were poorly water soluble and belonged to BCS Class II or IV . Yet, only 4% of the drugs surveyed were formulated as ASDs, and the majority of them were formulated as physical mixtures—i.e., the neat crystalline drugs were dry mixed or granulated with pharmaceutical excipients [13]. This suggests that a majority of orally administered anticancer drugs are suboptimally delivered, which indicates a substantial

opportunity for harnessing their complete therapeutic potential through the application of ASD technology.

Figure 1.3. Oral anticancer drugs, categorized based on type of formulation. “Reproduced from [13]. Reproduced with permission”.

From the above surveys, we can conclude there is a tremendous pool of existing approved and marketed oncology drug products that could benefit from ASD technology by converting their existing intravenous formulations into oral formulations or improving their current oral formulations.

7. CHALLENGES AND CONSEQUENCES ASSOCIATED WITH ORAL ADMINISTRATION OF POORLY WATER-SOLUBLE ANTICANCER DRUGS:

Based on the above analysis and discussion, it is evident that the oral administration of anticancer drugs is beneficial and there is a need for improved anticancer drug formulations. In order to develop superior formulations for harnessing more of the

therapeutic potential of anticancer drugs, it is imperative to understand the possible challenges that could arise. In addition to poor water solubility, several other challenges are associated with administering poorly water-soluble anticancer drugs orally [91].

7.1. Drug-Related Challenges:

7.1.1.Solid State:

For an oral anticancer drug to be effective, it must first dissolve in the gastrointestinal tract [98, 99]. As mentioned earlier, many anticancer drugs have poor water solubility, which limits their dissolution in gastrointestinal tract. The equilibrium solubility of anticancer drugs is not only dependent on interactions between the drug and solvent but also on intermolecular interactions within the solid state of the drug [100]. Most anticancer drugs are available in their crystalline state, which has strong intermolecular interactions. As discussed above, these intermolecular interactions lead to strong crystal packing, so they must be disrupted in order to solubilize the drug. Thus, crystal packing in the solid state of anticancer drugs is a major challenge that affects their solubility. Anticancer drugs can exist in multiple crystalline phases called polymorphs. These polymorphs have the same chemical composition but different internal structures [101]. Solvates or hydrates are formed when one or more solvents or water molecules are incorporated into a drug crystal lattice [102]. Thus, anticancer drugs can exist as different polymorphs, solvates, or hydrates in their solid state, which can affect their stability and solubility [103].

For example, axitinib, a kinase inhibitor, is used in the treatment of renal cell carcinoma [104]. Structurally, it has strong molecular flexibility, thus it can exist in 60 solvates, polymorphs of solvates, and five anhydrous forms, all of which have varied stability and solubility [105]. Depending on the method of synthesis, dasatinib can crystallize in several different solvate forms, each with varied solubility, thereby exacerbating the issue of its poor water solubility [106, 107]. Bicalutamide can exist in polymorphic Forms I 18

and II [108, 109]. While Form I of bicalutamide has higher stability, its unstable Form II is 2.4 times more soluble than Form I [108-110]. 6-mercaptopurine, an anticancer drug used in the treatment of leukemia, can exist in different polymorphic forms that have varied solubility [111-113]. Sorafenib tosylate exhibits polymorphism, and it can crystallize into three different polymorphs (i.e., Mod I, Mod II, and Mod III). Mod I is present in the corresponding commercial product Nexavar® [114]. Also, depending upon its method of preparation, Sorafenib tosylate can crystalize into different solvate forms that have varied solubility and chemical stability [115, 116]. It had been demonstrated that Form II of sorafenib tosylate has better solubility than Form I, but it was metastable and thus physically unstable in its neat form [117]. ARRY-380, also known as irbinitinib or tucatinib, is a selective herb2 inhibitor under investigation for treatment of breast cancer [118, 119]. During its polymorph screening, 29 crystal forms were discovered [118]. On further investigation of its seven solvate forms, it was found that the ethanol and tetrahydrofuran solvates of ARRY-380 have poor solubility. However, they were thermodynamically stable and hence used for further development [118, 120]. Ceritinib is a BCS Class IV drug used in the treatment of ALK-positive metastatic non-small cell lung cancer [121]. Its commercial formulation Zykadia® contains Form I/Form A of ceritinib [122]. Chennuru et al. discovered two novel forms of ceritinib: Form II, which is a hydrate form, and Form III, another polymorph. Form II and Form III have aqueous solubility of 0.9 mg/mL and 0.1 mg/mL, respectively, while Form I has an aqueous solubility of only 0.001 mg/mL [121]. Gefitinib is a kinase inhibitor used in the treatment of non-small cell lung cancer [123]. It exhibits polymorphism, with Form II being physically unstable [123, 124]. Vemurafenib is a potent kinase inhibitor used in the treatment of patients with unresectable or metastatic melanoma with the BRAF V600E mutation [125, 126]. During its first clinical trial, 100 mg and 300 mg capsules containing the crystalline Form I of vemurafenib were used. It was noted that Form I of vemurafenib slowly transformed into Form II, and the observed bioavailability was low during initial clinical trials [39, 125]. It was later discovered that Form I of vemurafenib was thermodynamically unstable, while Form II was thermodynamically stable but had 19

lower aqueous solubility [125]. Hence, further development using Form II of vemurafenib was conducted.

Thus, poorly water-soluble crystalline anticancer drugs can exhibit polymorphism. In general, if a metastable crystalline form of an anticancer drug is used, it has the propensity to transition into a thermodynamically stable form and thereby exacerbate the issue of poor water solubility.

7.1.2. Ionization:

In addition to crystallinity, the solubility of sparingly soluble ionizable anticancer drugs is also dependent on the pKa of the drug and the pH of the gastrointestinal tract [127- 129]. The pH, which varies throughout the gastrointestinal tract, is affected by factors such as bile secretion, pancreatic secretions, food content, other medications, and disease state [130, 131]. Overall, under fasting conditions, the pH of the stomach is between 1.0 and 2.5, the pH of the duodenum is between 5.8 and 6.5, and the pH of the jejunum is between 4.4 and 6.5 [132, 133]. Under non-fasting conditions, the pH of the stomach is between 4.3 and 5.4, the pH of the duodenum is between 3.1 and 6.7, and the pH of the jejunum is between 6.8 and 7.8 [133]. It is important to note that there is high variability in the reported pH values for the gastrointestinal tract. In general, the solubility of sparingly soluble acidic anticancer drugs decreases with a decrease in pH, since the drug largely remains unionized. The reverse is true for sparingly soluble basic anticancer drugs. Most anticancer drugs are absorbed from the small intestine; therefore, any changes in intestinal pH affects the ionization of the drug and thus its solubility and bioavailability [91]. It is also important to note that although the ionized form of drugs are more soluble, they are less permeable through nonpolar membranes [134]. Several sparingly water-soluble anticancer drugs have multiple ionizable groups, and they show pH-dependent solubility.

For example, lapatinib, a kinase inhibitor, is used in the treatment of breast cancer. Its solubility is 0.007 mg/mL in water and 0.001 mg/mL in 0.1 N HCl at 25 °C [135]. Dasatinib monohydrate is another kinase inhibitor used in the treatment of chronic myeloid leukemia. It is available as a crystalline white powder that exhibits significantly pH-dependent water solubility ranging from 18.4 mg/mL at pH 2.6 to 0.008 mg/mL at pH 6.0 [136]. Tamoxifen citrate, used in the treatment of breast cancer, has a solubility of 0.5 mg/mL in water and 0.2 mg/mL in 0.02 N HCl at 37 °C [137, 138]. Vismodegib, the first Hedgehog signaling pathway inhibitory agent used in the treatment of skin cancer, shows significant pH-dependent solubility ranging from 1 mg/mL at pH 1.0 to 0.0001 mg/mL at pH 7.0 [139]. Nilotinib is a potent kinase inhibitor used in the treatment of patients who are in the chronic and accelerated phases of Philadelphia chromosome‐positive chronic myeloid leukemia [140]. This drug is used as a hydrochloride monohydrate salt having pKa1 ~ 2.1 and pKa2 ~ 5.4 [140]. Nilotinib hydrochloride monohydrate is slightly soluble at pH 1 and practically insoluble at pH ≥ 4.5 [140]. Nintedanib, an anticancer drug used in the treatment of non-small cell lung cancer, also shows significant pH- dependent solubility, from 10 mg/mL at pH 2.0 to a dramatically decreased 0.001 mg/mL at pH 6.0 [141].

Thus, depending on the degree of ionization, an anticancer drug that is sparingly soluble at a given pH can become highly insoluble at another pH. Hence, pH-dependent solubility is another challenge associated with the oral administration of ionizable, poorly water-soluble anticancer drugs.

7.1.3. Stability:

The stability of anticancer drugs in the gastrointestinal content is a prerequisite for optimum delivery. The anticancer drug could undergo chemical hydrolysis, which is a degradation due to gastric juices containing hydrogen ions, bicarbonates, and chlorides [133, 142].

For example, chlorambucil, an alkylating agent used in the treatment of Hodgkin’s lymphoma, was found to undergo extensive degradation at higher pH and lower chloride concentrations in human gastric juices. It was found that 100% chlorambucil degraded over 180 min between pH 5.5 and 7.6 and chloride ion concentrations of 91–67 mmol/L [143]. Also, doxorubicin, a cytotoxic drug, was found to undergo gastric acid-catalyzed hydrolysis. It was estimated that in patients with a gastric pH of 1 and a gastric emptying time of 4 h, about 45% of the ingested doxorubicin would degrade in the patient’s stomach [144]. Etoposide, a topoisomerase inhibitor, is known to undergo degradation in gastric fluids [145, 146]. During the development of the stability-indicating HPLC method for 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid, a new anticancer drug under investigation, it was found that the drug underwent significant degradation at pH ≤ 3.5 [147]. This suggests potential instability of this drug in gastric media. BMS-753493 is a novel folate-targeted candidate being developed for the treatment of cancer. It was most stable between pH 6.0 and 7.0, and it showed instability at lower and higher pH values at 25 °C and 40 °C [148]. Thus, the instability of BMS-753493 at lower pH is a potential challenge for developing its oral formulation. Other factors (e.g., enzymatic degradation, food contents) also affect the stability of anticancer drugs in the gastrointestinal tract. These factors are discussed in subsequent sections.

7.1.4.Drug–drug interaction:

Co-therapy is common during cancer treatment, thus the risk of potential drug–drug interactions is high [149]. The prevalence of significant drug–drug interactions is higher among oral anticancer drugs than among intravenous anticancer drugs [149, 150]. Since we focus on oral administration in this paper, we discuss drug–drug interactions that have a negative impact on the oral absorption of anticancer drugs. Sometimes, other medications that are administered along with anticancer drugs may hinder the anticancer drugs’ oral bioavailability by affecting its solubility or permeability (a) through altering 22

the pH of the gastrointestinal tract, (b) by competing for transporters, or (c) by modifying metabolic enzyme activity [151].

As discussed above, several anticancer drugs exhibit pH-dependent solubility. Acid- reducing drugs, such as proton pump inhibitors, elevate gastric pH, thereby reducing the solubility of weakly basic anticancer drugs and thus reducing their oral bioavailability. From a clinical data survey of drug–drug interactions between anticancer drugs and proton pump inhibitors, it was found that the magnitude of the drug–drug interactions was largest for compounds whose in vitro solubility varied over the range of pH 1–4 [152]. Erlotinib, a kinase inhibitor, is used in the treatment of non-small cell lung cancer. In a study, it was found that the trough concentrations of erlotinib were significantly diminished when high doses of intravenous pantoprazole, a proton pump inhibitor, was administered simultaneously [153]. It was reported that when famotidine, an H2-receptor antagonist was administered 2 h post dasatinib, no impact on the exposure of dasatinib was seen. However, when the next dose of dasatinib was administered (i.e., 10 h post famotidine dose), a 60% reduction in AUC0–12 and Cmax for dasatinib was observed [154]. It was concluded that elevation of gastric pH by famotidine reduced the solubility of dasatinib and thus reduced its bioavailability [154]. Gefitinib also exhibits pH-dependent solubility [123]. In a retrospective study involving non-small cell lung cancer patients taking gefitinib, it was found that patients taking proton pump inhibitors had lower

AUC0–24 (median value 8,542 ng.hr/mL) compared to AUC0-24 (median value 13,103 ng.hr/mL) for patients taking only gefitinib [155]. Bosutinib, a kinase inhibitor used in the treatment of leukemia, shows pH-dependent solubility [156]. About a 38% reduction in Cmax and a 24% reduction in AUC were observed for bosutinib when it was coadministered with lansoprazole [156]. Pazopanib is a BCS Class II anticancer drug used in the treatment of soft-tissue sarcoma and renal cell carcinoma [157, 158]. It also exhibits pH-dependent solubility and is practically insoluble at pH > 4 [158].

A retrospective study evaluated treatment outcomes of patients with soft-tissue sarcoma who were treated with pazopanib. It was observed that for patients taking pazopanib 23

together with concomitant gastric acid suppressive agents, the median progression free survival (2.8 vs 4.6 months) and median overall survival (8.0 versus 12.6 months) were significantly lower than for patients who were taking only pazopanib [158]. It was suspected that long-term use of gastric acid suppressive agents leads to elevation of gastrointestinal pH. This leads to poor solubility of pazopanib, hence poor bioavailability and ultimately poor therapeutic outcomes [158]. It is interesting to note that another retrospective study involving renal cell carcinoma patients found that the concomitant use of pazopanib with gastric acid suppressive agents was not associated with progression- free survival or overall survival [157].

Abiraterone acetate is a prodrug used in the treatment of prostate cancer [159]. It is converted into its active metabolite, abiraterone, predominantly pre-systemically via esterases [160, 161]. In a pharmacokinetic study in dogs, it was observed that coadministration of orlistat with abiraterone acetate led to a decrease in plasma concentrations of abiraterone [160]. This is because orlistat inhibits esterases, thereby reducing the conversion of abiraterone acetate into abiraterone, thus reducing abiraterone supersaturation and absorption [160]. This drug–drug interaction between abiraterone acetate and orlistat requires further investigation.

Regorafenib is a kinase inhibitor used in the treatment of colorectal cancer [162]. Bile salt sequestering drugs such as cholestyramine and Cholestagel can form insoluble complexes with regorafenib, thereby reducing its solubility and in turn reducing its absorption and reabsorption [162, 163]. This further demonstrates that interactions between poorly water-soluble anticancer drugs and other drugs can negatively impact their solubility or permeability, which in turn alters the pharmacokinetics of poorly water-soluble anticancer drugs.

7.2. Physiology-Related Challenges: 7.2.1. Transmembrane efflux of drugs:

Certain proteinaceous transporters are located on the intestinal membrane, and they act as efflux pumps. These efflux pumps significantly reduce the oral bioavailability of anticancer drugs. When anticancer drugs successfully dissolve and permeate the membrane, these efflux pumps eject the anticancer drugs back into the intestinal lumen, so the anticancer drugs are ultimately eliminated through excretion.

The major efflux transporters belong to the ATP binding cassette (ABC) transporters family, and several anticancer drugs act as substrates for these transporters [164, 165]. The major efflux transporters of the ABC family include P-glycoprotein (P-gp, ABCB1), a breast cancer resistance protein (BCRP, ABCG2), and multidrug resistance- associated proteins (MRP2 , ABCC2) [166]. They are localized to barrier tissues of the body, such as the intestine, liver, kidneys, blood-brain barrier, and placenta. Table 1.2 lists the poorly water-soluble anticancer drugs that are substrates for major efflux pumps located on the intestinal membrane.

The P-glycoprotein transporter is one of the most widely studied efflux pumps. It has broad substrate specificity and is also known to act synergistically with cytochrome P450 enzymes [167]. By effluxing absorbed anticancer drugs back into intestinal lumen for reabsorption, P-gp efflux pumps permit significant time for cytochrome P450 enzymes to metabolize anticancer drugs, thereby significantly reducing their oral bioavailability [167]. One study found that the oral bioavailability of etoposide, a cytotoxic drug, was reduced by 26% due to P-gp efflux pumps [168]. Additionally, due to variations in P-gp efflux pump levels in patients, etoposide demonstrated high pharmacokinetic variability [169]. In preclinical studies in P-gp knockout mice, the apparent oral bioavailability of paclitaxel and docetaxel were increased by ~24% and ~19%, respectively as compared to the wild-type mice [170]. One study observed that the oral bioavailability of a topoisomerase inhibitor, topotecan, was increased from 40%

to 97% by the coadministration of a BCRP inhibitor [171]. It is interesting to note that while the oral bioavailability of several anticancer drugs is reduced because they act as substrates for efflux pumps, these anticancer drugs can also enhance the oral bioavailability of other drugs that have a comparatively lower affinity for efflux pumps [172]. This raises the possibility of a substantial risk of drug–drug interaction between different coadministered anticancer drugs, leading to a reduced oral bioavailability of one drug and an enhanced oral bioavailability of another drug.

Table 1.2. List of poorly water-soluble anticancer drugs that are substrates for major efflux pumps located on the intestinal membrane. [167, 172-174]:

Substrates Efflux Pump mitomycin c, vinblastine, vincristine, vinorebline, vindesine, etoposide, paclitaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, docetaxel, imatinib, irinotecan, SN-38 (7-ethyl-10- hydroxycampothecin), topotecan tamoxifen, methotrexate,

P-gp, mitoxantrone, amsacrine, 5-fluorouracil, actinomycin d, bisantrene, ABCB1 chlorambucil, cisplatin, cytarabine, gefitinib, teniposide, afatinib, alectinib (m-4), axitinib, bosutinib, ceritinib, cobimetinib, crizotinib, dabrafenib, dasatinib, erlotinib, lapatinib, lenvatinib, nilotinib, nintedanib, osimertinib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, trametinib, vemurafenib

doxorubicin, daunorubicin, epirubicin, idarubicinol, mitoxantrone, irinotecan, SN-38 (7-ethyl-10-hydroxycampothecin), imatinib, BCRP , methotrexate, topotecan, bisantrene, teniposide, topotecan, afatinib, ABCG2 axitinib, dabrafenib, dasatinib, erlotinib, gefitinib, lapatinib, lenvatinib, nilotinib, osimertinib, pazopanib, ponatinib, regorafenib, vemurafenib cabozantinib, sorafenib, cisplatin, vincristine, vinblastine, etoposide,

MRP2 , doxorubicin, daunorubicin, epirubicin, irinotecan, SN-38 (7-ethyl-10- ABCC2 hydroxycampothecin), methotrexate, mitoxantrone, mitomycin c, 5- fluorouracil

7.2.2.Pre-systemic metabolism:

The pre-systemic metabolism involves metabolism in the gastrointestinal lumen, the brush border metabolism, the intracellular metabolism, and the first-pass metabolism [164]. Enzymes such as amylases, lipases, esterases, and bacterial enzymes secreted by gut flora are present in the gastrointestinal lumen and are responsible for metabolizing anticancer drugs in the gastrointestinal tract [164, 175]. Brush border metabolism mainly occurs in the proximal small intestines and is caused by enzymes such as alkaline phosphatase, sucrase, and peptidases [176]. Intracellular metabolism in the gut is caused by extrahepatic microsomal enzymes of the cytochrome P450 3A family, mainly CYP 3A4 [177]. Thus, these enzymes are responsible for metabolizing the anticancer drugs before or during their absorption. Once the anticancer drugs are absorbed from the gastrointestinal tract, they can enter the liver via the hepatic portal vein. In the liver, a fraction of these absorbed drugs are metabolized by a plethora of enzymes. This is known as first-pass metabolism [164]. This means that even before an anticancer drug reaches systemic circulation, it can undergo metabolism that leads to the formation of inactive metabolites and thus low oral bioavailability. Some examples of poorly water-soluble anticancer drugs that undergo extensive pre-systemic metabolism include ibrutinib, flutamide, erlotinib, gefitinib, paclitaxel, lapatinib, tamoxifen, docetaxel, vincristine, vinblastine vindesine, vinorelbine, etoposide, sorafenib, sunitinib, imatinib, and everolimus [178-187]. Also, enzymes such as peptidases pose a significant challenge for the oral delivery of peptide anticancer drugs such as buserelin, triptorelin, gonadorelin, nafarelin, and leuprolide [188, 189].

Additionally, different levels of expression of metabolic enzymes in healthy subjects compared to cancer patients causes a vast amount of variability in pharmacokinetic data, specifically for anticancer drugs [190]. Also, polymorphism in genes that code for these metabolic enzymes causes high intersubject pharmacokinetic variability among anticancer drugs [191]. Since anticancer drugs act as substrates for metabolic enzymes, they have a high potential for drug–drug interactions with strong enzyme inducers and

inhibitors, thereby affecting their pharmacokinetics. For example, cabozantinib is a kinase inhibitor used in the treatment of thyroid cancer [192]. The enzyme inducer rifampin reduces the AUC of cabozantinib by 77% [192]. It is important to note that while pre-systemic metabolism reduces oral bioavailability for several anticancer drugs, it also helps activate some anticancer prodrugs such as abiraterone acetate and irinotecan [161, 193]. Hence, pre-systemic metabolism is a major challenge that affects the absorption of poorly water-soluble anticancer drugs, thereby leading to reduced bioavailability and increased pharmacokinetic variability.

7.2.3.Transporter saturation:

There are two main classes of transporters in the gastrointestinal tract: the solute carrier (SLC) family of transporters and the ATP-binding cassette (ABC) transporters [174]. The SLC transporters mainly play a role in drug absorption, while ABC transporters are mainly responsible for drug efflux, and these are discussed above. The most important SLC transporters on the intestinal membrane include the organic anion transporting polypeptide (OATP) family of transporters, the peptide transporter 1 (PEPT1), and the apical sodium/bile acid co-transporter (ASBT) [194].

The saturation of these compounds responsible for transporting some anticancer drugs into systemic circulation can lead to reduced bioavailability of anticancer drugs at high doses. Transporter saturation is also a cause of nonlinear pharmacokinetics. For example, melphalan is an anticancer drug with water solubility of < 1 mg/mL, and it is absorbed from the gastrointestinal tract via the amino acid transport system. At high doses of melphalan (> 0.75 mg/kg), significant reduction in AUC is observed due to the saturation of the amino acid transporters [195]. 5-aminolevulinic acid is used in the treatment of several malignant and premalignant conditions [196]. It is transported by PEPT1, and it has demonstrated concentration-dependent jejunal uptake in perfusion studies [196]. PEPT1 also plays a role in the intestinal uptake of peptide-like anticancer drugs [197].

Interestingly, the saturation of the aforementioned efflux drug transporters can increase the bioavailability of anticancer drugs at high doses [198]. Thus, transporter saturation can have a significant effect on the pharmacokinetics of substrate poorly water-soluble anticancer drugs, leading to dose-exposure nonlinearity.

7.3. Other Challenges:

7.3.1. Prandial state:

Anticancer drug solubility as well as absorption can be affected by the prandial status. For instance, in non-fasting conditions, the pH of the stomach may increase and thus alter anticancer drugs’ solubility and thereby their oral bioavailability. The composition of food (e.g., a high-fat meal) may enhance the solubility of a lipophilic drug and thereby increase its oral bioavailability. Under fasting conditions, gastric motility could be higher, and this can lead to anticancer drug formulation elimination, before drug release, dissolution, and absorption.

Also, certain components of food (e.g., flavonoids) may interact with anticancer drug transporters and metabolizing enzymes thereby affecting their bioavailability. Certain foods (e.g., grape fruit juice, wine, tea) demonstrate prominent food–drug interaction with anticancer drugs [199, 200]. Table 1.3 describes examples of poorly water-soluble anticancer drugs that demonstrate prominent food effects on their pharmacokinetics. It is evident from Table 1.3 that prandial state (fed versus fasted) and meal content can have considerable positive, negative, and even mixed effects on the pharmacokinetics of poorly water-soluble anticancer drugs.

Such food effects can make dosing-drug exposure unreliable, thereby warranting limitations such as fasting for certain periods or avoidance of certain meals while taking poorly water-soluble drugs orally. This can further affect dosage regimen designs. For instance, if a drug requires three hours of fasting, administering such a drug three times 30

daily is not practically possible. Also, patient adherence to medication can be negatively impacted. Thus, the effect of prandial state on the pharmacokinetics of poorly water- soluble anticancer drugs is a major challenge for delivering them orally.

Table 1.3. List of poorly water-soluble anticancer drugs demonstrating prominent food effects on their pharmacokinetics.

Pharmacokinetic Implication Anticancer Prandial State References Drug (compared to fasted state)

Cmax↑ 7-fold Fed: Low-fat meal Abiraterone AUC0-∞↑ 5-fold [201] acetate Cmax↑ 17-fold Fed: High-fat meal AUC0-∞↑ 10-fold Axitinib Cmax↓ 38% (Polymorph Fed AUC0-∞↓ 23% form IV) [202] Axitinib No clinically significant Fed: High and (Polymorph pharmacokinetic Moderate fat form XLI) implication.

Cmax↑ 1.8-fold Bosutinib Fed: High-fat meal [203] monohydrate AUC0-∞↑ 1.7-fold C ↑ 40.5% max [204] Cabozantinib Fed: High-fat meal AUC0-∞↑ 57%

Table 1.3 continued Pharmacokinetic Implication Anticancer Prandial State References Drug (compared to fasted state)

Cmax↑ 41% Fed: High-fat meal AUC0-∞↑ 73% [205] Ceritinib Cmax↑ 43% Fed: Low-fat meal AUC0-∞↑ 58%

C ↓ 51% max [206] Dabrafenib Fed: High-fat meal AUC0-∞↓ 31% C ↑ 36% max [207] Erlotinib Fed AUC0-24↑ 54%

Cmax↑ 2.42-fold Fed: Low-fat meal AUC0-∞↑ 2.67-fold [208] Lapatinib Cmax↑ 3.03-fold Fed: High-fat meal AUC0-∞↑ 4.25-fold

Cmax↓ 27% Fed: High-fat meal AUC0-∞↑ 59% [209] Midostaurin Cmax↓ 20% Fed: Standard meal AUC0-∞↑ 22% C ↑ 1.7-fold max [210] Neratinib Fed: High-fat meal AUC0-∞↑ 2.2-fold C ↑ 112% max [211] Nilotinib Fed: High-fat meal AUC0-∞↑ 82% C ↑ 20% max [212] Nintedanib Fed AUC0-∞↑ 20%

Table 1.3 continued Pharmacokinetic Implication Anticancer Prandial State References Drug (compared to fasted state)

Cmax↑ 2.10-fold Fed: Low-fat meal AUC0-72↑ 1.92-fold [213] Pazopanib Cmax↑ 2.08-fold Fed: High-fat meal AUC0-72↑ 2.34-fold

C ↑ 7.78-fold max [214] Sonidegib Fed: High-fat meal AUC0-∞↑ 7.38-fold C ↓ 68% max [215] Trametinib Fed AUC0-∞↓ 15%

7.3.2. Patient/Oncologist Related Challenges:

Other factors such as the patient’s age, patient’s comorbid condition, patient’s adherence, oncologist’s experience, and oncologist’s preferences may prove to be challenging for the oral administration of anticancer drugs [14, 216, 217]. Aging can lead to physiological changes such as reduction in enzyme activity, which may have prominent effects on anticancer drug pharmacokinetics [217]. Certain co-morbid conditions can alter the physiology of the gastrointestinal tract and hepatic function, thus affecting the bioavailability of the anticancer drug. Given et al. reported that surveyed cancer patients had 1–4 pills per day for oral cancer medications, in addition to 10–11 medications for their comorbid condition [218]. Such high pill burden can affect a patient’s adherence to the treatment regimen and thus affect the therapeutic outcome. Some oncologists may prefer IV anticancer drugs based on prior specific experiences and thus may avoid prescribing oral anticancer drugs. These miscellaneous challenges pose hurdles for the oral administration of poorly water-soluble anticancer drugs.

It is apparent from the above discussion that poorly water-soluble anticancer drugs do not face one or two unique challenges, but rather a multitude of interdependent challenges. This is true not only for approved poorly water-soluble anticancer drugs such as abiraterone, axitinib, erlotinib, and lapatinib, but also for newer anticancer drugs under investigation. For example, TAK-117 (also known as MLN1117 or serabelisib) is a new anticancer drug under investigation by Takeda for the treatment of advanced solid malignancies [219]. TAK-117 faces challenges such as poor water solubility, polymorphism, pH-dependent solubility, drug interaction with lansoprazole, and food effects; all of which change the pharmacokinetics of the drug [220, 221]. A complex interplay of these challenges leads to five major pharmacokinetic or pharmacodynamic consequences, as illustrated in Figure 1.4, which hinder the therapeutic outcomes of orally administered poorly water-soluble anticancer drugs.

Figure 1.4. Challenges and consequences associated with the oral administration of poorly water-soluble anticancer drugs.

8. ALLEVIATING THE CONSEQUENCES THROUGH ASDS:

ASDs can alleviate the consequences associated with the oral administration of poorly water-soluble anticancer drugs as follows:

Figure 1.5. Role of ASDs in alleviating the challenges and consequences associated with the oral administration of poorly water-soluble anticancer drugs, thereby leading to improved oncological therapeutic outcomes. [ASD–amorphous solid dispersions, MTC-minimum toxic concentration, MEC-minimum effective concentration, Cmax = maximum plasma drug concentration, Cmin = minimum plasma drug concentration, AUC = area under the plasma drug concentration time curve, t = duration of drug exposure] 36

8.1. Enhancing Oral Bioavailability:

Oral bioavailability is defined as the fraction of an orally administered drug that reaches systemic circulation. It can be determined by comparing the area under the curve (AUC) (of the plot of plasma drug concentration versus time) of an oral dose to the AUC of an intravenous dose [222]. ASD formulations can enhance the oral bioavailability of poorly water-soluble anticancer drugs when compared to conventional oral formulations such as physical mixtures of drugs and excipients (Figure 1.5a).

One of the major factors contributing to the poor oral bioavailability of anticancer drugs is their low water solubility. ASDs increase the rate of anticancer drug dissolution by increasing the apparent aqueous solubility of the drug in the gastrointestinal media [223]. Docetaxel is a microtubule inhibitor used in the treatment of a variety of cancers such as prostate cancer, breast cancer, lung cancer, gastric cancer, and head–neck cancer [224]. Currently, docetaxel is available as an injection for intravenous infusion, which is associated with acute hypersensitivity reactions [225]. Hence, an oral formulation of docetaxel is desirable. Docetaxel is practically insoluble in water (solubility < 0.1 mg/mL), is highly lipophilic (LogP ~4.2) and has bulky hydrophobic groups, all of which contribute to its poor oral bioavailability [224]. Lim et al. developed an ASD of docetaxel with Soluplus® (polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer) and increased docetaxel’s solubility in 0.1 M, pH 6.8 phosphate buffer by 93- fold, from 3.9 ± 0.2 µg/mL for neat crystalline docetaxel to 362.93 ± 11.01 µg/mL for a binary solid dispersion of docetaxel and Soluplus® [226]. Sawicki et al. reported the development of an ASD of paclitaxel or docetaxel with Povidone K30 (polyvinylpyrrolidone-PVPK30) and sodium dodecyl sulfate (SDS) at a 1:9:1 weight ratio [92, 227, 228]. These solid dispersions were reported to enhance the solubility of paclitaxel and docetaxel by 100 times and 40 times, respectively, compared to the crystalline forms of the drugs [227]. Chen et al. developed an emulsifying ASD of docetaxel that enhanced its solubility and dissolution by 34.2-fold and 12.7-fold, 37

respectively, compared to its crystalline form [229]. Piao et al. developed an ASD of paclitaxel with hydroxy propyl methyl cellulose acetate succinate (HPMCAS) and porous silicon dioxide [230]. This solid dispersion enhanced the solubility of paclitaxel and its bioavailability in rats, by 7-fold compared to neat paclitaxel [230]. Andrews et al. developed an ASD of bicalutamide with PVP K25 at drug:polymer weight ratios of 1:10, 2:10, and 3:10 [231]. They observed that physical mixture (1:10 w/w) enhanced the dissolution of bicalutamide by 2.31-fold compared to crystalline bicalutamide alone [231]. These ASDs were able to enhance the dissolution of bicalutamide by 7.53-, 8.05-, and 8.93-fold compared to crystalline bicalutamide, with an increasing concentration of PVP K25 [231]. Ren et al. reported development of an ASD of bicalutamide with PVP K30, and they saw similar trends in dissolution—i.e., the dissolution of ASD with higher PVP K30 > ASD with lower PVP K30 > physical mixture with higher PVP K30 > crystalline bicalutamide [232]. Several other studies have reported the development of solid dispersions of bicalutamide that exhibit reduced crystallinity, leading to improved apparent aqueous solubility of bicalutamide [233-236]. ASDs of ceritinib with polymers such as hydroxy propyl methyl cellulose (HPMC), hydroxypropyl cellulose (HPC), vinylpyrrolidone-vinyl acetate–Copovidone (PVPVA) and PVP have been reported to enhance the dissolution and bioavailability of ceritinib [237, 238]. Enzalutamide is a poorly water-soluble anticancer drug used in the treatment of castration-resistant prostate cancer [239]. Wilson et al. developed a binary ASD of enzalutamide with HPMCAS and PVPVA at drug loadings of 10% and 50% w/w. Interestingly, in this case, the dissolution enhancement of enzalutamide showed a trend of 50:50 %w/w enzaluatmide:HPMCAS ASD > 10:90 %w/w enzalutamide:PVPVA ASD > 50:50 %w/w enzalutamide:PVPVA ASD >>> 10:90 %w/w enzalutamide:HPMCAS ASD > crystalline enzalutamide [239]. It was inferred that 10:90 %w/w enzalutamide:HPMCAS ASD showed lower dissolution enhancement despite higher polymer content because it quickly precipitated into the crystalline form of the drug during dissolution. The 10:90 %w/w enzalutamide:PVPVA ASD showed slower precipitation into amorphous aggregates during dissolution, and it showed the highest bioavailability enhancement in rats for enzalutamide, ~7-fold [239]. 38

Shepard and Morgen developed a ternary ASD of erlotinib with poly[(methyl methacrylate)-co(methacrylic acid)] (Eudragit® L100 ) and HPMC. This ASD enhanced the dissolution of erlotinib and enhanced its bioavailability more than 10-fold [240]. Shah et al. demonstrated the ability of solid dispersion of etoposide with polyethylene glycol (PEG), in which etoposide in the amorphous state enhanced the dissolution rate of etoposide by 42-fold [241]. Song et. al. developed ASDs of the weakly basic drugs gefitinib and lapatinib with polystyrene sulfonic acid. This led to enhanced dissolution of both drugs [242]. Herbrink et al. developed an ASD of nilotinib with Soluplus®, which enhanced the solubility of nilotinib 630-fold compared to crystalline nilotinib in simulated intestinal fluids [243]. Oridonin is a poorly water-soluble drug that has marked anticancer effects [244]. Li et al. developed an ASD of oridonin with PVP K17 and were able to enhance its dissolution and bioavailability in dogs by 26-fold [244]. ASDs of tamoxifen citrate that enhance its dissolution and bioavailability have been reported [245, 246].

ASDs can eliminate poor water solubility that occurs due to polymorphic transitions of the drug. This is because the crystalline structure of the drug is disrupted, and the drug is converted into an amorphous form where it is stabilized by polymers or oligomers, thereby eliminating the possibility of transition into other polymorphs of the drug. Szafraniec et al. spray-dried neat bicalutamide and a mixture of bicalutamide with PVP [247]. While spray-dried neat bicalutamide showed polymorphic transitions in its solid state, the spray-dried bicalutamide-PVP mixture was stable, with bicalutamide being dispersed in an amorphous state in the PVP matrix [247]. Tres et al. prepared ASDs of bicalutamide with Kollidon VA 64 (Copovidone) at drug loadings of 5% w/w and 50% w/w [248]. As in previous cases, they observed that the dissolution of bicalutamide increased with higher concentrations of the polymer. After further investigation, they found that in ASDs loaded with 5% bicalutamide, the rate of drug and polymer dissolution was similar. However, in ASDs loaded with 50% bicalutamide, the polymer dissolved at a faster rate than the drug, leading to a rich layer of amorphous bicalutamide,

which precipitated to its polymorphic Form I and Form II, thereby lowering the overall dissolution [248]. These studies emphasized the importance of polymers and their concentration in ASDs in the prevention of polymorphic transitions of poorly water- soluble anticancer drugs, thereby enabling maximum stability and solubility and thus maximum bioavailability.

Although the focus of this paper is on poorly water-soluble anticancer drugs, it is important to recognize that, in addition to poor water solubility, several other factors hinder the oral bioavailability of poorly water-soluble anticancer drugs. Thus, it is imperative to discuss the role of ASDs in removing barriers to oral bioavailability that go beyond poor water solubility.

Another challenge leading to the low oral bioavailability of poorly water-soluble anticancer drugs is their instability in gastrointestinal media. Through a repurposing strategy, it was found that clarithromycin, an antibiotic, has promising anticancer activity [249]. However, its use in oncology is challenging, since it has poor oral bioavailability due to poor solubility at neutral intestinal pH, and high solubility coupled with high degradation in gastric pH [250, 251]. Pereira et al. proposed an ASD of clarithromycin with a hydrophobic cellulose derivative, cellulose acetate adipate propionate (CAAdP) polymer. They demonstrated the effect of the ASD in simultaneously enhancing the solubility of clarithromycin and preventing its degradation in gastric juices [252].

Poor oral bioavailability of anticancer drugs due to their interaction with other drugs can be avoided by emphasis on drug–drug interaction studies during early clinical development [253]. Moreover, the drug–drug interactions discussed above, which lead to reduced solubility of anticancer drugs due to gastrointestinal pH changes, can be avoided by the addition of acid-modifying agents in ASDs.

Many poorly water-soluble anticancer drugs also act as substrates for efflux transmembrane proteins, and this contributes to their poor oral bioavailability. For instance, docetaxel is a substrate for P-glycoprotein-mediated efflux pumps [254]. Song 40

et al. developed partial ASDs of docetaxel with Lutrol® F68 (poloxamer 188) alone and with Pluronic® P85 (poloxamer 235) and Lutrol® F68 (poloxamer 188). They found that both solid dispersions enhanced the dissolution of docetaxel; however, the bioavailability enhancement in rats, from the former was 1.39-fold, while the latter enhanced bioavailability by 2.97-fold [255]. This was because Lutrol ® F68 along with Pluronic® P85 synergistically exhibited P-glycoprotein efflux pump inhibitory activity. Miao et al. developed an ASD of paclitaxel with HPMCAS and Poloxamer 188 [256]. This ASD enhanced the solubility and permeability of paclitaxel, thereby enhancing its bioavailability by 1.78-fold compared to physical mixtures [256]. HM30181A enables the oral dosing of paclitaxel (Oraxol™), irinotecan (Oratecan™), and docetaxel (Oradoxel™), by specifically inhibiting P-glycoprotein efflux pumps [257]. Shanmugam et al. developed an ASD of paclitaxel and administered it with HM30181A and saw a further bioavailability enhancement of 25% greater than Oraxol™ in dogs [258].

Many poorly water-soluble anticancer drugs undergo significant first-pass metabolism by enzymes such as CYP3A. Thus, ASDs of anticancer drugs such as paclitaxel can be developed with—or administered with—CYP3A substrates such as cyclosporine A to enhance the drug’s apparent aqueous solubility and reduce first-pass metabolism [181]. Bohr et al. developed a co-amorphous formulation of docetaxel with bicalutamide at a 1:1 molar ratio [61]. This amorphous formulation can enhance the solubility of docetaxel and bicalutamide [61]. Moreover, bicalutamide was reported to inhibit efflux pumps as well as CYP enzymes, thereby enhancing the absorption of docetaxel. In a pharmacokinetic study in rats, this amorphous formulation showed a bioavailability enhancement of 15- fold for docetaxel and 3-fold for bicalutamide compared to their crystalline forms [61]. It is important to note that this amorphous formulation can have a risk of poor stability and can be further improved by formulating it as an ASD with the addition of polymers. Acetyl‐11‐keto‐beta‐boswellic acid (AKBA) is a poorly water-soluble drug as well as a CYP3A4 substrate, and it has demonstrated promising antitumor effects [259, 260]. Miller et al. developed ASDs of AKBA and Ritonavir, a CYP3A4 inhibitor, thereby

enhancing the dissolution , permeability, and thus the oral bioavailability of AKBA [259].

Majority of research has been focused on the ability of ASDs to enhance the aqueous solubility of poorly water-soluble anticancer drugs and their permeability enhancement is achieved by a general approach of co-administering permeation enhancing drugs. However, the role of ASDs in enhancing the permeability of poorly water-soluble drugs is not limited to only the addition or co-administration of efflux pump inhibitors or enzyme inhibitors. In fact, ASDs can simultaneously enhance solubility and permeability by supersaturating the dissolved drug in the gastrointestinal tract, thereby saturating the efflux pumps or enzymes, hence enabling higher drug permeation. For example, Beig et al. developed an ASD of etoposide with PVPVA and Eudragit L-100. They demonstrated this ASD’s ability not only to enhance etoposide’s dissolution, but also to enhance its permeability in rats, to levels comparable with the potent P-glycoprotein inhibitor GF120918 [261]. Exemestane is anticancer drug used in the treatment of breast cancer. It exhibits poor water solubility and poor permeability [262]. Kaur et al. developed an ASD of exemestane with phospholipids, bile salts, and cyclodextrins. This ASD exhibited a 5.2-fold dissolution enhancement, a permeability enhancement of 4.6-fold across Caco-2 cell line, and a 2.3-fold increase in bioavailability in rats [262]. In this case, the amorphization of exemestane led to this apparent increase in water solubility, and bile salts formed mixed micelles with phospholipids, which increased the transcellular uptake of exemestane [262]. Other attempts to simultaneously enhance exemestane’s solubility and permeability through ASDs have been reported [263, 264].

Thus, the rational design of ASDs of poorly water-soluble anticancer drugs can enhance the oral bioavailability of these drugs by increasing their apparent aqueous solubility, preventing polymorphic transitions, decreasing their degradation in gastric juices, decreasing their elimination by efflux proteins, and reducing their first-pass metabolism.

8.2. Reducing Pharmacokinetic Variability:

Pharmacokinetic variability describes the variations in a drug’s pharmacokinetic parameters. This variability results in fairly different plasma concentration–time profiles after the administration of the same dose to the same patient or different patients. Pharmacokinetic variability can be seen within the same patient (i.e., intrapatient or intrasubject pharmacokinetic variability) or it can be seen between different patients (i.e., interpatient or inter-subject pharmacokinetic variability).

For the purpose of bioequivalence studies, the FDA defines a drug to be highly variable when its intrasubject pharmacokinetic variability is ≥ 30% [265]. Pharmacokinetic variability for anticancer drugs could occur due to changes in absorption, metabolism, distribution, or excretion [266]. In this review, we focus only on the pharmacokinetic variability that occurs due to changes during the absorption process. In fact, one of the main sources of intrasubject and intersubject pharmacokinetic variability for anticancer drugs is the process of drug absorption. The estimated intersubject variability in the absorption (estimated as the first-order rate constant, Ka) for anticancer drugs ranges from 40% to > 100% [152, 266, 267].

Pharmacokinetic variability can have significant implications for anticancer drugs’ therapeutic outcomes. Several anticancer drugs have a narrow therapeutic window. Therefore, high pharmacokinetic variability can lead to plasma–drug concentrations above the minimum toxic concentrations, thereby leading to toxicity. Similarly, high pharmacokinetic variability can lead to insignificant drug exposures or plasma–drug concentrations below the minimum effective concentration, thereby leading to suboptimum therapy.

The pharmacokinetic variability of poorly water-soluble anticancer drugs can be reduced by ASD formulation compared to conventional oral formulations such as physical mixtures of the drug and excipients (Figure. 1.5b). As discussed earlier, there is significant variation in the pH of the gastrointestinal tract, and this variation can be 43

exacerbated due to several reasons, including differences in the disease state of patients (e.g., gastric ulcers), differences in patient age (e.g., age-related reduction in gastric secretions), prandial status, and the presence of any co-medications [266]. This variation in pH can alter drugs’ solubility, leading to significant pharmacokinetic variability.

Proxalutamide (GT0918) is a potent androgen receptor pathway inhibitor being developed for the treatment of prostate cancer [268]. It exhibits strong pH-dependent solubility, as can be seen in Figure 1.6a, and it has the potential to exhibit high pharmacokinetic variability [269]. Yang et al. screened several pH modifiers and designed an ASD of proxalutamide with polymer polyvinyl pyrrolidine and pH modifier citric acid. The rationale behind this design was that the pH modifier alters and maintains the microenvironmental pH around the proxalutamide dissolution boundary, thereby making its dissolution independent of changes in the pH of bulk dissolution media. Yang et al. carried out pharmacokinetic studies in dogs (Figure 1.6b) of the pH-modified ASD tablet versus the conventional tablet. They found that the co-efficient of variation (%CV) for the area under the plasma concentration–time curve (AUC0-∞) dropped from 52% to 21% [269].

Figure 1.6. (a) pH-solubility profile of GT0918 (proxalutamide) at 37 °C and (b) plasma concentration–time profiles of GT0918 (proxalutamide) in beagle dogs after a single-dose oral administration of pH-modified solid dispersion tablets (□) and conventional tablets (Δ). Reproduced with permission [269]. 44

Several anticancer drugs exhibit strong food effects on their pharmacokinetics, thereby leading to significant pharmacokinetic variability when prandial status and food content is not controlled. The rationale design of ASDs of such anticancer drugs can significantly lessen these food effects. For instance, if an anticancer drug shows significantly positive food effects with a high-fat diet, an ASD of such a drug with lipidic components and surfactants could eliminate the food effects and thus eliminate the variability. The food effects are eliminated because the lipids and surfactants help complete drug dissolution through emulsification, thereby eliminating the role of high-fat foods in the drug’s dissolution and absorption. Solyomosi et al. designed an amorphous system (presumably an ASD) of abiraterone acetate with Soluplus® and demonstrated an increase in the bioavailability of abiraterone, elimination of its positive food effects, and a reduction in its pharmacokinetic variability [270].

Other factors that can contribute to the pharmacokinetic variability of anticancer drugs between patients, including differences in the expression of efflux transporter proteins such as P-glycoprotein efflux pumps, due to genetic polymorphism or differences in the expression of metabolizing enzymes such as dihydropyrimidine dehydrogenase, which is also due to polymorphism in the genes responsible for coding for the enzyme [91, 266]. ASDs developed with excipients or other drugs that act as substrates or inhibitors of such efflux pumps or enzymes can eliminate the pharmacokinetic variability that arises from different expressions of such proteins or enzymes.

Moes et al. reported clinical testing of a docetaxel ASD, discussed above, along with administration of ritonavir, which is a CYP3A4 inhibitor [228]. Compared to a docetaxel premix solution, the docetaxel ASD showed a marked decrease in intersubject variability in cancer patients, with %CV reducing from 85% to 43% for AUC0-24 and 84% to 51% for Cmax [228]. Thus, the increase in the accuracy of dosing for docetaxel with reduced pharmacokinetic variability paved the way for its chronic use in metronomic dosing. Erlotinib shows a high intrapatient and interpatient pharmacokinetic variability due to its poor water solubility, its pre-systemic metabolism, its drug–drug interactions, and food 45

effects [271]. Truong et al. developed an ASD of erlotinib with self-emulsifying components and demonstrated a reduction in the intersubject variability of AUC0-∞ by

86% and of Cmax by 70% in rats[271]. Hence, ASDs can reduce the pharmacokinetic variability of poorly water-soluble anticancer drugs by reducing pH and ionization state effects on solubility, eliminating food effects, reducing efflux, and eliminating presystemic metabolism.

8.3.Enhancing Pharmacokinetic Linearity:

Drugs can exhibit linear or nonlinear pharmacokinetics. Linear pharmacokinetics implies that the area under the drug plasma concentration–time curve (AUC) is directly proportional to the dose [266, 272]. Therefore, when the dose is doubled, the AUC is expected to double. The linearity of a drug’s pharmacokinetics can be affected by absorption, distribution, metabolism, or elimination [272]. In this review, we will discuss the factors related only to the absorption process. Ideally, anticancer drugs should exhibit linear pharmacokinetics, so that it is easier for the oncologist to fine-tune the doses and control therapeutic outcomes without significant need for drug concentration monitoring. The pharmacokinetic linearity of poorly water-soluble anticancer drugs can be enhanced by ASD formulation compared to conventional oral formulations such as physical mixtures of a drug and excipients (Figure 1.5c).

The two main absorption-related factors that affect the pharmacokinetic linearity of poorly water-soluble anticancer drugs are the drug’s solubility and the saturation of drug transporters. For poorly water-soluble anticancer drugs, there is a limitation to its solubility in gastrointestinal media and thus its absorption. So, such drugs may show a linear increase in the area under the drug plasma concentration–time curve (AUC) with increase in the dose at lower levels, but the AUC will plateau at higher doses. Since ASDs typically exhibit high apparent aqueous solubility (i.e., high saturation solubility), their AUCs tend to be directly proportional to the drug dose, even at higher doses.

Elacridar is a potent inhibitor of P-glycoprotein (P-gp) and the breast cancer resistance protein (BCRP). It has the potential to be used as an absorption enhancer with several anticancer drugs [273]. However, its potential application is severely limited by its poor water solubility, and it also exhibits significant nonlinear pharmacokinetics [274]. Sawicki et al. developed binary and ternary ASDs of elacridar hydrochloride with polymers such as PVP, PVPVA, and surfactants such as sodium dodecyl sulfate. All solid dispersions demonstrated improvement in the dissolution of elacridar compared to physical mixtures with polymers and excipients. However, the ASD of elacridar with PVP and sodium dodecyl sulfate exhibited complete dissolution and was found to be thermodynamically stable [275]. They further developed tablets for oral administration with the elacridar ASD. The pharmacokinetics of these tablets were tested in an exploratory clinical trial in healthy volunteers at doses of 25 mg, 250 mg and 1,000 mg.

The maximum plasma concentration (Cmax) and the area under the drug plasma concentration–time curve (AUC) were found to increase linearly over the entire dose range (i.e., 25–1,000 mg) [274]. Thus, with application of ASDs, a linear and acceptable pharmacokinetic profile of elacridar was developed, thereby enabling its use in future clinical trials with anticancer drugs [274].

LY3009120 is a novel pan-RAF inhibitor being investigated in the treatment of cancer patients with the KRAS, NRAS, or BRAF mutations [276]. For exploratory studies, when LY3009120 was dosed as a conventional formulation to rats and dogs, it showed an oral bioavailability of < 4% and a lack of a dose–exposure relationship [276]. Thus, an ASD of LY3009120 was developed with PVP-VA and sodium lauryl sulfate (SLS) [276, 277]. This ASD enhanced bioavailability in dogs by an impressive 102-fold compared to conventional formulations [277]. Also, the ASD enabled pharmacokinetic linearity between dose and exposure at dose levels ranging from 10–100 mg/kg in rats [276].

When drug transporters are essential for drug absorption, and when the saturation of the former causes nonlinearity in the pharmacokinetics of a drug, these problems can be resolved with modification of the dosage forms or the dosage regimen. For instance, the 47

ASD can be used as a drug product intermediate and a controlled-release dosage form can be designed to synchronize with the saturation kinetics of drug transporters. Also, dosage regimens such as twice a day (B.I.D) or three times a day (T.I.D) can be designed to avoid saturation of drug transporters. Thus, ASDs can enhance the pharmacokinetic linearity of poorly water-soluble anticancer drugs, thereby enabling a linear dose– exposure relationship.

8.4. Enhancing Efficacy:

From the above discussion, it is evident that ASDs can enhance oral bioavailability, reduce pharmacokinetic variability, and enhance the pharmacokinetic linearity of poorly water-soluble anticancer drugs. These pharmacokinetic factors have a profound effect on the pharmacodynamics of anticancer drugs. The efficacy or the effectiveness of several anticancer drugs have demonstrated strong dependence on anticancer drug dose intensity and systemic exposure [278, 279].

For poorly water-soluble anticancer drugs that have a wide therapeutic window, their efficacy can be enhanced using an ASD formulation compared to conventional oral formulations when their efficacy is directly proportional to the maximum plasma drug concentration, the minimum plasma drug concentration at its steady state, the area under the plasma drug concentration–time curve, or the duration of drug exposure (see Figure 1.5d). Even when the poorly water-soluble anticancer drugs have narrow therapeutic windows, the therapeutic outcomes of such drugs can be improved by ASDs, which demonstrate controlled pharmacokinetics when their efficacy is dependent on the parameters described above. There remains a lack of literature that demonstrates the effect of ASDs in improving the pharmacodynamic efficacy of poorly water-soluble anticancer drugs.

For the anticancer drug abiraterone, which is used in the treatment of metastatic castration-resistant prostate cancer, it has been demonstrated that the prostate-specific 48

antigen (PSA) decay rate is directly proportional to the minimum plasma drug concentration at its steady state (i.e., Cminss) [280]. The treatment effect of abiraterone has been demonstrated to have a strong association with PSA kinetics, which in turn have a strong association with overall survival in prostate cancer patients [281]. Thus, elevating the Cminss for abiraterone led to a profound improvement in the therapeutic efficacy of abiraterone. For the current treatment with a conventional abiraterone formulation (Zytiga®), the average Cminss after three months of treatment initiation was found to be just 12 ng/mL [282]. Thus, if a formulation with an ASD of abiraterone is developed, it can elevate the plasma trough concentration of abiraterone and thereby elevate Cminss. This can improve the therapeutic efficacy of abiraterone.

Drug resistance in cancer treatment is another challenge that limits the efficacy of anticancer drugs [283]. Abiraterone exhibits its anticancer effect by the inhibition of the CYP17A1 enzyme. It was found that resistance to CYP17A1 inhibition can occur at standard doses—i.e., drug exposure of abiraterone after a certain period of treatment [159]. Li et al. demonstrated that this resistance can be reversed by increasing abiraterone exposure, thereby stimulating other modes of action for abiraterone [159]. Such an increase in drug exposure can be achieved by ASDs.

Gefitinib is an epidermal growth factor receptor inhibitor that shows potential for the treatment of several types of cancers. However, its efficacy is limited due to its poor oral bioavailability [284]. Godugu et al. developed an ASD of gefitinib that showed a 9.14- fold bioavailability enhancement in rats compared to its crystalline form [284]. Further, they showed significant tumor volume reduction in A431 xenograft tumor models in mice, by the gefitinib ASD compared to crystalline gefitinib [284]. Additionally, it was shown that the gefitinib ASD was able to increase its pharmacodynamic effects by significantly decreasing tumor progression biomarkers compared to crystalline gefitinib and the control arm [284]. This is yet another way in which ASDs can enhance the efficacy of poorly water-soluble anticancer drugs.

8.5. Reducing Toxicity:

The pharmacokinetics of poorly water-soluble drugs also affect the occurrence of toxicities. This is especially critical for anticancer drugs that have a narrow therapeutic window, and when their toxicities are directly proportional to the maximum plasma–drug concentration or the plasma–drug concentration alone. A high pharmacokinetic variability for such drugs means that at some points in time, their plasma–drug concentration could exceed the limit of safe concentrations and lead to toxicity, which can sometimes be life threatening for anticancer drugs [285].

The occurrence of toxicity for poorly water-soluble anticancer drugs can be reduced by using ASD formulations compared to conventional oral formulations, because the ASDs exert better control over the anticancer drug’s pharmacokinetics (Figure 1.5e). For sorafenib, an orally administered kinase inhibitor, high interpatient variability seemed to correlate with the occurrence of toxicity or subtherapeutic outcomes [21]. The interpatient variability range for the current conventional formulation of sorafenib (Nexavar®) is between 36% and 91% [13, 286]. Truong et al. developed an ASD of sorafenib with the polymer Soluplus® and the surfactant sodium lauryl sulfate (SLS). They demonstrated improvement in sorafenib’s bioavailability [19]. The pharmacokinetic studies for this ASD were performed in murine models, without actual dosage forms; therefore, they could not assess the apparent pharmacokinetic variability of sorafenib. However, this ASD formulation has the potential of reducing the pharmacokinetic variability of sorafenib, thus reducing sorafenib-associated toxicity. Thalidomide, a well- known example of a drug with high toxicity, is used in the treatment of certain cancers. It exhibits poor water solubility and high pharmacokinetic variability [287]. Barea et al. attempted to develop a solid dispersion of thalidomide with reduced crystallinity, and they demonstrated enhanced apparent solubility of thalidomide [287]. Thus, further attempts in the development of ASDs of thalidomide can reduce its pharmacokinetic variability and thus its potential toxicity. The concept of sustained-release ASDs can be 50

applied to toxicity associated with reduced high plasma–drug concentrations of poorly water-soluble anticancer drugs [288]. Nintedanib is a poorly water-soluble anticancer drug with a meagre oral bioavailability of 4.7% [289]. In order to decrease its side effects, prolong its drug release, and improve patient compliance, a sustained-release formulation with improved bioavailability of nintedanib is desirable [289]. Liu et al. developed an ASD of nintedanib with PVP K30 and soybean lecithin, then granulated it with HPMC for sustained release. The sustained-release ASD of nintedanib enhanced its bioavailability by 1.6-fold compared to its current commercial formulation in rats. In addition, this formulation had a minimal impact on Cmax [289]. This formulation would have the potential to reduce the side effects of nintedanib. Thus, the rational design of ASDs can reduce the toxicity associated with the oral administration of poorly water- soluble anticancer drugs. In addition to the pharmacokinetic and pharmacodynamic advantages mentioned above, ASD formulations of poorly water-soluble anticancer drugs have other advantages, such as reducing drug doses and patient pill burden, which ultimately improves patient compliance.

9. CASE STUDIES OF COMMERCIAL ONCOLOGY PRODUCTS BASED ON ASDS:

Table 1.4 lists commercially available oncology products based on ASDs [13, 40, 126, 290-295]:

Table 1.4. Commercial Oncology Products Based on Amorphous Solid Dispersions. Excipients used in Anti-cancer Water Structure Drug Product amorphous solid Drug Solubility dispersion

0.000362 Hypromellose acetate Vemurafenib Zelboraf®,Roche/Genentech mg/mL succinate

0.00102 Regorafenib Stivarga®, Bayer Polyvinyl pyrrolidone mg/mL

0.00163 Hydroxypropyl Everolimus Afinitor®, Novartis mg/mL methylcellulose

Vinylpyrrolidone- vinyl acetate, 0.000933 Venetoclax Venclexta®, AbbVie Polyoxyethylene mg/mL sorbitan monooleate, silica

0.119 Vinylpyrrolidone- Olaparib Lynparza®, AstraZeneca mg/mL vinyl acetate

9.1. Vemurafenib (Zelboraf®, Roche/Genentech):

Vemurafenib is a kinase inhibitor indicated for the treatment of patients with unresectable or metastatic melanoma with the BRAF V600E mutation [126]. Vemurafenib has poor water solubility (< 0.1 µg/mL) and a LogP of 3. During its initial Phase I studies, a conventional formulation with the crystalline form of vemurafenib was used. It underwent polymorphic transition, resulting in poor oral bioavailability of vemurafenib [39]. Moreover, for the Phase I trial of vemurafenib, a dose of 1,600 mg was administered, which correlates to 32 capsules of the conventional formulation [296]. Thus, efficient oral delivery of vemurafenib seemed unfeasible. However, promising results were obtained for vemurafenib. There was a statistically significant exposure– response relationship between progression-free survival and vemurafenib exposure (Cmin) (p < 0.0001) (see Figure 1.7a) [297]. Thus, in order to harness the therapeutic potential of vemurafenib, a more orally bioavailable formulation was required.

Shah et al. developed an ASD of vemurafenib with hypromellose acetate succinate using a solvent-controlled coprecipitation process [39]. The ASD of vemurafenib mitigated the risk of crystallization and polymorphic transition by stabilizing amorphous vemurafenib in the polymer matrix. In dissolution studies, the ASD of vemurafenib achieved 30 times more solubility than crystalline vemurafenib. Further, in human bioavailability studies, the ASD formulation of vemurafenib demonstrated a four- to five-fold increase in exposure compared to the crystalline drug (see Figure 1.7b) [39]. Ellenberger et al. has reported an ASD of vemurafenib formulated using a solvent-free process, KinetiSol® [298]. Ellenberger et al. reported greater oral bioavailability of vemurafenib in a murine model over the ASD developed by Shah et al. This indicates the potential for further enhancement of the oral delivery of vemurafenib. Additionally, the application of ASDs enabled a twice-daily dose of 960 mg of vemurafenib, which significantly reduced patient pill burden (i.e., four tablets twice daily) [296]. Ellenberger et al. demonstrated that the KinetiSol® processed ASD of vemurafenib also has potential for considerable reduction

in patient pill burden [298]. Thus, the ASD technology effectively enabled the therapeutic application of vemurafenib.

Figure 1.7. (a) Kaplan–Meier plots of overall survival data from trial 25026 showing a trend for exposure response. Low and high vemurafenib exposure were

9.2. Regorafenib (Stivarga®, Bayer):

Regorafenib is a kinase inhibitor indicated for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin-, and irinotecan-based chemotherapy, an anti-VEGF therapy, and—if the disease is the KRAS wild type—an anti-EGFR therapy [291]. Regorafenib is poorly water soluble (< 0.1 mg/mL) and has a LogP of 4.5. Due to its poor water solubility, an ASD of regorafenib with polyvinyl pyrrolidone (PVP K25) was developed. This

enhanced the dissolution of regorafenib by 4.5 times compared to physical mixture of regorafenib with PVP [9, 13]. In pharmacokinetic studies, regorafenib ASD enhanced the bioavailability of regorafenib seven fold compared to its conventional formulation [9, 13]. Other attempts to develop ASDs of regorafenib have been reported [299].

9.3. Everolimus (Afinitor®, Novartis):

Everolimus is a kinase inhibitor indicated for the treatment of patients with advanced renal cell carcinoma (RCC) after failure of treatment with sunitinib or sorafenib [290]. Everolimus has poor oral bioavailability due its low water solubility, instability in the gastrointestinal tract, and intestinal efflux by p-glycoprotein transporters [300]. An ASD of everolimus with hydroxypropyl methylcellulose was developed using spray drying technology [40]. It demonstrated four times more dissolution than crystalline powder [13]. Other solid dispersions of everolimus have been reported, which exhibit even better dissolution than Afinitor® [300, 301].

9.4. Venetoclax (Venclexta®, AbbVie):

Venetoclax is a BCL-2 inhibitor indicated for the treatment of patients with chronic lymphocytic leukemia (CLL) with 17p deletion [292]. Venetoclax is poorly water soluble (< 0.01 mg/mL) and has a logP of 6.9. Due to its low water solubility, venetoclax has poor oral bioavailability. This necessitated a solubility-enhancing formulation to deliver an effective dose [302].

In order to enhance the bioavailability of venetoclax, a lipid-based formulation and ASD formulations were developed. For ASD formulation venetoclax was first formulated with a polymer and surfactant. Thus two ASD formulations of venetoclax were made with polymer vinylpyrrolidone-vinyl acetate (Kollidon® VA 64) and surfactant polyoxyethylene sorbitan monooleate (Tween 80) in one formulation and the other

formulation contained Kollidon® VA 64 with surfactant D -α-Tocopherol polyethylene glycol 1000 succinate (Vitamin E TPGS) at same level. It was seen that the Tween 80 based ASD could achieve a higher drug loading than Vitamin E TPGS based ASD [302]. Drug loading in ASD formulation is a critical parameter since it ultimately affects the pill burden. Thus, final ASD formulation of venetoclax with Kollidon® VA 64, Tween 80, and silica (Aerosil) was developed. In a pharmacokinetic study in dogs, the performance of this ASD surpassed that of a lipid formulation by 61% in drug exposure and hence was selected for further development [302]. Thus, the application of ASD technology enabled the delivery of effective doses of venetoclax.

9.5. Olaparib (Lynparza®, AstraZeneca):

Olaparib is a poly (ADP-ribose) polymerase (PARP) inhibitor indicated for the maintenance treatment of adult patients with recurrent epithelial ovarian, fallopian tube, or primary peritoneal cancer who are in a complete or partial response to platinum-based chemotherapy. This drug is also indicated for the treatment of adult patients with deleterious or suspected deleterious germline BRCA-mutated advanced ovarian cancer who have been treated with three or more prior lines of chemotherapy [303].

Olaparib has poor water solubility and a LogP of 2.7. In order to improve its bioavailability, olaparib was initially developed and commercialized as a capsule formulation containing crystalline olaparib and lauroyl macrogol glycerides [295]. In order to deliver a dose of 400 mg twice daily, a total of 16 capsules were required. This negatively affected patient compliance [294, 295]. In order to reduce the dose and the pill burden, a more bioavailable and robust formulation was desirable. Thus, an ASD of olaparib with vinylpyrrolidone-vinyl acetate formulated using melt extrusion was developed [294, 295]. Compared to its capsule formulation, the ASD enhanced bioavailability by 2.65 fold. Thus, the dose could be reduced to 300 mg twice a day. The application of ASD formulations enabled the development of a tablet with 150 mg

olaparib. Hence, the pill burden was dramatically reduced from 16 capsules a day to four tablets a day [295]. Moreover, the olaparib ASD eliminated the food effect on drug absorption, as seen for the capsule formulation [295, 304]. The application of ASD formulations led to the development of a better product for olaparib, which improved patient compliance and hence improved therapeutic outcomes.

10. CONCLUSION:

Oral administration is the most preferred route for anticancer drug delivery. However, its application is limited owing to challenges associated with anticancer drugs’ physicochemical properties, predominantly poor water solubility, and the physiology of the gastrointestinal tract. ASD is a promising formulation development technology that can enhance the apparent aqueous solubility of poorly water-soluble anticancer drugs. ASD-based formulations can be adopted not only to improve the oral bioavailability of poorly water-soluble drugs but also to improve their pharmacokinetics and thus their efficacy and safety.

The early adoption of ASD technology would enable faster development of dose-linear exposure over a wide range of doses, thereby enabling faster identification of NOAELs (no observed adverse effect levels) preclinically and MTDs (maximum tolerated doses) clinically. This would avoid the issue of reaching an exposure plateau using a conventional formulation, and it would eliminate the need for reformulation to characterize the anticancer drug candidate’s effective and toxic dose.

Also, ASD technology would enable more consistent pharmacokinetics by reducing variability; therefore, statistically significant clinical signals could be achieved using fewer subjects and shorter trials. Early implementation of ASD technology in the oncology product development process could make lifesaving anticancer drugs available to patients more quickly. Additionally, early implementation of ASD technology could help mitigate the risk of false termination of lifesaving anticancer drug candidates due to 57

safety or efficacy issues that could simply be the result of erratic pharmacokinetics or poor bioavailability. The success of commercial ASD-based oncology products demonstrates that adopting ASD formulation technology leads to improved therapeutic outcomes in the field of oncology.

11. ACKNOWLEDGEMENTS AND DISCLOSURE: This chapter has been published : Gala, U.H., D.A. Miller, and R.O. Williams, Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2020. 1873(1): p. 188319.

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Chapter Two: Improved Dissolution And Pharmacokinetics Of Abiraterone Through KinetiSol® Enabled Amorphous Solid Dispersions

1. ABSTRACT: Abiraterone is a poorly water-soluble drug. It has a high melting point and limited solubility in organic solvents, making it difficult to formulate as an amorphous solid dispersion (ASD) with conventional technologies. KinetiSol® is a novel, high-energy, fusion-based, solvent-free technology that can produce ASDs. The aim of this study is to evaluate the application of KinetiSol® to make abiraterone ASDs. We developed binary KinetiSol® ASDs (KSDs) using both polymers and oligomers. For the first time, we demonstrated that KinetiSol® can process hydroxypropyl β cyclodextrin (HPBCD), which is an oligomer with a low molecular weight. We analyzed these binary KSDs using X-ray diffractometry, and we found them to be amorphous. Using in vitro dissolution analysis, we found that the maximum abiraterone dissolution enhancement was achieved using an HPBCD binary KSD; however, the KSD showed significant abiraterone precipitation in fasted state simulated intestinal fluid (FaSSIF) media. We identified hypromellose acetate succinate (HPMCAS 126G) as an abiraterone precipitation inhibitor. We developed an optimized ternary KSD using HPMCAS 126G. Our pharmacokinetic study revealed that HPBCD-based binary and ternary KSDs can enhance abiraterone bioavailability by 12.4-fold and 13.8-fold, respectively, compared to a generic abiraterone acetate tablet. Thus, this study is the first to demonstrate the successful production of an abiraterone ASD that exhibited substantial solubility and bioavailability enhancement.

2. GRAPHICAL ABSTRACT:

3. INTRODUCTION:

Abiraterone acetate is approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) and metastatic high-risk castration-sensitive prostate cancer (mHCSPC) [1]. Abiraterone acetate is a prodrug that is converted predominantly presystemically to its active metabolite abiraterone (see Figure 2.1) via esterase-catalyzed hydrolysis [2]. Abiraterone is a potent and selective irreversible inhibitor of the enzyme CYP17A1, which is required for androgen biosynthesis [3, 4]. It acts primarily by decreasing androgen production in the testicular, adrenal, and prostatic tumor tissues, thereby leading to slower disease progression [3, 5].

Figure 2.1. Structure of Abiraterone Acetate and Abiraterone. 88

Abiraterone has a water solubility of 3.05 µg/mL. It has low permeability, and it can be considered as a BCS (Biopharmaceutical Classification System) Class IV compound [6]. Abiraterone also has poor solubility in biorelevant media such as FaHIF (fasted-state human intestinal fluid) and FaSSIF (fasted-state simulated intestinal fluid) [2]. Thus, abiraterone demonstrates poor oral bioavailability without an enabling formulation. Moreover, the melting point of abiraterone is extremely high—around 501 K (i.e., 227.85 °C), and it is also poorly soluble in most organic solvents [6].

Because of these physicochemical properties, it is extremely challenging to develop an abiraterone formulation with optimal stability and bioavailability. On the other hand, abiraterone acetate has a slightly higher solubility than abiraterone in biorelevant media such as FaHIF and FaSSIF [2]. Hence, due to the challenges associated with formulating abiraterone as is, it was formulated into a tablet dosage form called Zytiga®, which contains abiraterone acetate. The oral dose of Zytiga is 1,000 mg of abiraterone acetate once daily, with 5 mg of prednisone once or twice daily [5].

Zytiga has poor oral bioavailability: less than 10%. It exhibits an extremely high food effect. One study reported that a high-fat meal increased systemic exposure to abiraterone by approximately 17- and 10- fold for Cmax and AUC0-∞, respectively [7, 8]. Even a low- fat meal had a substantial effect on Zytiga’s pharmacokinetics [7, 8]. Since there is significant variation in diets across the patient population, the administration of Zytiga with food can lead to variable abiraterone exposure. Thus, Zytiga is to be administered on an empty stomach. In addition to food effect, Zytiga also exhibits an extremely high pharmacokinetic variability. In a study, the intersubject variabilities in patients with mCRPC were found to be approximately 140% for Cmax and 107% for AUC0-24h for the single-dose Zytiga pharmacokinetics [7]. Moreover, the current Zytiga treatment results in subtherapeutic outcomes compared to the actual potential of abiraterone. It has been shown that increased exposure to abiraterone can lead to slow disease progression, longer overall survival, and a reversal of CYP17A1 inhibition resistance [9-11]. However, when the dosage of Zytiga is doubled from 1,000 mg to 2,000 mg, only an 8% increase in the 89

mean AUC was observed [5]. Thus, the current Zytiga formulation cannot deliver enough abiraterone to achieve the maximum therapeutic effect.

Despite the reported issues associated with Zytiga, only a few attempts have been made to enhance the solubility, dissolution, and bioavailability of abiraterone acetate. One such attempt was the development of the abiraterone acetate formulation Yonsa® using SoluMatrix Fine Particle Technology™ [12, 13]. Yonsa showed a modest improvement, by doubling the bioavailability of abiraterone [14]. Other reported attempts to improve the bioavailability of abiraterone include the development of nanoamorphous formulations, lipid-based formulations, nanoparticles, and self-microemulsifying formulations [15-20]. Unfortunately, none of these attempts could harness the complete therapeutic potential of abiraterone. Hence, there is still a need for an improved abiraterone formulation that enhances the dissolution and pharmacokinetics of abiraterone, ultimately leading to its improved therapeutic outcome.

An amorphous solid dispersion (ASD) is a formulation technique in which the drug, also known as the active pharmaceutical ingredient (API), is dispersed in an inert amorphous carrier such as a polymer [21]. ASDs aid the dissolution of poorly water-soluble drugs primarily by presenting the drug in an amorphous form, thereby lowering the total energy required for the solvation of the crystalline drug [22, 23]. Figure 2.2 illustrates the energetics involved in solubilizing a drug, both from its crystalline form and from an ASD. The process of drug solubilization generally involves three stages: (1) disruption of the physical form of the drug (an endothermic stage), (2) solvent cavitation (also an endothermic stage), and (3) solvation of the drug (an exothermic stage) [23]. For a drug in its crystalline form, the energy required to disrupt the crystalline lattice (Ea) is much higher than the energy required to disrupt the glass solution of a drug in an ASD form

(Ec). Also, the energy required for solvent cavitation in the solubilization of a crystalline solid (Eb) is higher than the energy required for solvent cavitation in the case of ASD solubilization (Ed), since the polymer/oligomer assists in higher solvent cavitation.

Thus, ASDs enhance the dissolution of poorly water-soluble drugs by lowering the energy barriers to solubilization. ASDs have been reported to enhance the pharmacokinetics of several anticancer drugs [24-26]. Also, ASDs have been reported to improve the therapeutic outcomes of anti-cancer drugs such as vemurafenib and gefitinib [26-28]. Hence ASD is a promising formulation technique to enhance the dissolution of abiraterone and improve its pharmacokinetic properties.

Figure 2.2. Activation energy diagram for the solubilization of a drug from a crystalline form (right) and from an ASD (left).

Several methods for manufacturing ASDs are based either on heating (e.g., hot melt extrusion) or on solvents (e.g., spray drying, conventional freeze drying, thin-film freezing, coprecipitation, electro-spinning) [29-31]. These methods are limited based on the drug’s physicochemical properties. For example, it has been reported that drugs with a high melting point ≥ 200 °C are difficult to render amorphous using the hot melt extrusion technique, thus these drugs typically lie outside its formulation space [32].

Also, since hot melt extrusion requires the application of external heat, attempts to formulate drugs with a high melting point can lead to drug and polymer degradation [32, 33]. Alternatively, the prerequisite for solvent-based ASD manufacturing techniques (e.g., spray drying, thin film freezing) is that the drug should be soluble in organic solvents or in a mixture of organic and aqueous solvents [34]. Also, solvent-based techniques pose challenges such as residual solvent toxicity and solvent explosion risks [32]. Thus, both the high melting point of abiraterone and its poor solubility in organic solvents have precluded the successful development of an abiraterone ASD, as evidenced by the absence of reports in the literature demonstrating abiraterone ASDs.

As we have previously reported, KinetiSol® technology, is a novel, high-energy, solvent- free, thermokinetic method for manufacturing ASDs. KinetiSol does not require the application of external heat [35, 36]. This technology can be used to develop ASDs of a wide range of drugs, including those with challenging physicochemical properties [35]. An ideal ASD containing a drug, is a system that not only achieves a high concentration of dissolved drug through the generation of a supersaturated state, but also maintains a high concentration of dissolved drug through the prevention or delay of drug precipitation. The generation of drug supersaturation is referred to as the spring effect, and its maintenance is referred to as the parachute effect [37]. Depending upon the drug’s properties, in certain cases, a binary ASD system containing the drug and a polymer (usually referred to as a primary polymer) is adequate to achieve the spring and parachute effects [38]. However, circumstances may require a ternary ASD system, which contains the drug, a primary polymer to induce the spring effect, and a secondary polymer to induce the parachute effect [39].

Thus, the objective of this work was to enable the development of an ideal ASD of abiraterone using KinetiSol® technology and thereby improve the dissolution and pharmacokinetic properties of abiraterone. To achieve this objective, we used long-chain polymers that are commonly reported in development of binary KinetiSol processed amorphous solid dispersions (KSDs). However, we also used a short-chain oligomer for 92

the first time. We identified the short-chain oligomer as an optimal solubility enhancer of abiraterone leading to its supersaturation (i.e., the spring effect) in acidic media. We also identified and optimized the level of a long-chain polymer to prevent abiraterone precipitation in neutral media (i.e., to create the parachute effect). We successfully developed both binary and ternary KSDs of abiraterone that improved its dissolution, increased its oral bioavailability, and reduced its pharmacokinetic variability. Therefore, the abiraterone KSD can ultimately deliver sufficient amounts of abiraterone to ensure the maximum therapeutic effect and thus improve therapeutic outcomes for prostate cancer patients.

4. MATERIALS AND METHODS:

4.1. Materials: Abiraterone API was purchased from Agno Pharma (New York, USA). Hydroxypropyl methylcellulose of varying viscosity grades (i.e., Methocel™ E3 Premium LV, Methocel™ E5 Premium LV, Methocel™ E15 Premium LV, and Methocel™ E50 Premium LV) were purchased from the Dow Chemical Company (Michigan, USA). Polyvinyl pyrrolidone of varying viscosity grades (i.e., Kollidon® 30 and Kollidon® 90) were supplied as gift samples by BASF (New Jersey, USA). Polyvinyl acetate phthalate (i.e., Phthalavin®) was supplied as gift sample by Colorcon (USA). Hydroxypropyl β cyclodextrin (i.e., Kleptose® HPB) was purchased from Roquette America (USA). Hydroxypropyl methylcellulose acetate succinate of varying degrees of acetate and succinate substitution (i.e., Affinisol™ HPMCAS 716G, Affinisol™ HPMCAS 912G, and Affinisol™ HPMCAS 126G) were supplied as gift samples by the Dow Chemical Company (Michigan, USA). Sodium carboxymethyl cellulose (i.e., Cellulose Gum 12M8P) was supplied as a gift sample by Ashland (New Jersey, USA). Methacrylic acid and ethyl acrylate copolymer (i.e., Eudragit® L 100-55) was purchased from Evonik Industries (New Jersey, USA). Microcrystalline cellulose (i.e., Avicel PH-102) was

purchased from the FMC Corporation (Pennsylvania, USA). Mannitol (i.e., Pearlitol 200SD) was purchased from Roquette America (USA). Cross-linked sodium carboxymethyl cellulose (i.e., Vivasol®) was purchased from JRS Pharma (New York, USA). Colloidal silicon dioxide (i.e., Aerosil® 200 P) was purchased from Evonik Industries (New Jersey, USA). Magnesium stearate was purchased from Peter Greven (Muenstereifel, Germany). The FaSSIF dissolution media were prepared using FaSSIF/FeSSIF/FaSSGF powder purchased from Biorelevant.com (Surrey, UK). Generic abiraterone acetate tablets (i.e., Zelgor® (250mg abiraterone acetate)) were purchased from a pharmacy in India, manufactured by Sun Pharmaceutical India Ltd. (Mumbai, India). The solvents used for HPLC analysis were of HPLC grade. All other chemicals and reagents used for dissolution and HPLC analysis were of ACS grade.

4.2. Methods:

4.2.1.Development of KSDs:

4.2.1.1. KinetiSol® Processing:

Abiraterone ASDs were prepared using a KinetiSol® small-scale compounder (formulator) designed and manufactured by DisperSol Technologies LLC (Texas, USA). Before compounding, the API and polymer/oligomer excipients (Table 2.2 and Table 2.4) were accurately weighed, dispensed into a polyethylene bag, and hand-blended for 2 min to prepare physical mixtures (PMs). These physical mixtures were charged into the KinetiSol formulator chamber. Inside the formulator chamber, a shaft with protruding blades was rotated at varying incremental speeds ranging from 4,000 rpm to 6,000 rpm without the addition of external heat in order to impart frictional and shear forces to the sample material. The temperature of the mass was monitored using an infrared probe. When the molten mass temperature reached 160 °C, the mass was rapidly ejected, collected, and pressed between two stainless steel plates to rapidly quench the sample.

4.2.1.2. Milling:

The quenched mass obtained after KinetiSol processing was milled using a lab scale rotor mill (i.e., IKA tube mill 100 (IKA Works GmbH & Co. KG, Staufen, Germany)). For milling, the fragments of quenched mass were loaded into a 20 mL grinding chamber, which was operated for 60 s with a grinding speed between 10,000 and 20,000 rpm. This milled material was subsequently passed through a #60 mesh screen (≤ 250 μm). Material retained above the screen (i.e., > 250 μm) was cycled through the mill with the same parameters. This process of milling and sieving was repeated until all material passed through the screen. The resultant material (< 250 μm) was labeled as KSD.

4.2.2. Physicochemical characterization of KSDs:

4.2.2.1. X-Ray Powder Diffraction:

X-Ray powder diffraction (XRPD) analysis was conducted using a Rigaku MiniFlex600 II (Rigaku Americas Corporation, Texas, USA) instrument equipped with a Cu-Kα radiation source generated at 40 kV and 15 mA. The API, PM, and KSD samples were loaded into an aluminum pan, leveled with a glass slide, then analyzed in the 2-theta range between 2.5 °C and 35.0 °C while being spun. The step size was 0.02 °C, and the scanning rate was set to 5.0 °C/min. The following additional instrument settings were used: Slit condition: variable + fixed slit system; soller (incident): 5.0 degrees; IHS: 10.0 mm; DS: 0.625 degrees; SS: 8.0 mm; soller (receiving): 5.0 degrees; RS: 13.0 mm (open); and monochromatization: kb filter (× 2). The data were collected using Miniflex Guidance software (Rigaku Corporation, Tokyo, Japan) and processed using PDXL2 software (Rigaku Corporation, Tokyo, Japan).

4.2.2.2. Modulated Differential Scanning Calorimetry:

Thermal analysis was conducted with modulated differential scanning calorimetry (mDSC) using a differential scanning calorimeter model Q20 (TA Instruments, Delaware, 95

USA) equipped with a refrigeration-based cooling system and an autosampler. The API and KSD samples were prepared by weighing 5–10 mg of the material and loading it into a Tzero pan. The pan was sealed with a Tzero lid using a Tzero press. Following the sample equilibration at 30 °C for 5 min, the temperature was ramped at 5 °C/min up to 250 °C with a modulation of ±1 °C every 60 s. Nitrogen was used as the sample purge gas at a flow rate of 50 mL/min. The data were collected using TA Instruments Explorer software (TA Instruments, Delaware, USA) and processed using Universal Analysis software (TA Instruments, Delaware, USA).

4.2.2.3. HPLC analysis:

A high-performance liquid chromatography (HPLC) method was developed for the chemical analysis of abiraterone KSDs. An Agilent HPLC system-1260 Infinity (Agilent, California, USA) was used for reverse phase HPLC analysis. The HPLC column was a Zorbax C18 extend (150 mm x 4.6 mm, 3.5 µm) (Agilent, California, USA). Mobile phase A was 0.1% formic acid, and mobile phase B was degassed acetonitrile. A gradient profile was designed with an initial higher amount of aqueous phase, followed by a gradual increase in the organic phase. The flow rate was 0.5 mL/min and the run time was 40 min. The column was held at 25 °C, and the data were collected at a single wavelength of 254 nm. Samples were prepared at a nominal concentration of 0.1 mg/mL level with 7:2:1 acetonitrile:methanol:formic acid (0.1%) as the standard or sample diluent. All samples were filtered through 0.45 μm nylon syringe filters (GE Healthcare Bio-Sciences, Pennsylvania, USA), before analysis. Sample chromatography was analyzed using Empower software, version 3.0 (Waters, Massachusetts, USA).

4.2.2.4. Dissolution:

An in vitro non-sink gastric transfer dissolution method was developed to analyze the dissolution of abiraterone API, the generic abiraterone acetate tablets, the binary KSDs, the select ternary component for KSDs, as well as the dissolution of the ternary KSDs. 96

For the dissolution analysis of abiraterone API and the binary and ternary KSDs, the samples (equivalent to 31mg of abiraterone API) were loaded in an Erlenmeyer flask (dissolution vessel) containing 35 mL of 0.01N HCl (pH 2.0), placed in an incubator– shaker (Excella E24 (New Brunswick Scientific, New Jersey, USA)) set to 37 °C and a rotational speed of 180 rpm. After 30 min, 35 mL of FaSSIF (prepared in a 50 mMol phosphate buffer at pH 6.8) was added to the dissolution vessel. At predetermined time points, samples were drawn from the dissolution vessel and centrifuged using an ultracentrifuge (Airfuge™, Beckman, Indianapolis, USA). The supernatants were further diluted using the HPLC diluent and analyzed using the HPLC method mentioned above. For the generic abiraterone acetate tablets, the whole tablet was analyzed and the dissolution media volumes were scaled accordingly. For the selection of the ternary component, 35 mg of secondary polymer candidates (see Table 2.3) were added in 35 mL of 0.01N HCl (pH 2.0), and the dissolution of the binary KSD was conducted using the method described above. The area under the drug dissolution curve (AUDC) was calculated using the linear trapezoidal method.

4.2.2.5. Tableting:

The KSD and tableting excipients (Avicel PH-102, Pearlitol 200SD, Vivasol, Aerosil 200 P and magnesium stearate) were accurately weighed and dispensed. Aerosil 200 P was sieved through #40 mesh (420 µm) until all material passed through the sieve. The KSD and all tableting excipients, except magnesium stearate, were loaded in a vial and mixed using a vortex mixer (Thermo Scientific, Massachusetts, USA). Magnesium stearate was then added to the vial and blended using a spatula. The resultant tableting blend was then dispensed in aliquots equivalent to 44.6 mg of abiraterone. Each aliquot was loaded in the tablet die and compressed using a single-station hand tablet press (BVA Hydraulics, Missouri, USA) with a target hardness of 8–12 kP.

4.2.2.6. Pharmacokinetic Study in Beagle Dogs:

An in vivo pharmacokinetic study in fasted non-naïve male beagle dogs was carried out at Charles River Laboratories (Massachusetts, USA). This animal study was conducted according to an approved Charles River Laboratories IACUC protocol (#20111395). The 44.6 mg equivalent abiraterone tablets were analyzed along with a generic 250 mg equivalent abiraterone acetate tablet. Each study arm for each formulation consisted of five dogs. The dogs were fasted overnight before dosing, and the food was returned after 4 h post dosing. Each dog was administered a single tablet of the respective formulation (as per the study arm) along with a post dose flush of 40 mL sterile water adjusted to pH 2.0. At predefined time points of 0.5, 1, 1.5, 2, 3, 4, 6, 8, and 10 h post dose, 1 mL blood samples were drawn from each dog using venipuncture of a peripheral vessel and placed into tubes containing sodium heparin anticoagulant. The blood samples were centrifuged to isolate the plasma. The plasma samples were then analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for abiraterone content.

4.2.2.7. Pharmacokinetic Analysis:

Pharmacokinetic parameters were estimated using Watson pharmacokinetic software version 7.3.0.01 (Thermo Fisher Scientific, Massachusetts, USA) using a noncompartmental approach consistent with the oral route of administration. The area under the plasma concentration–time curve (AUC) was calculated using the linear trapezoidal method. The relative bioavailability (i.e., the F value) “…. was calculated using the following formula.”

퐴푈퐶(0−10hr)(test abiraterone tablet) × 퐷표푠푒(abiraterone )(generic abiraterone acetate tablet) 퐹 = 퐴푈퐶(0−10hr)( generic abiraterone acetate tablet) × 퐷표푠푒(abiraterone ) (test abiraterone tablet)

Statistical analysis was performed using JMP® 14.3.0 software (SAS, North Carolina, USA). The two groups were compared consecutively using Student’s t-test (α = 0.05). 98

5. RESULTS AND DISCUSSION:

5.1. Development of binary KSDs:

In order to develop binary KSDs of abiraterone, we selected five polymers/oligomers that vary in their chemistry, architecture, molecular weight, and viscosity (see Table 2.1). We selected the primary polymers/oligomers with varying chemistry (long-chain linear and short-chain cyclic architectures) to enable all possible noncovalent interactions between the primary polymer/oligomer and abiraterone in the binary KSDs. This was done to determine the most suitable primary polymer/oligomer that would form a stable, molecularly dispersed abiraterone KSD and prevent abiraterone recrystallization. We selected primary polymers/oligomers that have a wide range of molecular weights and viscosity grades to explore the thermokinetic processing space of KinetiSol technology. Table 2.2 lists the binary KSDs’ composition, processing parameters, and appearance after KinetiSol processing. All primary polymer compositions (i.e., the Lots 1, 2, 3, and 4 PMs) were found to be processable using KinetiSol technology. Ellenberger et al. reported a list of polymers used for KinetiSol processing [35]. They observed that all the reported polymers had high molecular weights. It is commonly believed that high molecular weight and linear or branched polymers are necessary for friction generation and KinetiSol processing. However, in the current study, it was observed for the first time that the Lot 5 PM containing HPBCD, which is a cyclic oligomer with low molecular weight, was also well processed using KinetiSol technology. The total processing time for all the lots was less than 45 s. The processing time at elevated temperatures for the Lots 1–4 KSDs was less than 15 s, while the Lot 5 KSD required less than 7.5 s. Discoloration was observed in the Lots 1–4 KSDs but not in the Lot 5 KSD. This indicates that the discoloration was most likely due to long-chain polymer processing at elevated temperatures. The Lot 1 and Lot 2 KSDs showed slight discoloration and were yellowish in color, while the Lot 3 KSD also showed slight discoloration but was light

brown in color. None of these KSDs showed high levels of discoloration consistent with polymer degradation, as was seen during hot melt extrusion of these polymers [40, 41]. The Lot 4 KSD showed significant discoloration and was brown in color. This could be due to PVAP degradation at elevated temperatures and also due to subsequent drug degradation due to interaction with polymer degradants. All the lots showed an opaque appearance, as expected, due to trapped air in the ejected KSD masses. The binary KSD masses for the Lots 1–4 KSDs were more agglomerated and less brittle than the mass of the Lot 5 KSD, since long-chain linear polymers were used in the Lots 1–4 KSDs, which imparted a more rigid network of polymers within which abiraterone was dispersed. Nonetheless, all the binary KSD masses were successfully milled to yield a binary KSD powder with a particle size of < 250 µm.

100

< 1.5 <

1,399

chain Cyclic Cyclic chain

44]

Oligomer

(HPBC

cyclodextrin

Glucose based Glucose

Kleptose® HPB Kleptose®

(mPa.s : 10% w/v w/v :10% (mPa.s °C) at25 inwater

Hydroxy propyl β β propyl Hydroxy

Short

[35, 42 [35,

–

~60,700

(PVAP)

Phthalavin®

chain Linear Polymer Linear chain

Phthalate based Phthalate

(mPa.s: in water at 25 °C) 25 at in water (mPa.s:

Polyvinyl acetate phthalate acetate Polyvinyl

Long

8.5

chain chain

–

based

Linear Linear

20 °C) 20

5.5

~50,000

Polymer

(PVP 30) (PVP

Polyvinyl Polyvinyl

Long

pyrrolidone pyrrolidone

Pyrrolidone Pyrrolidone

(mPa.s : 10% :10% (mPa.s

Kollidon® 30 Kollidon®

w/v in water at at in w/v water

6.0

–

chain Linear Linear chain

4.0

~28,700

Polymer

cellulose cellulose

(HPMC E5) (HPMC

Premium LV Premium

water at 20 °C) 20 at water

Methocel™ E5 E5 Methocel™

Cellulose based Cellulose

Long

(mPa.s : 2% w/v in :2% w/v (mPa.s

Hydroxy propyl methyl propyl Hydroxy

3.6

chain chain

–

°C)

2.4

~20,000

Long

Primary polymers/Oligomers selected for development of binary KSDs of abiraterone of abiraterone KSDs ofbinary development for selected polymers/Oligomers Primary

(HPMC E3) (HPMC

Premium LV Premium

in water at 20 at20 inwater

Methocel™ E3 E3 Methocel™

Linear Polymer Linear

Cellulose based Cellulose

Hydroxy propyl propyl Hydroxy

methyl cellulose cellulose methyl

(mPa.s : 2% w/v :2% w/v (mPa.s

Table 2.1. 2.1. Table

Weight

Viscosity

Polymer/

Oligomer

Structure

Monomer Monomer

Molecular Molecular

Chemistry

Commercial Commercial

Architecture Productused

101

Table 2.2. Binary KSD compositions, processing parameters, and their corresponding appearance. Composition Primary Batch Processing Shear Stress *Processing Lot Drug (% Polymer/ Size Temperature (Rotational Time Appearance No. wt) Oligomer (g) (°C) speed (rpm)) (seconds) (% wt)

Abiraterone HPMC E3 4,000, 5,000, 10 + 10 + 1 10 160 (10) (90) 6,000 6.9

Abiraterone HPMC E5 4,000, 5,000, 10 + 10 + 2 10 160 (10) (90) 6,000 7.1

Abiraterone PVP K30 4,000, 5,000, 10 + 10 + 3 10 160 (10) (90) 6,000 3.7

Abiraterone PVAP 4,000, 5,000, 4 10 160 10 + 10 +23 (10) (90) 6,000

Abiraterone HPBCD 4,000, 5,000, 5 10 160 10+10+6.3 (10) (90) 6,000

*Each processing time number corresponds to the time spent on the corresponding shear stress stage (i.e., the rotation speed stage). 102

5.2.Physicochemical characterization of binary KSDs:

Burke et al. reported that abiraterone API exists in a crystalline state, and its crystal conformation is stabilized by hydrogen bonding between the nitrogen atom of the pyridine ring and the hydroxyl group [45]. The X-ray diffractogram of abiraterone showed sharp diffraction peaks (see Figure 2.3), indicating its crystalline state. Three main diffraction peaks for abiraterone were observed at 8.38°, 16.46° and 19.27° that were similar to those reported by Solymosi et al. [6]. However, certain peak positions and relative peak intensities of our abiraterone API X-ray diffractogram differed from that reported by Solymosi et al. [6]. This could be due to differences between the various polymorphs of abiraterone.

These diffraction peaks of abiraterone were also observed in the X-ray diffractogram (XRD) of binary PMs (see Figure 2.3), thus indicating their crystalline nature. The X-ray diffractograms of all the binary KSDs showed a halo pattern and no abiraterone diffraction peaks (see Figure 2.3), thus indicating their amorphous nature. In the diffractograms of the Lots 1 and 2 KSDs, a very small diffraction peak was observed around 31.5–31.7° . This peak was also observed in the diffractograms of the Lots 1 PM, 2 PM, HPMC E3, and HPMC E5 (data not shown). We noted that both HPMC E3 and HPMC E5 contain ≤ 1% sodium chloride [46]. Lee et al. observed a sharp diffraction peak at 31.5 ° in the X-ray diffractogram of sodium chloride [47]. Therefore, the diffraction peak seen around 31.5–31.7 ° in the diffractograms of the Lots 1 and 2 KSDs can be attributed to the presence of sodium chloride.

103

Figure 2.3. X-Ray diffractograms of abiraterone API (top) and abiraterone binary PMs, KSDs (bottom).

The mDSC thermogram of abiraterone (see Figure 2.4), showed a sharp melting endotherm at 228.71 °C. This confirmed a melting point and melting range of 501 K (227.85 °C) and 228–230 °C, respectively, which has been reported in the literature [6,

104

48]. None of the mDSC thermograms of binary KSDs showed any melting endotherms (see Figure 2.4). In particular, there was no melting endotherm at 228 °C. This further substantiates that KinetiSol processing rendered abiraterone amorphous in all the binary KSDs.

The Lots 1–4 KSDs showed a glass transition temperature (Tg) of 111.14, 118.32, 149.01, and 120.14 °C, respectively. No other Tg event was observed for these lots. Thus, we can infer that these KSDs are single-phase systems in which abiraterone APIs are uniformly and homogeneously dispersed in their respective polymers. For the Lot 1 and Lot 2 KSDs, a small thermal event was observed at 192.35 °C and 191.41 °C, respectively. The magnitude of these thermal events was negligible, and they may be an artifact of the experimental parameters. For the Lot 5 KSD, no thermal event was observed, which indicates that a single-phase amorphous system was formed.

105

Figure 2.4. mDSC thermograms of abiraterone API (top) and abiraterone binary KSDs (bottom).

HPLC analysis showed that the KSDs of Lots 1, 2, 3, and 5 had purities of 99.2, 99.2, 97.5, and 99.3%, respectively. The KSDs of Lots 1, 2, and 5 showed no individual impurity ≥ 0.5%. The Lot 3 KSD showed two unknown impurities of > 0.5% but < 1.0%. These data demonstrate that no significant degradation of abiraterone API occurs during KinetiSol processing. The Lot 4 KSD showed total impurities of > 5.0%. This could be due to PVAP having undergone significant degradation, which led to the formation of phthalic acid and acetic acid, thus causing abiraterone degradation [49]. A polymer

106

similar to PVAP (e.g., cellulose acetate phthalate) is known to undergo thermal degradation at 105 °C to form phthalic acid and acetic acid[50].

5.3. Dissolution of binary KSDs:

For dissolution testing, we employed a two-stage, non-sink, gastric transfer dissolution method in which the sample is first exposed to acidic media, simulating fasted stomach content, followed by exposure to neutral-to-basic media, simulating fasted intestinal content. Figure 2.5 illustrates the in vitro, non-sink, gastric transfer dissolution profile of abiraterone API, the generic abiraterone acetate tablet, and the binary KSDs.

With the exception of the Lot 4 KSD, all other binary KSDs were able to enhance the overall dissolution of abiraterone compared to the neat crystalline abiraterone API. After integrating the total area under the drug dissolution curve (AUDC Total ), we found that the relative AUDC Total for the generic abiraterone acetate tablet was 219.8% relative to neat abiraterone API. This difference occurs because abiraterone acetate is more soluble than abiraterone in biorelevant media [2, 6].

The relative AUDC Total for the Lot 1 KSD, the Lot 2 KSD, and the Lot 3 KSD was 288.8%, 316.0%, and 255.1%, respectively, relative to neat abiraterone API. This shows that the KSDs based on the polymers HPMC E3, HPMC E5, and PVP K30 can enhance abiraterone dissolution even more than its prodrug abiraterone acetate due to abiraterone amorphization. The relative AUDC Total for the Lot 4 KSD was just 55.1% as compared to neat abiraterone API, indicating that PVAP-based KSDs showed lower abiraterone dissolution than even the crystalline abiraterone API. However, this is due to the higher impurities in Lot 4 KSD. As discussed above, the PVAP was degraded, leading to a potential loss of ionizable groups, which in turn lowers both PVAP solubilization and abiraterone solubilization. The relative AUDC Total for the Lot 5 KSD was dramatically higher (i.e., 1,173.4%) relative to abiraterone API. This indicates that the oligomer HPBCD-based KSD far surpassed the dissolution enhancement of neat abiraterone API

107

when compared not only to its prodrug abiraterone acetate but also to its polymer-based KSDs.

HPBCD has a hydrophobic inner cavity and a hydrophilic outer surface. It can form an inclusion complex with poorly water-soluble drugs by interacting with the hydrophobic groups of the drug and including them within its cavity. This interaction improves the drug’s physicochemical properties and thus increases the drug’s aqueous solubility [51]. Oligomer-based KSD dissolution performance surpassed that of polymer-based KSDs likely because in addition to abiraterone amorphization in the Lot 5 KSD, HPBCD further enhanced abiraterone solubility by forming inclusion and non-inclusion complexes with abiraterone, likely in KSD and during dissolution [51, 52]. Moreover, the oligomer HPBCD has a more hydrophilic outer surface than the other polymers tested, thereby leading to higher abiraterone hydration and solubilization. Similarly, Verma and Kumar reported higher gliclazide dissolution in the HPBCD solid dispersion than the PVP K30 solid dispersion [53].

Abiraterone API was more soluble in acidic media (i.e., in 0.01N HCl) and less soluble in neutral to basic media (i.e., when FaSSIF was added). This is because abiraterone is weakly basic in nature. Solymosi et al. also observed that abiraterone was more soluble in simulated gastric fluid (pH 1.6) than FaSSIF[6]. The generic abiraterone acetate tablet is more soluble than abiraterone API in acidic media. After closer examination, it is evident that Lots 1, 2, 3, and 5 KSDs have higher abiraterone dissolution, meaning they can attain more supersaturation of abiraterone compared to neat abiraterone API and the generic abiraterone acetate tablet in 0.01N HCl.

However, rapid abiraterone precipitation occurs for KSDs in FaSSIF media. This means that HPMC E3, HPMC E5, PVP K30, and HPBCD were good spring agents in 0.01N HCl but were poor parachute agents in FaSSIF media. It can be assumed that a majority of the abiraterone will precipitate as crystalline abiraterone due to its higher crystallization tendency. However, some of the abiraterone will precipitate into a crystal-

108

amorphous aggregate form or an amorphous aggregate form. For example, during the dissolution of enzalutamide ASDs, the formation of drug crystals and amorphous drug aggregates have been observed [54]. The nature of abiraterone precipitate needs further investigation. The degree of precipitation from 0.01N HCl to FaSSIF for all the KSDs discussed above has a similar range, from 92.0–94.0%. This suggests that neither HPMC E3, HPMC E5, PVP K30, nor HPBCD were good precipitation inhibitors. This could be due to (a) the low viscosity grades of HPMC E3, HPMC E5, and PVP K30 and (b) the low molecular weight and cyclic architecture of HPBCD. The drug concentration of the generic abiraterone acetate tablets in FaSSIF media was 25.74 µg/mL on average, which is similar to the abiraterone acetate FaSSIF solubility value of 64.6 ± 5.2 µM (i.e., ~25.29 µg/mL) reported in the literature [2]. Interestingly, in another study, the FaSSIF solubility of abiraterone acetate from Zytiga was reported to be only 18 µg/mL [15]. We observed that the average solubility of abiraterone API in FaSSIF was 11.33 µg/mL, which conflicts with the value of 12.7 ± 4.2 µM (i.e., ~4.43 µg/mL) reported in the literature [2]. This is likely due to differences in abiraterone polymorphs, as discussed above. Of all KSDs, in FaSSIF media, only the Lot 5 KSD had a higher drug concentration than abiraterone API. But, when compared to the drug concentration of generic abiraterone acetate tablets in FaSSIF media, the Lot 5 KSD exhibited higher drug concentration only up to 90 min.

Thus, it was determined that of all the binary KSDs tested, the Lot 5 KSD containing HPBCD showed the highest overall dissolution enhancement for abiraterone. HPBCD showed a significant spring effect in 0.01N HCl but showed a poor parachute effect in FaSSIF media. Hence, the precipitation of abiraterone in FaSSIF media must be inhibited or reduced.

109

Figure 2.5. In vitro, non-sink, gastric transfer dissolution profiles of neat abiraterone API, generic abiraterone acetate tablets, and binary KSDs. The red region: 0.01N HCl. The blue region: FaSSIF. The inset represents the enlargement of the dissolution profile in FaSSIF.

5.4. Selection of a suitable ternary component for KSDs:

The main goal of adding a ternary component (i.e., a secondary polymer) to the KSDs in the current study is to prevent abiraterone precipitation in FaSSIF media. For this reason, we decided to explore two concepts.

First, we selected secondary polymer candidates that dissolve immediately and impart high viscosity in the microenvironment of the dissolving drug at lower polymer concentrations. The rationale here was that polymers that have a higher viscosity grade would maintain supersaturated drug concentrations and prevent the drug’s precipitation in FaSSIF.

110

It has been reported that an increase in the viscosity of the media reduces molecular mobility, thereby interfering with drug nucleation and crystallization [55, 56]. Moreover, higher-viscosity grade polymers tend to have higher molecular weights and more functional groups that interact with the precipitated hydrophobic crystalline drug surface, thus preventing further drug crystallization [55].

Hence, we selected the following: (a) hydroxypropyl methylcellulose (Methocel™ E15 Premium LV (HPMC E15)), (b) Methocel™ E50 Premium LV (HPMC E50), (c) polyvinyl pyrrolidone (Kollidon® 90 PVP K90), and (d) sodium carboxymethyl cellulose (Cellulose Gum 12M8P (Na CMC)). A 2% w/v aqueous solution of HPMC E15 and HPMC E50 imparts a viscosity of 15 mPa·s and 50 mPa·s, respectively [42]. A 10% w/v aqueous solution of PVP K 90 can impart a viscosity of 300–700 mPa·s [42]. A 1% w/v aqueous solution of Na CMC can impart a viscosity of about 2,000 mPa·s [42].

Second, we selecetd polymers that have pH-dependent solubility and are soluble at pH 5 and above. The rationale here was that these polymers would dissolve in FaSSIF, then increase viscosity in the microenvironment of the dissolving drug and thus prevent drug precipitation. Therefore, we selected hydroxypropyl methylcellulose acetate succinate with varying degrees of acetate and succinate substitution (i.e., Affinisol™ HPMCAS 716G, Affinisol™ HPMCAS 912G, and Affinisol™ HPMCAS 126G). Polyvinyl acetate phthalate (i.e., Phthalavin® (PVAP)) and methacrylic acid and ethyl acrylate copolymer (i.e., Eudragit® L 100-55). HPMCAS 716 G, HPMCAS 912 G, and HPMCAS 126 G dissolve at a pH of ≥ 5.5, ≥6.0, and ≥ 6.8, respectively [35, 57]. PVAP dissolves at a pH of ≥ 5.0, and Eudragit® L 100-55 dissolves at pH of ≥ 5.5 [42].

Figure 2.6 shows the in vitro, non-sink, gastric transfer dissolution profiles of the Lot 5 KSD with different secondary polymers. Table 2.3 lists the values for the relative area under the drug dissolution curve. It is important that secondary polymers prevent abiraterone precipitation in FaSSIF media, but it is also important that they do not significantly decrease abiraterone supersaturation in 0.01N HCl.

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It has been reported that the addition of a hydrophilic polymer typically increases a medium’s ability to dissolve cyclodextrin [58]. However, from the AUDC 0.01N HCl values relative to the Lot 5 KSD, it is evident that the addition of secondary polymer candidates reduced abiraterone dissolution by ≤ 22.0%. This could result from the hydrophilic groups of these polymers interacting with the outer surface of HPBCD, thereby reducing its ability to enhance dissolution. Interestingly, even for polymers dissolving at pH ≥ 5, there was a reduction in AUDC 0.01N HCl values relative to the Lot 5 KSD. The cause of this needs further investigation.

From the inset of Figure 2.6, we can see that most secondary polymer candidates can prevent abiraterone precipitation in FaSSIF media. However, HPMCAS 126G was the most capable of preventing abiraterone precipitation. The AUDC FaSSIF value of HPMCAS 126G was 244.8% relative to Lot 5 KSD, thereby making HPMCAS 126G the best precipitation inhibitor (or the best parachute agent) among the secondary polymer candidates that were tested. This could result from HPMCAS 126G dissolving above pH ≥ 6.8, thereby dissolving only in FaSSIF media to exert its effect.

HPMCAS is amphiphilic. Its hydrophobic regions interact with abiraterone, and its hydrophilic regions interact with FaSSIF media and permit the stabilization of abiraterone [59]. Also, among the various grades of HPMCAS tested, HPMCAS 126G was the most hydrophobic. Its relatively higher substitution with hydrophobic methoxy and acetate groups can interact with the hydrophobic regions of abiraterone and prevent further recrystallization [60]. It has been reported that HPMCAS maintains drug supersaturation and prevents drug precipitation by reducing molecular mobility for nucleation, thus prolonging the time required for nucleation and re-dissolving precipitated aggregates by interacting with the hydrophobic groups on the surface of the drug, hence interfering with drug crystallization [61-63]. So, it was determined that HPMCAS 126 G was a suitable secondary polymer for the development of a ternary abiraterone KSD.

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Figure 2.6. In vitro, non-sink, gastric transfer dissolution profile of abiraterone API, generic abiraterone acetate tablets, Lot 5 KSD and Lot 5 KSD with different secondary polymer candidates. Red region: 0.01N HCl. Blue region: FaSSIF. The inset represents the enlargement of the dissolution profile in FaSSIF.

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Table 2.3. Relative area under the drug dissolution curve for the selection of suitable ternary components (i.e., secondary polymers).

Percent Rel. Percent Rel. Percent Rel. AUDC Total AUDC 0.01N HCl AUDC FaSSIF Sample (Relative to Lot (Relative to Lot 5 (Relative to Lot 5 5 KSD) KSD) KSD)

Lot 5 KSD 100.0 100.0 100.0

Abiraterone API 8.4 1.8 46.2 Generic Abiraterone 18.4 3.6 104.1 Acetate Tablet Lot 5 KSD with 89.0 83.1 109.1 HPMC E15 Lot 5 KSD with 89.2 82.1 113.8 HPMC E50 Lot 5 KSD with 91.8 87.9 105.0 PVP K90 Lot 5 KSD with HPMCAS 108.9 83.5 244.8 126 G Lot 5 KSD with HPMCAS 85.9 86.1 77.4 716 G Lot 5 KSD with HPMCAS 88.1 79.7 134.6 912G Lot 5 KSD with Na 85.4 85.8 79.1 CMC Lot 5 KSD with 80.4 78.0 85.3 PVAP Lot 5 KSD with Eudragit L- 93.8 90.3 107.5 100 55

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5.5. Development of ternary KSDs:

We developed several compositions of ternary KSDs to identify the suitable concentration of HPBCD and HPMCAS 126 G to achieve the highest dissolution of abiraterone. Table 2.4 provides the composition of the ternary KSDs, along with their processing parameters and appearance. We observed that ternary KSD compositions (i.e., the Lots 6, 7, and 8 PMs) could be processed using KinetiSol® technology. The Lot 9 PM appeared to be underprocessed in the defined conditions, likely because the Lot 9 PM contained more HPMCAS 126G, leading to more friction and faster attainment of the set temperature of 160 °C. Thus, the Lot 9 KSD had the lowest processing time, which may not be enough for fusion of the KSD components. However, this assumption is based only on appearance and would be verified by XRPD analysis.

Overall, the shear stress stages and processing times for all ternary KSDs were less than the binary KSD (i.e., the Lot 5 KSD) (see Table 2.2). The total processing time of the Lots 6 to 9 KSDs was less than 20 s, and the processing time at elevated temperature was less than 5 s. The Lots 6, 7, 8, and 9 KSDs showed slight discoloration and were light brown in color. As expected, these lots were opaque due to air entrapment in the KSD masses. All the ternary KSD masses were successfully milled to yield ternary KSD powders with a particle size of < 250 µm.

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Table 2.4. Ternary KSD composition, processing parameters, and appearance.

Composition Shear Primary Batch Processing Stress *Processing Lot Secondary API (% Cyclic Size Temperature (Rotational Time Appearance No. Polymer Wt) Oligomer (g) (°C) speed- (seconds) (% Wt) (% Wt) rpm) HPMCAS Abiraterone HPBCD 4,000, 6 126 G 10 160 10 + 3.6 (10) (80) 5,000 (10)

Abiraterone HPBCD HPMCAS 4,000, 7 10 160 10 + 6.3 (10) (70) 126 G (20) 5,000

Abiraterone HPBCD HPMCAS 4,000, 8 10 160 10 + 5.2 (10) (60) 126 G (30) 5,000

Abiraterone HPBCD HPMCAS 9 10 160 4,000 9.6 (10) (50) 126 G (40)

*Each processing time number corresponds to the time spent on the corresponding shear stress stage (i.e., the rotation speed stage).

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5.6. Physicochemical characterization of ternary KSDs:

The X-ray diffractograms of the Lots 6, 7, and 8 KSDs (see Figure 2.7) showed a halo pattern and no abiraterone diffraction peaks, indicating their amorphous nature. The X- ray diffractogram of the Lot 9 KSD showed characteristic abiraterone peaks at 16.65° and 19.55°, but did not show the peak at 8.38°, which suggests a crystalline nature or a partially amorphous nature. The X-ray diffractogram of the Lot 9 KSD also showed small peaks at 13.08, 15.47, and 23.82° which are also present in the crystalline abiraterone API diffractogram (Figure 2.3). Thus, as noted above, (based on appearance) the Lot 9 KSD was under processed.

Figure 2.7. X-ray diffractograms of abiraterone ternary KSDs.

We analyzed only the Lots 6, 7, and 8 KSDs for purity because they were amorphous. The HPLC analysis showed that the Lots 6, 7, and 8 KSDs had purities of 98.1%, 98.2%, and 98.5%, respectively. The Lots 6, 7, and 8 KSDs showed no individual impurity ≥ 0.5%. The total impurity level of these lots were only slightly higher than the Lot 5 KSD. This is could occur because at higher temperatures, HPMCAS 126G can degrade to form acetic acid and succinic acid [57]. In the case of KinetiSol® processing, the amount of 117

HPMCAS 126G degradation would have been minimal due to only transient exposure to high temperatures. Thus, low levels of acid would be released, leading to a negligible level of abiraterone impurity formation.

5.7. Dissolution of ternary KSDs:

Initially, we conducted dissolution of the ternary KSDs (i.e., the Lots 6 and 7 KSDs) and compared this to the binary KSDs (i.e., the Lot 5 KSD) (see Figure 2.8). The overall dissolution of the Lot 6 KSD > Lot 5 KSD > Lot 7 KSD. The initial supersaturation of abiraterone in 0.01N HCl was higher for the Lot 6 KSD > Lot 5 KSD > Lot 7 KSD. The reason for this can be attributed to the mole ratio of abiraterone:HPBCD present in the KSDs. The mole ratio of abiraterone:HPBCD in the Lots 5 and 6 KSDs was 1.00:2.25 and 1.00:2.00, respectively, while the mole ratio for the Lot 7 KSD was 1.00:1.75. Thus, in the Lot 7 KSD, there were insufficient moles of HPBCD to interact with or include the abiraterone molecule, leading to lower supersaturation levels. The exact nature of the interaction between abiraterone and HPBCD in KSDs needs further investigation. Contrary to the dissolution behavior observed in 0.01N HCl during the selection of the ternary component (see Figure 2.6), the addition of HPMCAS 126 G within the ternary KSD led to better supersaturation in 0.01N HCl, as was expected [58]. This occurs because HPMCAS 126G was added externally to the KSD during the process of selecting the ternary component, but in the ternary KSDs, HPMCAS 126G was included in the KSD, where it could interact with abiraterone and aid in dissolution. From the dissolution in FaSSIF, it is evident that increasing the concentration of HPMCAS 126G did not further increase precipitation inhibition or the parachute effect. Thus, it was determined that the ternary KSD composition of 10:80:10 % w/w of abiraterone:HPBCD:HPMCAS 126 G was the most suitable formulation to enhance abiraterone dissolution.

Sarode et al. reported that the thermal processing (hot melt extrusion) of HPMCAS-H grade (similar to HPMCAS 126G) had a negative impact on drug dissolution due to the degradation of HPMCAS-H and the release of free acids. Since KinetiSol processing of 118

HPMCAS 126G had no negative impact on abiraterone dissolution, it can be concluded that minimal to no degradation of HPMCAS 126G takes place during KinetiSol processing.

Figure 2.8. In vitro, non-sink, gastric transfer dissolution profiles of the Lots 5, 6, and 7 KSDs. Red region: 0.01N HCl. Blue region: FaSSIF. The inset represents the enlargement of the dissolution profile in FaSSIF.

5.8. Pharmacokinetic Study in Beagle Dogs:

Cyclodextrins are known to enhance the oral bioavailability of drugs by enhancing their solubility. However, when cyclodextrins are used in excess in oral formulations, they can hamper drug absorption due to constant binding with the free drug [64]. Thus, in addition to in vitro analysis, it is imperative to study the effect of KSDs in vivo. So, we decided to study polymer-based KSDs along with oligomer-based KSDs in vivo. Moreover, we decided to study binary KSDs along with ternary KSDs to evaluate the impact of the addition of a ternary component.

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In order to develop a viable dosage form for abiraterone delivery the KSDs were compressed into immediate release tablet formulation. Three formulations containing Lot 2 KSD, Lot 5 KSD and Lot 6 KSD were compressed into Lot 2 Tablet, Lot 5 Tablet and Lot 6 Tablet respectively. Each tablet contained 44.6mg of abiraterone. All tablets had optimum hardness, assay, purity, acceptable friability, disintegration time and dissolution. These tablets were tested and compared against generic abiraterone acetate tablets containing 250 mg of abiraterone acetate. Figure 2.9 shows the in vivo plasma concentration versus time profiles of these tablets, and Table 2.5 lists the pharmacokinetic parameters. It can be seen that the Lots 5 and 6 tablets containing

HPBCD achieved a higher maximum abiraterone plasma concentration (i.e., Cmax at one fifth the dose compared to the generic abiraterone acetate tablets). This proves that (a) the Lots 5 and 6 KSDs can deliver high amounts of abiraterone and (b) the concentration of HPBCD in these lots had no negative impact on the abiraterone absorption process.

If we dose adjust Lot 2 Tablet Cmax, then it too is higher than the Cmax of the generic abiraterone acetate tablets. The time to achieve Cmax (i.e., the Tmax) of the generic abiraterone acetate tablet and the Lot 2 tablet were comparable, but the Tmax of the Lots 5 and 6 tablets were lower. This suggests that HPBCD-based tablets can achieve faster abiraterone supersaturation, as observed in the in vitro dissolution study. Although not statistically significant, but polymer-based KSD (i.e., the Lot 2 Tablet) was able to enhance the abiraterone bioavailability by 3.4-fold. The Lots 5 and 6 tablets can enhance abiraterone bioavailability with statistical significance by 12.4-fold (p = 0.0210) and 13.8-fold (p = 0.0020), respectively.

It is interesting to note that although not statistically significant, the AUC0-10hr of ternary KSD based the Lot 6 Tablet was higher than binary KSD based Lot 5 Tablet, thereby indicating the positive effect of the addition of a ternary component. Also, the Lots 5 and

6 tablets were able to significantly reduce the intersubject variability (i.e., % CV for Cmax and AUC0-10hr) compared to generic abiraterone acetate tablets. This is because ASDs

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entail more consistent and complete dissolution of drugs [26]. Overall, pharmacokinetics of abiraterone were drastically improved by HPBCD-based KSDs.

Figure 2.9. In vivo plasma concentration versus time profiles from oral dosing of the generic abiraterone acetate tablet, the Lot 2 Tablet, the Lot 5 Tablet, and the Lot 6 Tablet in fasted non-naïve male beagle dogs.

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Table 2.5. Results from the in vivo pharmacokinetic (PK) study in male beagle dogs.

Generic abiraterone Lot 2 Tablet Lot 5 Tablet Lot 6 Tablet acetate tablet (44.6 mg (44.6 mg (44.6 mg (250 mg abiraterone) abiraterone) abiraterone) abiraterone acetate) PK Units Average %CV Average %CV Average %CV Average %CV parameters

Cmax ng/mL 86.32 66.57% 53.58 58.84% 280.00 33.51% 305.00 20.72%

Tmax hr 1.20 37.27% 1.20 55.90% 0.80 34.23% 0.70 39.12%

T1/2 hr 4.78 50.70% 3.12 42.20% 2.30 32.30% 3.47 28.90%

AUC0-10hr ng*hr/mL 177.29 77.53% 122.21 25.64% 438.01 34.29% 487.58 14.11%

F Value (Dose unitless 1.0 3.4 12.4 13.8 Adjusted)

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6. CONCLUSIONS:

From this study, we found that KinetiSol technology enabled the development of abiraterone ASDs that can enhance the dissolution and pharmacokinetics of abiraterone. Also, this study demonstrates for the first time that KinetiSol technology can process both long-chain linear polymers and short-chain cyclic oligomers with low molecular weights. Overall, HPBCD-based KSDs improved the dissolution and pharmacokinetics of abiraterone. These KSDs have a potential to eliminate food effects, enhance abiraterone efficacy, reverse resistance to abiraterone in prostate cancer patients. These formulations should be explored further to investigate their impact on therapeutic outcomes.

7. ACKNOWLEDGEMENTS AND DISCLOSURE:

Gala acknowledges and thanks Charles River Laboratories, Massachusetts for their support with the animal study. Williams acknowledges financial support for Gala from DisperSol Technologies, LLC.

Parts of this manuscript were presented in a symposium at the Annual Meeting of the American Association of Pharmaceutical Scientists (AAPS), 2017, San Diego and in a poster at AAPS PharmSci 360 Meeting, 2018, Washington D.C. Gala and Miller are coinventors on intellectual property related to this work.

8. REFERENCES:

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Chapter Three: The Effect Of Drug Loading On The Properties Of KinetiSol® Processed Abiraterone–Hydroxypropyl β Cyclodextrin Solid Dispersions

1. ABSTRACT:

Abiraterone is a poorly water-soluble drug used in the treatment of prostate cancer. In our previous study, we reported that KinetiSol® processed solid dispersions (KSDs) based on oligomer hydroxypropyl β cyclodextrin (HPBCD) improved the dissolution and pharmacokinetics of abiraterone. However, we have not previously reported the effect of drug loading on the physicochemical properties and in vivo performance of HPBCD- based KSDs. We hypothesize that increasing the drug loading beyond an optimal point reduces the in vitro and in vivo performance of KSDs. To confirm our hypothesis, we developed KSDs with 10–50% w/w drug loading and analyzed these KSDs using X-ray diffractometry and modulated scanning calorimetry. We found that KSDs containing 10– 30% drug loading were amorphous. Solid state interaction studies using nuclear magnetic resonance spectroscopy and Raman spectroscopy indicated that the maximum abiraterone–HPBCD interaction occurred within the 10% drug-loaded KSD. This is likely because the molar ratio of abiraterone:HPBCD was 1:2 in this KSD. The solution state phase solubility profile for abiraterone–HPBCD was A-type, meaning that the amount of solubilized abiraterone was directly proportional to the concentration of HPBCD. At elevated temperatures, the 10% and 20% drug-loaded KSDs were chemically stable, while the 30% drug-loaded KSD showed recrystallization of abiraterone. As the drug loading increased in the KSD, the in vitro dissolution performance and the in vivo pharmacokinetic performance decreased. This can be attributed to reduced abiraterone– HPBCD interaction with increased drug loading. Overall, the 10% drug-loaded KSD showed a dissolution enhancement of 15.7-fold compared to neat abiraterone API, and it showed a bioavailability enhancement of 3.9-fold compared to the commercial abiraterone acetate tablet Zytiga®. Thus, this study confirms for the first time that KinetiSol, a high-energy, solvent-free technology, forms an optimally performing 10%

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abiraterone–HPBCD complex in KSDs in terms of improved in vitro and in vivo performance.

2. GRAPHICAL ABSTRACT:

3. INTRODUCTION:

Abiraterone (Figure 3.1) is a poorly water-soluble drug used in the treatment of prostate cancer [1]. Currently, it is used in the form of an acetate prodrug which has slightly higher solubility than abiraterone in biorelevant media [2]. The prodrug is available in the tablet dosage form Zytiga®, which contains abiraterone acetate in crystalline form. This dosage form exhibits poor oral bioavailability of less than 10%, a high food effect, and high pharmacokinetic variability [3, 4]. Another tablet dosage form, Yonsa®, was approved in 2018. It contains abiraterone acetate in fine particulate form, and it exhibits a modest bioavailability improvement of only two fold in humans [5, 6]. Thus, there is a need for an improved abiraterone formulation that significantly enhances abiraterone’s bioavailability and improves its pharmacokinetics. Additionally, it has been shown that an increase in abiraterone exposure can lead to slower disease progression [7-9]. Hence, an improved abiraterone formulation with high oral bioavailability could ultimately improve therapeutic outcomes.

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Cyclodextrins (CDs) are cyclic oligomers containing at least six D-(+)-glucopyranose units attached by α(1→4) glycosidic bonds [10, 11]. The glucopyranose units in CDs are present in chair conformation, thereby giving CDs a truncated cone-like structure [11]. The outer surface of CDs has secondary hydroxyl groups that extend from the wider edge and primary hydroxyl groups that extend from the narrow edge of the cone, which imparts a hydrophilic nature to the outer surface [12]. On the other hand, the inner cavity of CDs contains skeletal carbons with hydrogen atoms and oxygen bridges, which imparts a lipophilic nature to the inner cavity [12].

The most common natural CDs are α-CD, β-CD, and γ-CD, which contain six, seven, and eight glucopyranose units, respectively [11]. The random substitution of the hydroxy groups of CDs with hydrophobic or hydrophilic groups imparts higher aqueous solubility to the CDs by interrupting their intermolecular hydrogen bonding. For example, the hydroxypropyl substitution of β-CD to form hydroxy propyl β cyclodextrin (HPBCD) (Figure 3.1) increases its water solubility from 18.5 mg/ml to > 600 mg/ml, respectively [11]. CDs can form inclusion complexes with the entire drug or a part of the drug by including the drug in its lipophilic central cavity through noncovalent interactions [13]. By forming partial or complete inclusion complexes with drugs, CDs can impart higher aqueous solubility to the drug and enhance its oral bioavailability [13]. Hence, CDs are used as carriers in the solid dispersion development of poorly water-soluble drugs [14, 15].

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Figure 3.1. Structure of abiraterone and hydroxypropyl β cyclodextrin.

KinetiSol® is a novel, high-energy, solvent-free, fusion-based, thermokinetic method for manufacturing solid dispersions of poorly water-soluble drugs [16]. In our prior study, we enabled the development of abiraterone solid dispersions using KinetiSol technology [17]. We reported that the oligomer HPBCD-based KinetiSol processed solid dispersions (KSDs) outperformed polymer-based KSDs in terms of dissolution enhancement, bioavailability enhancement, and improvement in the pharmacokinetics of abiraterone [17]. Based on a risk–benefit analysis, we selected the binary KSD containing 10% w/w abiraterone and 90% w/w HPBCD as the lead KSD because (a) it was similar to the ternary KSD (containing 10% w/w abiraterone, 80% w/w HPBCD, and 10% w/w hypromellose acetate succinate (HPMCAS 126G)) in terms of in vivo pharmacokinetic performance in male beagle dogs and (b) it reduces the risk of acid-catalyzed abiraterone degradation [17].

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Drug loading, or the amount of drug incorporated in a solid dispersion, is a critical parameter that affects the processability, physicochemical properties, stability, and the in vitro and in vivo performance of solid dispersions as well as the strength of the final dosage form [18-20]. In general, for solid dispersions, when the drug loading is less than the equilibrium solubility of the crystalline drug in the polymer carrier, the system is thermodynamically stable and the drug is molecularly dispersed in the polymer carrier matrix, forming a homogenous system [20]. Such a system is considered an ideal solid dispersion; however, it may not be practical because this would occur at extremely low drug loadings for most drug–polymer carrier systems [20]. When the drug loading is high in solid dispersions (especially when the drug loading is higher than the equilibrium solubility of the amorphous drug in the polymer carrier), such systems are highly unstable and can lead to spontaneous phase separation and crystallization, thereby negatively affecting the stability and performance of solid dispersions [18].

Usually, less drug can be loaded when polymers or oligomers with low molecular weights are used as carriers. For instance, Moya-Ortega et al. found that less drug could be loaded into oligomer γ-CD compared to a polymer polyethylene-glycol-γ-CD [21]. Moreover, when oligomers such as CDs are carriers for solid dispersions, it cannot not be assumed that low drug loading is necessarily beneficial. This is because low drug loading would require more CD, which can hamper drug absorption in the gastrointestinal tract and lead to lower bioavailability [22, 23]. In addition, the problem of high drug loading discussed above also applies to drug–CD solid dispersions. Thus, the identification of optimal drug loading becomes even more critical when the carrier is an oligomer such as HPBCD.

We hypothesize that increasing the drug loading beyond an optimal point reduces the in vitro and in vivo performance of KSDs. To the best of our knowledge, the literature has not reported on the impact of drug loading (in drug–CD compositions prepared using a high-energy, solvent-free method) on several aspects of formulation development ranging from the processing of the formulation to its performance in an animal model. 135

The objective of our current study is to investigate the effect of drug loading on the processability, physicochemical properties, solid-state interactions, stability, in vitro dissolution, and in vivo pharmacokinetic performance of abiraterone:HPBCD KSDs, with the aim of maximizing abiraterone bioavailability and ultimately improving its therapeutic efficacy.

4. MATERIALS AND METHODS:

4.1. Materials:

Abiraterone active pharmaceutical ingredient (API) was purchased from Attix Pharma (Ontario, Canada). Hydroxypropyl β cyclodextrin (i.e., Kleptose® HPB) was purchased from Roquette America (USA). Microcrystalline cellulose (i.e., Avicel PH-102) was purchased from the FMC Corporation (Pennsylvania, USA). Mannitol (i.e., Pearlitol 200SD) was purchased from Roquette America (USA). Cross-linked sodium carboxy methyl cellulose (i.e., Vivasol®) was purchased from JRS Pharma (New York, USA). Hypromellose acetate succinate HMP-grade (i.e., AQOAT®) was purchased from Shin- Etsu (New Jersey, USA). Colloidal silicon dioxide (i.e., Aerosil® 200 P) was purchased from Evonik Industries (New Jersey, USA). Magnesium stearate was purchased from Peter Greven (Muenstereifel, Germany). The fasted-state simulated intestinal fluid (FaSSIF) dissolution media was prepared using FaSSIF/FeSSIF/FaSSGF powder purchased from Biorelevant.com (Surrey, UK). Abiraterone acetate tablets (Zytiga®, 250 mg abiraterone acetate) were purchased from Myoderm (Pennsylvania, USA). These tablets were manufactured for Janssen Biotech (Pennsylvania, USA). The solvents used for HPLC analysis were HPLC grade. All other chemicals and reagents used for the dissolution and HPLC analysis were ACS grade.

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4.2. Methods:

4.2.1. KinetiSol® Processing:

Abiraterone KSDs with different drug loadings were prepared using KinetiSol technology. Initially, all KSDs were prepared using a research-scale compounder (formulator) designed and manufactured by DisperSol Technologies LLC (Texas, USA). Later, amorphous KSDs were prepared using a manufacturing-scale compounder (manufacturing compounder) designed and manufactured by DisperSol Technologies LLC (Texas, USA).

Before compounding, the drug abiraterone and the oligomer HPBCD (Table 3.1) were accurately weighed and thoroughly mixed to prepare physical mixtures (PMs). These physical mixtures were charged into the KinetiSol compounder chamber. Inside the chamber, a shaft with protruding blades was rotated at varying incremental speeds ranging from 500 rpm to 7,000 rpm, without external heat addition, to impart frictional and shear forces to the sample material. The temperature of the mass was monitored using an infrared probe. When the molten mass temperature reached 150–180 °C, the mass was rapidly ejected, collected, and pressed between two stainless steel plates to rapidly quench the sample.

4.2.2.Milling:

The quenched mass obtained after KinetiSol processing was milled in small batches using a lab-scale rotor mill (i.e., IKA tube mill 100 (IKA Works GmbH & Co. KG, Staufen, Germany)). For milling, the fragments of quenched mass were loaded into a 20 mL grinding chamber operated with a grinding speed between 10,000 and 20,000 rpm for 60 s. The milled material was subsequently passed through a #60 mesh screen (≤ 250 μm). The material retained above the screen (i.e., > 250 μm) was cycled through the mill with the same parameters, and this process of milling and sieving was repeated until all

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material passed through the screen. The resultant material (< 250 μm) was labeled as a KSD.

4.2.3.Melt-quenching Abiraterone:

Abiraterone was melt-quenched to provide a neat, amorphous abiraterone reference sample for Raman spectroscopy. A small quantity of abiraterone (< 0.5 g) was added to an open scintillation vial and blowtorched for a few seconds until the entire quantity of abiraterone melted. The scintillation vial containing the molten mass was immediately submerged in liquid nitrogen. When the intensity of the nitrogen boiling subsided, the vial was transferred to a vacuum desiccator, and a vacuum was applied for about 2 h. The vacuum was released after 2 h, and the vial was removed from the desiccator. The quenched abiraterone mass was scraped from the vial, lightly grounded using a mortar and pestle, and sieved using a #60 mesh screen (< 250 μm). The melt-quenched abiraterone was placed in a freezer until required for further use.

4.2.4.X-Ray Powder Diffraction:

X-ray powder diffraction (XRPD) analysis was conducted using a Rigaku MiniFlex600 II (Rigaku Americas, Texas, USA) instrument equipped with a Cu-Kα radiation source generated at 40 kV and 15 mA. The API, KSDs, and the melt-quenched abiraterone samples were loaded into an aluminum pan, leveled using a glass slide, and analyzed in the 2-theta range between 2.5° and 35.0° while being spun. The step size was 0.02°, and the scanning rate was set to 5.0°/min. The following additional instrument settings were used: Slit condition: variable + fixed slit system; soller (incident): 5.0°; IHS: 10.0 mm; DS: 0.625°; SS: 8.0 mm; soller (receiving.): 5.0°; RS: 13.0 mm (Open); and monochromatization: kb filter (× 2). The data were collected using Miniflex Guidance software (Rigaku Corporation, Tokyo, Japan) and processed using PDXL2 software (Rigaku Corporation, Tokyo, Japan).

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4.2.5.Modulated Differential Scanning Calorimetry:

Thermal analysis was conducted with modulated differential scanning calorimetry (mDSC) using a differential scanning calorimeter model Q20 (TA Instruments, Delaware, USA) equipped with a refrigeration-based cooling system and an autosampler. The API and KSD samples were prepared by weighing 5–10 mg of the material and loading it into a Tzero pan. The pan was sealed with a Tzero lid using a Tzero press. Following the sample equilibration at 30 °C for 5 min, the temperature was ramped to 250 °C at 5 °C/min with a modulation of ±1 °C every 60 s. Nitrogen was used as the sample purge gas at a flow rate of 50 mL/min. The data were collected using TA Instrument Explorer software (TA Instruments, Delaware, USA) and processed using Universal Analysis software (TA Instruments, Delaware, USA).

4.2.6.HPLC Analysis:

A stability-indicating, high-performance liquid chromatography (HPLC) method was developed for the chemical analysis of abiraterone KSDs. A Dionex Ultimate 3000 HPLC system (ThermoFisher Scientific, Massachusetts, USA) was used for reverse phase HPLC analysis. The HPLC column was a Kinetex® XB C18, 150 mm x 4.6 mm and 2.6 µm (Phenomenex, California, USA). Mobile phase A was a 20 mM ammonium formate buffer (pH 3), and mobile phase B was degassed acetonitrile. We designed an initial gradient profile with a higher amount of aqueous phase followed by a gradual increase in the organic phase. The flow rate was 0.9 mL/min, and the run time was 42 min. The column was held at 35 °C, and the data were collected at a single wavelength of 254 nm. Samples were prepared at a nominal concentration of 0.5 mg/mL with 7:2:1 methanol: isopropyl alcohol: tetrahydrofuran as the standard/sample diluent. All samples were filtered through 0.45 μm PVDF syringe filters (GE Healthcare Life-Sciences, Pennsylvania, USA), before analysis. Chromatography samples were analyzed using Chromeleon™ software, version 7.0 (ThermoFisher Scientific, Massachusetts, USA).

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4.2.7. Solid-state Nuclear Magnetic Resonance Spectroscopy:

One-dimensional (1D) 13C solid-state nuclear magnetic resonance spectroscopy (ssNMR) was conducted in the NMR Lab at the University of Texas at Austin (Texas, USA). The 13C ssNMR spectra were collected using a Bruker AVANCE™ III HD 400 MHz instrument (Bruker Corporation, Massachusetts, USA). A cross-polarization experiment was conducted using 4 mm MAS probe, and the 13C frequency employed was 100.62 MHz. The contact time was set to 2 ms, the spin rate was set to 10 KHz, and the relaxation delay ranged from 2–30 s. The temperature was set to 300.0 K. The chemical shift reference of standard adamantane 38.48 ppm was used. The data were collected using Bruker NMR software (Bruker Corporation, Massachusetts, USA) and processed using MNOVA software, version 14 (Santiago de Compostela, Spain).

Two-dimensional (2D) 13C–1H heteronuclear correlation (HETCOR) spectra were acquired using a Bruker AVANCE™ III HD 400 triple-resonance spectrometer operating at a 1H frequency of 400.13 MHz in the Biopharmaceutical NMR Laboratory (BNL) of Preclinical Development at Merck Research Laboratories (Merck & Co., Inc., West Point, PA). We utilized an experimental temperature of 298 K and a MAS frequency of 12 kHz. All data were processed using Bruker TopSpin software. The 13C–1H HETCOR experiments were conducted using a CP contact time of 2 ms and a recycle delay of 2 s.

4.2.8.Raman Spectroscopy:

Raman spectroscopy was conducted using a HyperFlux™ PRO Plus (HFPP) Raman spectroscopy system (Tornado Spectral Systems, Ontario, Canada). The API, PM, KSDs, and melt-quenched abiraterone samples were loaded onto an aluminum stage. The samples were subjected to a laser beam with a wavelength of 785 nm and power of 200 mW. We collected 50 exposures per spectrum, and three spectra were collected per sample. An exposure time of 100 ms was employed. We enabled cosmic ray removal and dark spectral correction. The spectral data were collected using SpectralSoft software (Tornado Spectral Systems, Ontario, Canada). The spectral data pre-processing and 140

multivariate analysis was conducted using Unscrambler X software (Camo Analytics, Oslo, Norway).

4.2.9.Phase Solubility Analysis:

A phase solubility analysis was conducted in two separate media: 0.01N HCl (pH 2.0) and FaSSIF, prepared in a 50 mMol phosphate buffer (pH 6.8). Solutions of HPBCD ranging from 0–600 mg/mL were prepared in each medium in scintillation vials. An excess of abiraterone was added to each vial, and the vials were sonicated for 30 min and placed on a bench. Samples were drawn from each vial at time points of 48 h and 7 days. The samples were centrifuged using an ultracentrifuge (Eppendrof, Hamburg, Germany). The supernatants were further diluted using the HPLC diluent and analyzed using the HPLC method mentioned above to find the concentration of abiraterone.

4.2.10. Stability Analysis:

Stability analysis was performed at elevated temperatures. KSD samples were loaded into a scintillation vial and heated for 6 h on a hot plate set at 90°C. The samples were then analyzed using XRPD as stated above. Upon XRPD analysis, the samples were reheated on a hot plate set at 150 °C for 6 h, and the samples were reanalyzed using XRPD.

4.2.11. In Vitro Dissolution Study:

An in vitro, non-sink, gastric transfer dissolution method was developed to analyze the dissolution of the abiraterone API and KSDs. For dissolution analysis, samples equivalent to 44.6 mg of abiraterone API were loaded in an Erlenmeyer flask (dissolution vessel) containing 50 mL 0.01N HCl (pH 2.0), placed in an Excella E24 incubator-shaker (New Brunswick Scientific, New Jersey, USA) set to 37 °C and a rotational speed of 180 rpm.

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After 30 min, 50 mL of FaSSIF (prepared in 50 mMol phosphate buffer (pH 6.8)) was added to the dissolution vessel. At predetermined time points, samples were drawn from the dissolution vessel and centrifuged using an ultracentrifuge (Eppendorf, Hamburg, Germany). The supernatants were further diluted using the HPLC diluent and analyzed using the HPLC method mentioned above. The area under drug dissolution curve (AUDC) was calculated using the linear trapezoidal method.

4.2.12. Tableting:

The KSD and tableting excipients (Avicel PH-102, Pearlitol 200SD, Vivasol, Kleptose, AQOAT, Aerosil 200 P, and magnesium stearate) were accurately weighed and dispensed. Aerosil 200 P was sieved through #40 mesh (420 µm) until all material passed through the sieve. The KSD and all tableting excipients except magnesium stearate were loaded in a vial and mixed using a vortex mixer (Thermo Scientific, Massachusetts, USA). Magnesium stearate was then added to the vial and blended using a spatula. The resultant tableting blend was then dispensed in aliquots equivalent to 50 mg abiraterone. Each aliquot was loaded in the tablet die and compressed using a single-station hand tablet press (BVA Hydraulics, Missouri, USA) with a target hardness of 8–12 kP.

4.2.13. In Vivo Pharmacokinetic Study in Beagle Dogs:

An in vivo pharmacokinetic study in fasted, non-naïve, male beagle dogs was conducted at Pharamaron (Ningbo, China). The animal study was conducted according to an approved Pharmaron IACUC protocol #PK-D-06012018. We analyzed the 50.0 mg equivalent abiraterone tablets along with a 250 mg equivalent abiraterone acetate tablet (Zytiga). Each study arm for each formulation consisted of three dogs.

The dogs were fasted overnight before dosing, and the food was returned 4 h post dosing. Each dog was administered a single tablet of the respective formulations (per the study arm) along with a post-dose flush of 40 mL sterile water. At predefined time points of 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 18, 24, 36, and 48 h post dose, 1 mL blood 142

samples were drawn from each dog using venipuncture of a peripheral vessel. These samples were placed in tubes containing sodium heparin anticoagulant then centrifuged to isolate the plasma. The plasma samples were analyzed for abiraterone content using liquid chromatography with tandem mass spectrometry (LC-MS/MS).

4.2.14. Pharmacokinetic Analysis:

Pharmacokinetic parameters were estimated using Phoenix™ WinNonlin software, version 6.1 (Certara, New Jersey, USA) using a non-compartmental approach consistent with the oral route of administration. The area under the plasma concentration–time curve (AUC) was calculated using the linear trapezoidal method. The relative bioavailability (i.e., F value) was calculated using the following formula:

퐴푈퐶(0−48hr)(test abiraterone tablet) × 퐷표푠푒(abiraterone )(ref. abiraterone acetate tablet) 퐹 = 퐴푈퐶(0−48hr)( ref. abiraterone acetate tablet) × 퐷표푠푒(abiraterone ) (test abiraterone tablet)

5. RESULTS:

5.1.Development of KSDs:

Table 3.1 lists the KSDs composition and processing parameters. The Lots 1 to 5 PMs were all processible using KinetiSol technology. Initially, all lots were processed on a research-scale compounder. Later, to yield more material for further testing, the Lots 1 to 3 KSDs were processed using a manufacturing-scale compounder. The total processing time for the Lots 1 to 4 KSDs was less than 20 s, while the Lot 5 KSD had a processing time of 41.5 s. We observed a considerable amount of material sticking for the Lot 4 and Lot 5 KSDs. All lots were easily milled and sieved using #60 mesh to yield KSD powders with a particle size of < 250µm.

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Table 3.1. KSD composition and processing parameters.

Shear Composition Batch Processing Stress Total Lot KinetiSol® Size Temperature (Rotational Processing No. Drug (% Compounder Oligomer (g) (°C) speed- Time (s) wt) (% wt) rpm) Abiraterone HPBCD Manufacturing 2,400, 1 90 150 16 (10) (90) Compounder 2,700

Abiraterone HPBCD Manufacturing 2,400, 2 90 180 18 (20) (80) Compounder 2,700

Abiraterone HPBCD Manufacturing 500, 2,700, 3 90 180 16.5 (30) (70) Compounder 3,000 Abiraterone HPBCD 1,000, 4 Formulator 10 160 19.3 (40) (60) 6,000 1,000, Abiraterone HPBCD 5 Formulator 10 160 6,000, 41.5 (50) (50) 7,000

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5.2.Physicochemical analysis of KSDs:

Figure 3.2 illustrates the X-ray diffractograms of neat abiraterone API, melt-quenched abiraterone API, HPBCD, Lot 1 PM, and Lots 1 to 5 KSDs. The X-ray diffractogram of neat abiraterone API showed sharp characteristic diffraction peaks at 8.43°, 16.54°, and 19.31°. The melt-quenched abiraterone API X-ray diffractogram showed no sharp diffraction peaks and displayed a halo pattern. The X-ray diffractogram of HPBCD also displayed a halo pattern. The X-ray diffractogram of the Lot 1 PM showed sharp diffraction peaks at 8.70°, 16.72°, and 19.55°, corresponding to the characteristic peaks of neat abiraterone API. Additionally, Lot 1 PM displayed peaks at 12.08°, 15.51°, 17.29°, 21.48°, and 23.98°. These positions differ slightly from the peaks observed in the neat abiraterone API diffractogram. The X-ray diffractograms of the Lots 1 to 3 KSDs showed a complete halo pattern and no diffraction peaks. The X-ray diffractograms of the Lot 4 and Lot 5 KSDs showed sharp diffraction peaks at 8.52°, 16.54°, and 19.60°, corresponding to the characteristic peaks of neat abiraterone API. Additionally, the Lot 4 and Lot 5 KSDs displayed certain smaller diffraction peaks that were also observed in the neat abiraterone API diffractogram.

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Figure 3.2. X-ray diffractograms of (Top) neat abiraterone API, melt-quenched abiraterone API and HPBCD; (Bottom) for the Lot 1 PM and the Lots 1 to 5 KSDs. The dotted circles in the top figure indicate the characteristic peaks of abiraterone API. The dotted lines in the bottom figure indicate the peak position regions of these characteristic peaks. 146

The mDSC thermograms of the Lot 1 PM and the Lots 1 to 5 KSDs are illustrated in Figure 3.3. The Lot 1 PM showed a sharp melting endotherm at 227.25 °C. The Lots 2 and 3 KSDs showed no significant thermal events at evaluated mDSC run conditions. The Lot 3 KSD showed a small thermal event at 216.20 °C and a melting endotherm at 224.72 °C. The Lot 4 KSD showed a broad melting endotherm at 219.99 °C. Similarly, the Lot 5 KSD showed a melting endotherm at 223.62 °C.

Figure 3.3. mDSC thermogram of the Lot 1 PM and the Lots 1 to 5 KSDs.

The HPLC analysis shows that the Lots 1 to 3 KSDs had total impurities of 0.28%, 0.37%, and 0.38%, respectively. None of these lots had an individual unknown impurity of ≥ 0.2%.

5.3.Solid-state interaction between abiraterone and HPBCD in KSDs:

Figure 3.4 illustrates the 13C ssNMR spectra of neat abiraterone API, HPBCD, the Lot 1 PM and the Lots 1 to 5 KSDs. The 1D 13C ssNMR spectrum of neat abiraterone API

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showed 21 sharp signals: 11 signals between 20 and 60 ppm, one signal at about 72 ppm, and nine signals between 120 and 160 ppm. HPBCD showed six broad signals at about 19 ppm, 61 ppm, 67 ppm, 73 ppm, 82 ppm, and 102 ppm. The Lot 1 PM showed a mix of sharp and broad signals, which corresponds to both neat abiraterone API and HPBCD. The Lots 1 to 3 KSDs showed major signals corresponding to HPBCD. They showed some broad signals between 20 and 60 ppm, corresponding to neat abiraterone API; however, the signals between 120 and 160 ppm, which correspond to neat abiraterone API, were absent or extremely broadened in the 13C ssNMR spectra of the Lots 1 to 3 KSDs. The Lot 4 and Lot 5 KSDs showed a mix of sharp and broad signals, corresponding to both neat abiraterone API and HPBCD.

Figure 3.5 illustrates 2D 13C–1H HETCOR spectra of neat abiraterone API, HPBCD, and the Lot 3 KSD. 1H cross sections at 103.9 ppm in the 13C dimension are shown in the 2D spectra.

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Figure 3.4. 13C ssNMR spectra of neat abiraterone API, HPBCD, the Lot 1 PM, and the Lots 1 to 5 KSDs. The dotted rectangles indicate the regions of sp3 hybridized carbon atoms, the C3 carbon atom, and sp2 hybridized carbon atoms of neat abiraterone API.

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Figure 3.5. 2D 13C–1H HETCOR spectra of neat abiraterone API (black), HPBCD (red), and the Lot 3 KSD (blue). 1H cross sections at 103.9 ppm in the 13C dimension are shown in the 2D spectra.

The raw Raman spectra of melt-quenched abiraterone API and KSDs showed interference due to fluorescence. Hence, spectral preprocessing was conducted by converting the raw spectra into second derivatives using a second-degree polynomial and 150

a Savitsky–Golay 31-point smoothing filter. The second derivative spectra were then scatter-corrected by applying a standard normal variate transformation. Table 3.2 lists the Raman peak positions for neat abiraterone API, melt-quenched abiraterone API, the Lot 1 PM, and the Lots 1 to 5 KSDs. Table 3.3 lists Raman peak shifts for melt-quenched abiraterone API, the Lot 1 PM, and the Lots 1 to 5 KSDs. Specifically, the table lists the Raman peak positions corresponding to pyridine ring vibrations, steroid moiety vibrations, and C=C vibrations in the B and D ring of abiraterone (see Figure 3.1). For the melt-quenched API, the peak shifts with respect to neat abiraterone API are listed. For the Lot 1 PM and the Lots 4 and 5 KSDs, peak shifts with respect to neat abiraterone API are calculated, since they all primarily contain abiraterone in crystalline form (Figure 3.2). For the Lots 1 to 3 KSDs, the peak shifts with respect to melt-quenched abiraterone API are calculated, since they all primarily contain abiraterone in amorphous form (see Figure 3.2).

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Table 3.2. Raman peak positions for neat abiraterone API, melt-quenched abiraterone API, Lot 1 PM, and Lots 1 to 5 KSDs.

melt- neat Lot Lot Lot Lot Lot Lot quenched Abiraterone 1 1 2 3 4 5 Theoretical Abiraterone API PM KSD KSD KSD KSD KSD assignments API (a) (b) (c) (d) (e) (f) (g) (h) Peak Positions [Wavenumber (cm-1)]

Pyridine ring and steroid moiety 1024 1025 1024 1028 1027 1027 1025 1025 vibrations

Whole abiraterone 1049 1048 1049 1050 1050 1050 1049 1049 molecule vibrations

C=C (D-ring) + 1584 1586 1586 1586

pyridine 1593 1599 1594 1603 1602 1601 1594 1594

C=C (B-ring) 1663 1667 1663 1671 1669 1668 1665 1665

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Table 3.3. Raman peak shifts for melt-quenched abiraterone API, Lot 1 PM, and Lots 1 to 5 KSDs.

melt- Lot Lot Lot Lot Lot Lot quenched 1 1 2 3 4 5 Theoretical Abiraterone PM KSD KSD KSD KSD KSD assignments API (b-a) (c-a) (d-b) (e-b) (f-b) (g-a) (h-a) Peak Shifts [Wavenumber (cm-1)] Pyridine ring and steroid 1 0 3 2 2 1 1 moiety vibrations Whole abiraterone -1 0 2 2 2 0 0 molecule vibrations C=C (D-ring) 2 2 2 + pyridine 6 1 4 3 2 1 1 C=C (B-ring) 4 0 4 2 1 2 2

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5.4.Solution-state phase solubility profile:

Figure 3.6 shows the phase solubility profiles for abiraterone–HPBCD in 0.01N HCl and FaSSIF. In both phases (i.e., 0.01N HCl and FaSSIF), as the concentration of HPBCD increased, the solubility of abiraterone also increased. The solubility of abiraterone in 0.01N HCl was much higher than its solubility in FaSSIF, specifically in the HPBCD concentration range of 71.48–428.88 µM/mL.

Figure 3.6. Phase solubility profiles for abiraterone–HPBCD in 0.01N HCl and FaSSIF.

5.5.Stability of KSDs:

Figure 3.7 displays X-ray diffractograms of the Lots 1 to 3 KSDs at 90 °C and 150 °C. At both 90 °C and 150 °C, the Lot 1 and 2 KSDs showed a complete halo pattern with no sharp diffraction peaks that correspond to neat abiraterone API (Figure 3.2). The Lot 3 KSD showed sharp diffraction peaks corresponding to neat abiraterone API (Figure 3.2) at both 90 °C and 150 °C. Specifically, the Lot 3 KSD showed characteristic abiraterone peaks at 16.65° and 19.66°, but the peak at 8.43° was absent.

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Figure 3.7. X-ray diffractograms of Lots 1 to 3 KSDs at 90 °C and 150 °C. The dotted lines indicate the peak position region of abiraterone characteristic peaks.

5.6.In Vitro and In Vivo Performance of KSDs:

Figure 3.8 illustrates the in vitro, non-sink, gastric transfer dissolution profiles of neat abiraterone API and the Lots 1 to 5 KSDs. The Lots 1 to 5 KSDs showed abiraterone dissolution enhancement of 15.7-fold, 12.1-fold, 7.2-fold, 5.0-fold, and 3.1-fold, respectively, compared to neat abiraterone API. All KSD lots showed higher abiraterone dissolution in 0.01N HCl compared to FaSSIF. Among the KSDs, the abiraterone dissolution enhancement trend was: Lot 1 KSD > Lot 2 KSD > Lot 3 KSD > Lot 4 KSD > Lot 5 KSD. After integrating the total area under the drug dissolution curve (AUDC

Total ), the relative AUDC Total for the Lots 2 to 5 KSDs was 76.9%, 45.6%, 32.0%, and 19.5%, respectively, compared to the Lot 1 KSD.

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Figure 3.8. In vitro, non-sink, gastric transfer dissolution profiles of neat abiraterone API and Lots 1 to 5 KSDs. The red region indicates 0.01N HCl, and the blue region indicates FaSSIF.

In order to develop a viable dosage form for abiraterone delivery, the KSDs were compressed into immediate-release tablet formulations. Three tablet formulations containing a 10% drug loaded KSD (Lot 1 KSD), a 20% drug loaded KSD (Lot 2 KSD), and a 30% drug loaded KSD (Lot 3 KSD) were compressed into the Lot 1 Tablet, the Lot 2 Tablet, and the Lot 3 Tablet, respectively, with each tablet containing 50.0 mg abiraterone. All tablets had optimum hardness, assay, purity, acceptable friability, disintegration time, and dissolution. These tablets were tested and compared against the Zytiga tablet containing 250 mg abiraterone acetate.

Figure 3.9 shows in vivo average plasma concentration versus the time profiles from the oral dosing of Zytiga and the Lots 1 to 3 Tablets in fasted, non-naïve, male beagle dogs. Table 3.4 lists the pharmacokinetic (PK) parameters. Zytiga showed extremely variable plasma concentrations versus the time profiles between all three animals. One of the animals (#JA0167) in the Zytiga study arm (individual animal data not shown) showed a 156

maximum abiraterone plasma concentration (i.e., a Cmax of 78.50 ng/mL at 1 h and another Cmax of 115.00 ng/mL at 10 h), which contributed to higher drug exposure (i.e.,

AUC(0–48 h) for Zytiga). The Lots 1 to 3 tablets showed lower drug exposure variability

(i.e., lower %CV for AUC(0–48 h) compared to Zytiga. Zytiga showed an extremely high variability of 121.39% for Tmax. Overall, the Lots 1 to 3 tablets were able to enhance the bioavailability of abiraterone by 3.9-fold, 2.7-fold, and 1.7-fold, respectively, compared to Zytiga.

Figure 3.9. In vivo average plasma concentration vs. time profiles from oral dosing of Zytiga® and the Lots 1 to 3 Tablets in fasted, non-naïve, male beagle dogs.

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Table 3.4. Results from in vivo pharmacokinetic (PK) study in male beagle dogs.

Zytiga® (250 mg Lot 1 Tablet (50 Lot 2 Tablet (50 Lot 3 Tablet (50 abiraterone mg abiraterone) mg abiraterone) mg abiraterone) acetate)

Average %CV Average %CV Average %CV Average %CV

Cmax ng/mL 153.00 28.24 311.67 32.61 221.90 54.72 119.87 33.77

Tmax h 4.17 121.39 0.83 34.64 1.00 0.00 0.83 34.64

AUC(0–48 ng·h/mL 523.78 46.39 451.44 32.36 319.91 42.93 195.90 35.51 hr) F Value (Dose unitless 1.0 3.9 2.7 1.7 Adjusted)

In Figure 3.10, we plot the in vitro and in vivo relative performance percentages of KSDs with various drug loadings. Both in vitro performance (in terms of AUDC Total) and in vivo performance (in terms of AUC(0–48 h)) decreased as the drug loading of the KSDs increased. Interestingly, the relative performance trends for both the in vitro and in vivo studies were similar.

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Figure 3.10. In vitro and in vivo relative performance percentages of KSDs with various drug loadings.

6. DISCUSSION:

6.1.Development of KSDs:

Conventionally, drug–CD pharmaceutical compositions have been prepared using solvents (e.g. organic solvents, carbon dioxide) based technologies such as spray drying, freeze drying, solvent evaporation, or kneading using a solvent and a supercritical fluid process [24-29]. Several disadvantages are associated with these technologies, such as limited scalability, high energy consumption, the required use of toxic organic solvents, challenging solvent removal, and the potential for drug degradation [27].

The use of certain solvent-free technologies such as microwave irradiation, sealed heating, and hot melt extrusion have also been reported for the preparation drug–CD 159

pharmaceutical compositions [30-34]. The major drawback of these technologies is drug degradation due to microwave irradiation or heating [27].

Grinding is another solvent-free method that has been used to prepare drug–CD pharmaceutical compositions [35, 36]. The typical grinding time for drug–CD compositions ranges from several minutes to hours [27]. Subjecting drugs to high physical stress over longer time periods, which is typical of the grinding process, can lead to significant drug degradation [37].

KinetiSol is a thermokinetic, solvent-free technology that does not require the application of external heat, and its typical processing times are in the order of seconds (< 30 s) [16]. Thus, KinetiSol is a promising technology for processing drug–CD pharmaceutical compositions. Using KinetiSol technology, abiraterone–HPBCD KSDs with 10–50% drug loadings were first developed on a research scale compounder (i.e., a formulator). The formulator is suitable for formulation screening and feasibility studies on a small scale, since it can process up to 15 g of material per batch [16]. However, the manufacturing scale compounder, also known as the manufacturing compounder, is suitable for preparing preclinical and early-stage clinical supplies, since it can process up to 350 g of material per batch [16].

Since the Lots 1 to 3 KSDs were to be tableted for animal studies, these lots were produced again using a manufacturing compounder to yield higher KSD quantities. The processing parameters of the Lots 1 to 3 PMs were easily transferred from the formulator to the manufacturing compounder using scaling factors, thereby demonstrating that KinetiSol is a scalable technology. We observed that the 10–40% drug loadings (i.e., the Lots 1 to 4 KSDs) required less total processing time (< 20 s) to reach the target temperature compared to the time required for the 50% drug loaded Lot 5 KSD (41.5 s) (see Table 3.1). This could be the result of more shear stress over a longer duration, which is needed to thermokinetically process abiraterone which has a high melting point and is present in larger quantity.

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The processing temperatures for all five lots were comparable and ranged from 150–180 °C (see Table 3.1). For the Lots 3 to 5 KSDs, a lower rotational speed was initially adopted (i.e., 500 rpm, 1,000 rpm, and 1,000 rpm, respectively), but a higher rotational speed was adopted afterward (i.e., 2,700 rpm, 6,000 rpm, and 6,000 rpm, respectively) to ensure uniform mixing of the drug, since these lots contained higher drug loadings (see Table 3.1). The Lots 4 and 5 KSDs contained more molten abiraterone, which led to material sticking. This issue can be easily solved by the addition of a lubricant such as sodium stearyl fumarate.

Overall, KSDs with drug loadings of 10–50% w/w were processible using KinetiSol technology and were easily quenched, milled, and sieved to yield KSD powders.

6.2.Physicochemical analysis of KSDs:

The X-ray diffractogram (Figure 3.2) of neat abiraterone API showed sharp diffraction peaks, thus indicating its crystalline nature. The characteristic peaks of the neat abiraterone API were similar to 8.38°, 16.46°, and 19.27°. These peaks were reported in the previous study, and this indicates that the same polymorphs were used in both studies, even though they had different sources [17]. However, the neat abiraterone API polymorph used in this study differed from that reported by Solymosi et al. [1].

The melt-quenched abiraterone API showed a halo XRPD pattern (see Figure 3.2), which suggests that abiraterone was converted into its neat amorphous form. It is well known that neat amorphous APIs are highly unstable and have a strong tendency to recrystallize [38]. Thus, in order to prevent recrystallization, melt-quenched abiraterone API was stored in a freezer below 0 °F.

The X-ray diffractogram (Figure 3.2) of HPBCD showed a halo pattern similar to that reported by Gala and Ren et al. This indicates that HPBCD was amorphous in state [26, 39]. Native CDs are crystalline in nature due to their intermolecular hydrogen bonding [11, 26]. However, due to alkyl substitution, their intermolecular hydrogen bonding is 161

disrupted. This gives rise to the amorphous nature of modified CDs such as HPBCD [11, 12].

The Lot 1 PM displayed sharp diffraction peaks (see Figure 3.2) thus indicating its crystalline nature. The slight difference in the XRPD peak positions of the Lot 1 PM compared to neat abiraterone API is due to the contribution of the amorphous halo pattern of HPBCD in the Lot 1 PM. The Lots 1 to 3 KSDs showed a halo XRPD pattern (see Figure 3.2), thus indicating that abiraterone API was converted into its amorphous form in these KSDs. However, sharp diffraction peaks were observed in the X-ray diffractograms (Figure 3.2) of the Lots 4 and 5 KSDs, which indicates that abiraterone API in these KSDs was mostly in crystalline form. Also, the diffraction peaks observed for the Lots 4 and 5 KSDs largely correspond to the peaks of neat abiraterone API, so it can be inferred that KinetSol technology did not change the polymorphic form of abiraterone API in these KSDs.

The melting peak observed in the mDSC thermogram (Figure 3.3) of the Lot 1 PM corresponds to the melting point of abiraterone (around 228 °C) reported in the literature [1, 17]. No melting endotherms that correspond to abiraterone API were observed in the mDSC thermograms of the Lots 2 and 3 KSDs, and this further substantiates the claim that abiraterone API was converted to its amorphous form in these lots.

Interestingly, the two small thermal events observed in the mDSC thermogram (Figure 3.3) of the Lot 3 KSD indicates that some amount of abiraterone in the Lot 3 KSD remains in crystalline form. This, however, conflicts with the XRPD pattern of the Lot 3 KSD. Thus, we analyzed the non-reversible heat flow versus temperature profile (data not shown) of the Lot 3 KSD mDSC thermogram, and we observed two broad recrystallization endotherms before the melting endotherms observed in the reversible heat flow versus temperature profile. This shows that the melting events were largely due to the mDSC run parameters.

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The broad melting endotherms seen in the mDSC thermogram (Figure 3.3) of the Lots 4 and 5 KSDs indicate the melting of crystalline abiraterone API. These melting endotherms also substantiate this observation in their XRPD diffractograms (see Figure 3.2). It should be noted that the melting endotherms observed for the Lots 4 and 5 KSDs occurred at a temperature lower than the melting point of abiraterone. This could result from eutectic phenomena due to the interaction between abiraterone and HPBCD that results from intimate abiraterone–HPBCD mixing and thermokinetic processing by KinetiSol technology [40].

Since the total purity of abiraterone in all the amorphous KSDs was > 99.50%, it can be concluded that abiraterone did not degrade during KinetiSol processing.

Hence, overall, KinetiSol technology was able to render KSDs with drug loadings of 10– 30% w/w to their physically amorphous and chemically stable form.

6.3.Solid-state interaction between abiraterone and HPBCD within KSDs:

The 1D 13C ssNMR spectra (see Figure 3.4) of neat abiraterone API, Lot 1 PM, and Lots 4 and 5 KSDs showed extremely sharp signals, which indicates their crystalline nature. On the other hand, the 13C ssNMR spectra of HPBCD and the Lots 1 to 3 KSDs showed primarily broad signals, which indicates their amorphous nature. This further validates our inferences based on X-ray diffractometry and modulated differential scanning calorimetry.

In order to understand the solid-state interactions within KSDs, we first assigned the 13C ssNMR signals to carbon atoms in abiraterone and HPBCD. In the 13C ssNMR spectrum of neat abiraterone API, the signals from 20–60 ppm can be assigned to sp3 hybridized carbon atoms of abiraterone (see Figure 3.1), which are C1, C2, C4, C6, C8, C9, C10, C11, C12, C13, C14, C15, C23, and C24. The signal at about 72 ppm can be assigned to C3. The signals between 120 ppm and 160 ppm can be assigned to sp2 hybridized carbon atoms of abiraterone (see Figure 3.1), which are C5, C7, C16, C17, C18, C19, C20, C21 163

and C22. In the 13C ssNMR spectrum of HPBCD, the signal assignments are 19 ppm (hydroxypropyl group), 61 ppm (C6), 67ppm (hydroxypropyl group), 73 ppm (C2, C3, C5), 82 ppm (C4), and 102 ppm (C1) of glucopyranose unit in HPBCD (see Figure 3.1). A similar 13C ssNMR spectrum of HPBCD has been reported in literature [41].

Since the 13C ssNMR spectrum of the Lot 1 PM showed additive signals from both neat abiraterone API and HPBCD, it can be inferred that no interaction occurred between abiraterone and HPBCD in the PM.

The absence of abiraterone sp2 hybridized carbon signals between 120 ppm and 160 ppm in the Lot 1 KSD 13C ssNMR spectrum indicates that the B-ring, D-ring, and pyridine ring of all abiraterone, interact with HPBCD and are likely covered or included in the HPBCD cavity. For the Lots 2 and 3 KSDs, these signals have broadened, thus indicating some interaction between HPBCD and the B-ring, D-ring, and pyridine ring of abiraterone. However, unlike the Lot 1 KSD, not all the amorphous abiraterone in these lots interacts with HPBCD. The absence of an abiraterone C3 signal and the broadening of abiraterone sp3 hybridized carbon signals between 20 ppm and 60 ppm in the 13C ssNMR spectra of the Lots 1 to 3 KSDs suggest an interaction between HPBCD and the A-ring and C-ring of abiraterone in these lots.

The 13C ssNMR spectra of the Lots 4 and 5 KSDs show peaks corresponding to both abiraterone and HPBCD with minimal broadening, thus it can be concluded that minimal to no interaction occurs between abiraterone and HPBCD in these KSD lots.

We utilized 2D 13C–1H HETCOR (see Figure 3.5) to further investigate the abiraterone– HPBDC interaction at a higher resolution. Most recently, this 2D heteronuclear correlation spectroscopy has successfully identified the enriched API–polymer interactions in ASDs [42, 43]. For example, various kinds of hydrogen bonding, electrostatic interactions, and hydrophobic interactions have been discovered between posaconazole and different polymers, including HPMCAS and HPMCP (hypromellose

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phthalate) [43]. In one study, higher energy input has been found to enhance the molecular interaction in a ternary ASD [42].

In the current study, Figure 3.5 shows the 2D spectra of crystalline abiraterone (black), HPBCD (red), and the Lot 3 KSD (blue). The enlarged spectra in the 13C regions at 90– 160 ppm include aromatic peaks of abiraterone at 120–150 ppm and a peak of anomeric carbon atoms of HPBCD at approximately 103 ppm [44]. Abiraterone exhibits well- resolved carbon resonances in the crystalline reference (black) and fewer peaks in the KSD due to line broadening and peak overlapping, which agrees well with the analysis of the 1D 13C spectra. The HPBCD reference spectrum (red) shows one broad 1H peak where the protons are bonded to the anomeric carbon atoms. Interestingly in KSD spectrum (blue), the anomeric carbon atoms show a new correlation with a proton peak at approximately 6.4 ppm, which can presumably be assigned to the aromatic protons of abiraterone. This new cross peak suggests an intermolecular interaction between the aromatic region of abiraterone and the anomeric protons of HPBCD.

The peak assignments (see Table 3.2) for the Raman spectrum of neat abiraterone API was conducted based on the Raman characteristic group frequencies reported in literature [45, 46]. The peak shifts (see Table 3.3) for melt-quenched abiraterone API are pronounced due to hydrogen bonding disruption in amorphous abiraterone, thereby changing its chemical environment. No major peak shifts are observed for the Lot 1 PM, which again confirms a lack of interaction between abiraterone and HPBCD in the PM.

Among KSDs, the highest peak shifts are observed for the Lot 1 KSD, which suggests a maximum interaction between abiraterone and HPBCD in the Lot 1 KSD. We focused on the region between 1,535 cm−1 and 1,700 cm−1 because it shows peaks related only to abiraterone API with no interference due to HPBCD. The peak shifts related to C=C vibrations in the B-ring, D-ring, and pyridine ring of abiraterone are of high magnitude in the Lot 1 KSD, suggesting that these rings have interacted with HPBCD and are likely included in its hydrophobic pocket. These peak shifts reduce in magnitude from the Lot 1

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KSD to the Lot 3 KSD (i.e., as drug loading increases). This suggests a reduced interaction between abiraterone and HPBCD in the Lots 2 and 3 KSDs.

For the Lots 4 and 5 KSDs, small peak shifts are observed due only to C=C vibrations in the B-ring, which suggests minimal partial interaction between abiraterone and HPBCD in these KSDs. It can be assumed that little interaction occurs between abiraterone and the outer surface of HPBCD in the KSDs due to the hydrophilic nature of the HPBCD outer surface. However, there remains a possibility of hydrogen bonding between abiraterone -OH and that of HPBCD when abiraterone is not completely included within HPBCD. This non-included abiraterone likely has less apparent aqueous solubility than abiraterone that is included within HPBCD. Gong and Zhu reported that abiraterone acetate and abiraterone can form complexes with β-cyclodextrin [47].

Overall, ssNMR spectroscopy and Raman spectroscopy suggest that (a) all amorphous abiraterone in the Lot 1 KSD is included or complexed within HPBCD, (b) the Lots 2 and 3 KSDs contain amorphous abiraterone that is partially complexed within HPBCD, and (c) the Lots 4 and 5 KSDs contain mostly crystalline abiraterone, which has minimal interaction with HPBCD. The abiraterone:HPBCD molar ratios for the Lots 1 to 5 KSDs are 1.0:2.2, 1.0:1.0, 1.0:0.6, 1.0:0.4, and 1.0:0.2, respectively. Thus, as drug loading increases, the number of molecules of HPBCD available to interact with abiraterone decreases. This leads to a reduced interaction between abiraterone and HPBCD.

The stoichiometry of abiraterone:HPBCD in these KSDs cannot be stated based on these technqiues. However, it appears that the ideal stoichiometry for abiraterone:HPBCD is 1:2. This can be further substantiated based on theoretical dimensions of abiraterone and HPBCD. The inner cavity diameter of HPBCD is 0.62– 0.78 nm. It is partially shielded, and its length is 0.79 nm [48-50]. Abiraterone is considered a pyridyl derivative of pregnenolone, which has a reported length of 13 Å (1.3 nm) [51]. The kinetic diameter of pyridine is 5.7 Å (0.57 nm) and that of cyclohexane (similar to A-ring - cyclohexanol of abiraterone) is 0.69 nm [52]. Thus, abiraterone can be included in the HPBCD cavity

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from either end, but the entire length of abiraterone cannot be covered by a single molecule of HPBCD. Hence, theoretically, 2 moles of HPBCD are necessary for complete abiraterone inclusion. This conforms to the results of solid-state interaction studies, suggesting that the 10%w/w drug loaded Lot 1 KSD contains complete abiraterone complexation with HPBCD.

6.4.Solution-state phase solubility profile:

Solution-state phase solubility profiles are usually generated to understand drug–CD complexation dynamics for preparing complexes using solvent-based methods. However, in this study, we generated such a profile to understand its impact on KSD dissolution. In the solution state, CDs can form complexes with free solubilized drug through driving forces such as the release of enthalpy-rich water molecules from the CD cavity, van der Waals interactions, electrostatic interactions, hydrogen bonding, and hydrophobic interactions [11].

Based on the Higuchi and Connors classification, it can be seen that the A-type solution- state phase solubility profiles are generated for abiraterone and HPBCD in both 0.01N HCl and FaSSIF media (see Figure 3.6) [11, 13]. This means that as the concentration of HPBCD increases, the amount of solubilized abiraterone increases. Thus, upon KSD dissolution, the re-solubilization of unabsorbed abiraterone would depend on the concentration of HPBCD. Hence, the highest viable HPBCD concentration is preferred for abiraterone solubilization.

Additionally, higher abiraterone solubilization was seen in 0.01N HCl compared to FaSSIF because the intrinsic solubility of abiraterone is higher in 0.01N HCl, hence more abiraterone solubilizes and forms complexes with HPBCD in 0.01N HCl compared to FaSSIF. It should be noted that usually unionized drug forms more stable complexes with CDs than ionized drugs [11]. Since the pKa of abiraterone is 4.81, it forms more stable complexes in FaSSIF [53].

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6.5.Stability of KSDs:

We evaluated the stability of KSDs at elevated temperatures. We observed that abiraterone in the Lots 1 and 2 KSDs remained amorphous at both 90 °C and 150 °C, while the Lot 3 KSD showed abiraterone recrystallization at both elevated temperatures (see Figure 3.7). This could be explained by solid-state interactions between abiraterone and HPBCD in these KSDs. In both the Lot 1 and Lot 2 KSDs, each molecule of abiraterone is complexed with at least one molecule of HPBCD, so abiraterone is thermally and kinetically stabilized in these KSDs. This inhibits their recrystallization when heated. In the Lot 3 KSDs, only a fraction of abiraterone molecules are complexed with HPBCD, thus the free uncomplexed abiraterone molecules recrystallize when heated, thus destabilizing the KSD.

6.6.In Vitro and In Vivo Performance of KSDs:

In the in vitro dissolution study (see Figure 3.8), we observed that all KSDs enhanced the dissolution of abiraterone. The relative in vitro dissolution performance of KSDs decreased as the drug loading increased. This can be attributed to decreased abiraterone– HPBCD complexation, which decreases the enhancement of abiraterone solubility as the drug loading is increased. It should be noted that dissolution performance can be increased or decreased as drug loading is increased. This is specific to the drug, the type of CD, the method of preparation, and the dissolution medium [54-56]. As seen in the solution-state phase solubility profiles, the KSDs also exhibited higher abiraterone dissolution in 0.01N HCl compared to FaSSIF media. In FaSSIF media, the dissolution of the Lot 1 KSD was higher compared to other KSDs, since the rate of abiraterone precipitation is lower in the Lot 1 KSD due to complete abiraterone complexation. Overall, the 10% w/w drug loaded Lot 1 KSD showed the highest in vitro dissolution enhancement of abiraterone.

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In the in vivo pharmacokinetic study (see Figure 3.9 and Table 3.4), it was observed that all the KSD tablets enhanced the exposure of abiraterone compared to Zytiga on a dose- adjusted basis. Compared to the drug exposure (i.e., the AUC) of the generic abiraterone acetate tablet Zelgor® reported in the previous study, the drug exposure of Zytiga was higher at the same dose of 250 mg abiraterone acetate [17]. After investigating this anomaly, we found that the disintegration time of Zytiga was greater than that of Zelgor. Thus, it is likely that Zytiga resided for a longer time in the animal’s stomach, at a lower pH where the drug’s solubility is greater, leading to higher abiraterone acetate dissolution and higher abiraterone exposure compared to Zelgor.

Another reason for the higher Zytiga exposure is its variability and the dual significant absorption events seen in one animal. These dual absorption events observed in one animal in the Zytiga test arm could be due to reingestion of Zytiga through coprophagia (i.e., the consumption of feces). Such instances of coprophagia during pharmacokinetic studies using beagle dogs have been reported in the literature [57].

The enterohepatic recirculation of abiraterone acetate has not been reported in mass balance studies in humans, but it has been hypothesized to a play a role in pharmacokinetic studies in animals [58, 59]. Williams et al. reported the AUC(0–24 h) values of 475 ± 518 h·ng/mL, 652 ± 661 h·ng/mL, and 413 ± 378 h·ng/mL for Zytiga 250 mg abiraterone acetate in fasted male beagle dogs [60, 61]. This further highlights the high pharmacokinetic variability of Zytiga.

At approximately one fifth the dose, the Lots 1 and 2 Tablets showed higher Cmax than

Zytiga. On a dose-adjusted basis, the Lots 1, 2, and 3 KSDs showed higher AUC(0–48 h) than Zytiga, hence all three KSD tablets could enhance the bioavailability of abiraterone.

As the drug loading increased in the KSDs from the Lots 1, 2, and 3 Tablets, the Cmax and the AUC(0–48 h) decreased, hence the bioavailability enhancement also decreased. This can be attributed to reduced abiraterone–HPBCD interaction and reduced dissolution after increased drug loading.

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Also, the Lot 1 Tablet contained the most HPBCD, yet it showed the highest abiraterone exposure among the KSD tablets; therefore, we can infer that HPBCD had no negative effect on abiraterone dissolution. As reported in another previous study, the drug exposure variability was reduced in the KSD tablets, thus improving abiraterone pharmacokinetics [17]. Overall, the Lot 1 KSD at 10% w/w drug loading showed the best in vivo pharmacokinetic performance.

Figure 3.10 demonstrates that in vitro dissolution performance correlates well with the in vivo pharmacokinetic performance of KSDs and KSD tablets. From Figure 3.9, one can extrapolate that 5% w/w drug loaded KSDs could have shown even better performance. However, this may not be true, since at 10% w/w drug loading, abiraterone is completely complexed with HPBCD, and additional HPBCD may not cause proportional performance enhancement. Additionally, a 5%w/w drug loaded KSD would cause a pill burden issue. Therefore, we conclude that the optimal drug loading for KSDs is 10% w/w.

7. CONCLUSIONS:

From this study, we conclude that KinetiSol is as an efficient, high-energy, solvent-free technology for the production of abiraterone–HPBCD compositions with drug loading ranging from 10–50% w/w. As drug loading increases, (a) the interaction between abiraterone and HPBCD in the KSD decreases, (b) the dissolution enhancement of abiraterone decreases, and (c) the bioavailability enhancement of abiraterone decreases. The 10% w/w drug loaded KSD is stoichiometrically balanced for complete interaction with abiraterone to produce maximum in vitro and in vivo performance. Thus, the 10% w/w drug loaded KSD has the potential to improve therapeutic outcomes in prostate cancer patients.

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8. ACKNOWLEDGEMENTS AND DISCLOSURE:

Gala acknowledges and thanks Ms. Angela Spagenberg (Dispersol Technologies LLC), Mr. Steven Sorey (UT Austin) and Dr. Yongchao Su (Merck) for their support of ssNMR and Raman Studies. Gala acknowledges and thanks Pharmaron, China for their support with the animal study. Williams acknowledges financial support for Gala from DisperSol Technologies, LLC. Gala, Miller and Williams are coinventors on intellectual property related to this work.

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38. Knapik-Kowalczuk, J., et al., Physical Stability and Viscoelastic Properties of Co-Amorphous Ezetimibe/Simvastatin System. Pharmaceuticals (Basel, Switzerland), 2019. 12(1): p. 40. 39. Ren, k., et al., Physicochemical characteristics and oral bioavailability of andrographolide complexed with hydroxypropyl-β-cyclodextrin. Die Pharmazie, 2009. 64: p. 515-20. 40. Gala, U., H. Pham, and H. Chauhan, Pharmaceutical applications of eutectic mixtures. J Dev Drug, 2013. 2: p. 1-2. 41. Pessine, F., A. Calderini, and G. Alexandrino, Review: cyclodextrin inclusion complexes probed by NMR techniques, magnetic resonance spectroscopy. Dong- Hyun Kim editor, 2012. 42. Hanada, M., et al., Predicting physical stability of ternary amorphous solid dispersions using specific mechanical energy in a hot melt extrusion process. Int J Pharm, 2018. 548(1): p. 571-585. 43. Lu, X., et al., Molecular Interactions in Posaconazole Amorphous Solid Dispersions from Two-Dimensional Solid-State NMR Spectroscopy. Mol Pharm, 2019. 16(6): p. 2579-2589. 44. O’Brien, E.P. and G. Moyna, Use of 13C chemical shift surfaces in the study of carbohydrate conformation. Application to cyclomaltooligosaccharides (cyclodextrins) in the solid state and in solution. Carbohydrate Research, 2004. 339(1): p. 87-96. 45. Long, D.A., Infrared and Raman characteristic group frequencies. Tables and charts George Socrates John Wiley and Sons, Ltd, Chichester, Third Edition, 2001. Price £135. Journal of Raman Spectroscopy, 2004. 35(10): p. 905-905. 46. Stolarczyk, E.U., et al., Design and Molecular Modeling of Abiraterone- Functionalized Gold Nanoparticles. Nanomaterials (Basel), 2018. 8(9). 47. Gong, A. and X. Zhu, beta-Cyclodextrin sensitized spectrofluorimetry for the determination of abiraterone acetate and abiraterone. J Fluoresc, 2013. 23(6): p. 1279-86. 175

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58. Belfort, G.M.H., Boyd L. and Botella, GabrielMartinez Abiraterone Acetate (Zytiga®): An Inhibitor of CYP17 as a Therapeutic for Castration-Resistant Prostate Cancer, in Successful Drug Discovery. 2016. p. 115-135. 59. Acharya, M., et al., A phase I, open-label, single-dose, mass balance study of 14C-labeled abiraterone acetate in healthy male subjects. Xenobiotica, 2013. 43(4): p. 379-89. 60. Hywel Williams, Prashant Agarwal, and E. Jule, Abiraterone acetate lipid formulations. 2016. 61. Williams, H.M., Michael; Vodak, David; Jule, Eduardo; Benameur, Hassan, M1130-04-28 - Improving Oral Drug Absorption – Which Technology to Select? A Spray-Dried Dispersion and Lipid-Based Formulation Case Study Using Abiraterone Acetate. 2019.

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Chapter Four: Comparative Evaluation Of KinetiSol® Processed Amorphous Solid Dispersions Of Abiraterone Acetate And Abiraterone: Is The Prodrug Necessary?

1. ABSTRACT:

The prodrug abiraterone acetate, known commercially as Zytiga®, is widely used in the treatment of prostate cancer. It has been reported that rapid presystemic conversion of abiraterone acetate to the active metabolite abiraterone generates supersaturated concentrations of abiraterone in the gastrointestinal lumen, which leads to increased oral absorption. In our previous studies, we reported the development of KinetiSol® processed solid dispersions (KSD) of abiraterone with substantially improved pharmacokinetics. The aim of the current study is to evaluate abiraterone acetate KSDs to determine whether the prodrug form further enhances performance. It was hypothesized that the magnitude of the solubility enhancement imparted by a KSD formulation would far exceed the additional solubility benefit of the prodrug form, thus KSDs of abiraterone and abiraterone acetate would exhibit similar in vitro and in vivo performance. Extensive KinetiSol formulation space was explored, and the optimal abiraterone acetate KSD formulation was determined to have a 10% w/w drug loading and hydroxy propyl β- cyclodextrin (HPBCD) as the oligomer; mirroring prior formulation development results with abiraterone. Comparative physicochemical stability analysis of 10% w/w abiraterone acetate and abiraterone KSDs with HPBCD indicated that both formulations were physically stable, but chemical analysis revealed that abiraterone acetate was chemically unstable in its amorphous KSD formulation. The results of non-sink, gastric transfer dissolution analysis showed comparable in vitro performance for both KSDs. Both KSDs exhibited similar oral bioavailability enhancement, with abiraterone acetate and abiraterone KSDs increasing bioavailability by 3.4-fold and 3.8-fold over Zytiga, respectively. The results of this study thus demonstrate that the solubility enhancement achieved via the utilization of an amorphous solid dispersion formulation can far outweigh the solubility improvements achieved through chemical modification of the

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active moiety. Further, this study suggests that amorphous solid dispersions of abiraterone acetate are chemical unstable; therefore, direct formulation of the active moiety, abiraterone, is required to achieve a viable amorphous drug product.

2. GRAPHICAL ABSTRACT:

3. INTRODUCTION:

Prodrugs are compounds that have little or no pharmacological activity and are converted to the pharmacologically active parent drug in vivo through enzymatic or chemical reactions [1]. It has been estimated that about 12% of all new, small-molecule chemical entities approved by the US Food and Drug Administration (US FDA) between 2007 and 2018 were prodrugs [1]. One of the main goals of designing a prodrug for oral delivery is to improve the physicochemical characteristics of the active parent drug and thereby improve its solubility or permeability to ultimately achieve the desired pharmacokinetics [2].

Abiraterone acetate is a prodrug that is converted predominantly presystemically by esterase enzymes to its active moiety, abiraterone (see Figure 4.1). It has been reported

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that the ester hydrolysis of abiraterone acetate occurs largely in the gastrointestinal lumen and additionally in the gastrointestinal tissues and the liver (see Figure 4.1)[3–5]. In clinical studies, the plasma concentration of abiraterone acetate after administration of the commercial product Zytiga® was reported to be below detectable levels (< 0.2 ng/mL) in more than 99.0% of analyzed samples [6].

In a study by Stappaerts et al., a single tablet of Zytiga (250 mg abiraterone acetate) was administered to healthy volunteers. Their duodenal fluids were then aspirated at an interval of 10–15 min for a period of 4 h. At all timepoints, the concentration of abiraterone acetate was minimal, thereby suggesting that the prodrug is rapidly cleaved intraluminally by esterases to form abiraterone [3].

Figure 4.1. The presystemic conversion of abiraterone acetate to abiraterone.

Just like abiraterone, abiraterone acetate has a poor water solubility of < 0.1 mg/mL [7]. The apparent permeability of abiraterone acetate is < 3.69×10−6 cm/s and that of abiraterone is < 3.46×10−6 cm/s through the Caco-2 cell line, thus both compounds are considered poorly permeable [6]. Considering that the prodrug form provides minimal 180

solubility or permeability benefits, it is not readily apparent why this form of abiraterone was selected for commercialization.

An early report on the development of novel steroidal inhibitors indicates that the prodrug abiraterone acetate was developed due to its ease of formulation compared to abiraterone [8]. Bryce et al. reported that abiraterone acetate was developed to enhance the oral bioavailability of abiraterone [9]. Stappaerts et al. reported that abiraterone acetate has higher solubility than abiraterone in biorelevant media [3]. These authors conducted in vitro, in situ, and in vivo studies that suggested that rapid conversion of abiraterone acetate to abiraterone in the gastrointestinal tract generates abiraterone concentrations that exceed its intrinsic solubility, thus leading to higher abiraterone flux and enhanced oral absorption [3]. This indicates that the prodrug advantage of abiraterone acetate stems from its ability to produce supersaturated concentrations of abiraterone.

Despite the aforementioned prodrug advantage, Zytiga has poor oral bioavailability, a substantial positive food effect, and high pharmacokinetic variability [6, 10]. Zytiga is indicated to be administered on an empty stomach due to variations in diet across the patient population and to reduce the risk of increased pharmacokinetic variability [11]. While investigating the effect of prandial status on the intraluminal behavior of abiraterone acetate, Geboers et al. concluded that the poor oral bioavailability of Zytiga in the fasted state is due to transient supersaturation of abiraterone that results from the ester hydrolysis of the acetate drug, which is then followed by rapid precipitation [12].

An amorphous solid dispersion (ASD) is a formulation approach used to enhance the solubility of poorly water soluble drugs [13]. ASDs achieve aqueous supersaturation of insoluble drugs by presenting the compound in an amorphous form, thereby lowering the energy required for solvation of the drug [14, 15]. ASDs have been reported to improve the pharmacokinetics and pharmacodynamics of numerous anticancer drugs [16]. Unlike the drug supersaturation achieved by ester hydrolysis of a prodrug, the supersaturation 181

achieved by ASDs can be considered more robust since it is achieved through the intrinsic properties of the composition, independent of extrinsic factors such as enzyme concentration.

Williams et al. reported the development of an amorphous solid dispersion of abiraterone acetate with hydroxypropyl methylcellulose acetate succinate (HPMCAS), which enhanced the bioavailability of abiraterone by 2.3-fold in the fasted state [17]. Solymosi et al. reported the development of a nano-amorphous formulation of abiraterone acetate with polyethylene glycol, polyvinyl acetate, and a polyvinylcaprolactame-based graft copolymer (Soluplus®), which also improved the bioavailability of abiraterone [18]. Basa-Denes et al. investigated the mechanism behind the rapid absorption of the above nano-amorphous formulation of abiraterone acetate. They concluded that the ester hydrolysis of abiraterone acetate alters the pharmacokinetics of the formulation and leads to a more rapid absorption rate of abiraterone [19].

Prodrugs and ASDs may be considered complementary approaches, specifically if they serve two different purposes, i.e., the prodrug may enhance the permeability of the active drug, while the ASD of the prodrug enhances solubility [20, 21]. The only commercial products in which both these strategies have been applied are two drug combination products, Harvoni® and Epclusa®. However, in both these products, the prodrug strategy is applied to one drug and the ASD strategy is applied to the other [22, 23]. There is an absence of literature regarding instances in which both a prodrug and an ASD approach are applied for the same purpose of enhancing the apparent solubility of the active drug. Therefore, it is unclear whether these strategies would in fact act synergistically to enhance the apparent aqueous solubility of a poorly water-soluble active drug.

Several challenges are associated with the synthesis and formulation of prodrugs. Among these challenges, the most formidable are (a) complicated and costly synthesis processes and (b) chemical instability [24, 25]. Hence, it is imperative to carefully evaluate the

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benefits and risks of developing a prodrug, specifically when it is designed to enhance the apparent solubility of the active drug.

In Chapter 2 and 3, it was shown that the optimal abiraterone KSD formulation (abiraterone:HPBCD (1:9 w/w)) provided pharmacokinetic performance that was superior to crystalline abiraterone acetate. The aim of the current study is to investigate ASD formulations of abiraterone acetate and evaluate them against the abiraterone ASD discussed in Chapter 2 and 3, to test the hypothesis that abiraterone acetate provides no additional performance benefit because the magnitude of solubility enhancement achieved from an optimal ASD far exceeds the solubility improvement achieved from the prodrug form. If this hypothesis is affirmed, this study would indicate that abiraterone acetate has limited value in maximizing abiraterone’s oral bioavailability and improving therapeutic outcomes for prostate cancer patients.

4. MATERIALS AND METHODS:

4.1.Materials:

Abiraterone acetate active pharmaceutical ingredient (API) was supplied as a gift sample by Teva API/TAPI (New Jersey, USA) and later purchased from Attix Pharma (Ontario, Canada). Both abiraterone acetate APIs were of the same polymorph. Abiraterone API was purchased from MSN Laboratories Pvt. Ltd. (Telangana, India). Hydroxypropyl methyl cellulose (Methocel™ E5 Premium LV) was purchased from Dow (Michigan, USA). Hydroxy propyl methylcellulose acetate succinate (Affinisol™ HPMCAS 126G) was supplied as a gift sample by Dow (Michigan, USA). Hydroxypropyl methylcellulose acetate phthalate (hypromellose phthalate) was supplied as a gift sample by Shin-Etsu (New Jersey, USA). Polyvinyl pyrrolidone (Kollidon® 30), vinylpyrrolidone–vinyl acetate copolymer (Kollidon® VA64), polyethylene glycol, polyvinyl acetate, and polyvinylcaprolactame-based graft copolymer (Soluplus®) were supplied as gift samples

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by BASF (New Jersey, USA). Methacrylic acid and ethyl acrylate copolymer (Eudragit® L 100-55) was purchased from Evonik Industries (New Jersey, USA). Hydroxypropyl β- cyclodextrin (Kleptose® HPB) was purchased from Roquette America (USA). Microcrystalline cellulose (Avicel PH-102) was purchased from FMC Corporation (Pennsylvania, USA). Mannitol (Pearlitol 200SD) was purchased from Roquette America (USA). Crosslinked sodium carboxymethyl cellulose (Vivasol®) was purchased from JRS Pharma (New York, USA). Hypromellose acetate succinate, HMP grade (AQOAT®) was purchased from Shin-Etsu (New Jersey, USA). Colloidal silicon dioxide (Aerosil® 200 P) was purchased from Evonik Industries (New Jersey, USA). Magnesium stearate was purchased from Peter Greven (Muenstereifel, Germany). The fasted state simulated intestinal fluid (FaSSIF) dissolution media was prepared using FaSSIF/FeSSIF/FaSSGF powder purchased from Biorelevant.com (Surrey, UK). Abiraterone acetate tablets (Zytiga®, 250 mg abiraterone acetate) were purchased from Myoderm (Pennsylvania, USA) and manufactured for Janssen Biotech (Pennsylvania, USA). The solvents used for HPLC analysis were of HPLC grade. All other chemicals and reagents used for dissolution and HPLC analysis were of ACS grade.

4.2.Methods:

4.2.1.KinetiSol® Processing:

Abiraterone acetate KSDs were prepared using KinetiSol® technology. Initially, all KSDs were prepared using a research-scale compounder (the Formulator) designed and manufactured by DisperSol Technologies LLC (Texas, USA). Later, selected abiraterone acetate KSDs were prepared using a manufacturing-scale compounder (the Manufacturing Compounder) designed and manufactured by DisperSol Technologies LLC (Texas, USA).

The manufacturing compounder was operated in batch mode. Before compounding, abiraterone acetate and the excipient carrier (see Tables 4.1 and 4.2) were accurately

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weighed and thoroughly mixed to prepare physical mixtures (PMs). These physical mixtures were charged into the KinetiSol compounder chamber. Inside the chamber, a shaft with protruding blades was rotated at varying incremental speeds ranging from 2,400 rpm to 7,000 rpm, without external heat, to impart frictional and shear forces to the sample material. The temperature of the mass was monitored using an infrared probe. When the temperature reached a value of 120–150 °C, the molten mass was rapidly ejected, collected, and pressed between two stainless steel plates to rapidly quench the sample.

The abiraterone KSD was prepared according to the method described in the previous study [26]. Briefly, before compounding, 10% w/w abiraterone and 90% w/w HPBCD were accurately weighed and thoroughly mixed to prepare a physical mixture (see Table 4.3). This physical mixture was divided into sub-lots and charged into the KinetiSol manufacturing compounder chamber, then processed in semi-continuous mode. Inside the chamber, a shaft with protruding blades was rotated at varying incremental speeds ranging from 2,400 rpm to 2,700 rpm, without external heat, to impart frictional and shear forces to the sample material. The temperature of the mass was monitored using an infrared probe. When the temperature reached a value of 180 °C, the molten mass was rapidly ejected, collected, and pressed between two stainless steel plates to rapidly quench the sample.

4.2.2.Milling:

The quenched mass obtained after KinetiSol processing was milled in small batches using a lab scale rotor mill, (i.e., an IKA tube mill 100 (IKA Works GmbH & Co. KG, Staufen, Germany)). For milling, the fragments of the quenched mass were loaded into a 20 mL grinding chamber operated with a grinding speed between 10,000 and 20,000 rpm for 60 s. The milled material was subsequently passed through a #60 mesh screen (≤ 250 μm). The material that remained above the screen (i.e., > 250 μm) was cycled through the mill using the same parameters. This process of milling and sieving was repeated until all 185

material passed through the screen. The resultant material, with a particle size less than 250 μm, was labeled as a KSD.

4.2.3.X-Ray Powder Diffraction:

X-Ray powder diffraction (XRPD) analysis was conducted using a Rigaku MiniFlex600 II (Rigaku Americas, Texas, USA) instrument equipped with a Cu–Kα radiation source generated at 40 kV and 15 mA. The APIs and KSD samples were loaded into an aluminum pan, leveled with a glass slide, and analyzed in the 2-theta range from 2.5– 35.0° while being spun. The step size was 0.02°, and the scanning rate was set to 5.0 °/min. The following additional instrument settings were used: slit condition: variable + fixed slit system, soller (incident): 5.0°, IHS: 10.0 mm, DS: 0.625°, SS: 8.0 mm, soller (receiving): 5.0°, RS: 13.0 mm (open), and monochromatization: kb filter (× 2). The data were collected using Miniflex Guidance software (Rigaku Corporation, Tokyo, Japan) and processed using PDXL2 software (Rigaku Corporation, Tokyo, Japan).

4.2.4.HPLC Analysis:

A stability-indicating, high-performance liquid chromatography (HPLC) method was developed for the chemical analysis of the abiraterone acetate KSDs and the abiraterone KSD. A Dionex Ultimate 3000 HPLC system (ThermoFisher Scientific, Massachusetts, USA) was used for reverse-phase HPLC analysis. The HPLC column was a Kinetex® XB C18, 150 mm x 4.6 mm and 2.6 µm (Phenomenex, California, USA). Mobile phase A was a 20 mM ammonium formate buffer (pH 3), and mobile phase B was degassed acetonitrile. A gradient profile was designed with an initially higher amount of aqueous phase followed by gradual increase in the organic phase. The flow rate was 0.9 mL/min, and the run time was 42 min. The column was held at 35 °C, and the data were collected at a single wavelength of 254 nm. Samples were prepared at a nominal concentration of 0.5 mg/mL with 7:2:1 methanol:isopropyl alcohol:tetrahydrofuran as the standard/sample diluent. All samples were filtered through 0.45 μm PVDF syringe filters (GE Healthcare

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Life-Sciences, Pennsylvania, USA), before analysis. The sample chromatography was analyzed using Chromeleon™ software, version 7.0 (ThermoFisher Scientific, Massachusetts, USA).

4.2.5.Physicochemical Stability Analysis:

A thermal analysis of the APIs was conducted using differential scanning calorimetry (DSC) with a model Q20 differential scanning calorimeter (TA Instruments, Delaware, USA) equipped with a refrigeration-based cooling system and an autosampler. The API samples were prepared by weighing 5–10 mg of the material and loading it into a Tzero pan. The pan was sealed with a Tzero lid using a Tzero press. Following the sample equilibration at 30 °C for 5 min, the temperature was increased at a rate of 5 °C/min up to 250 °C. The sample was then cooled at a rate of 5 °C/min down to −40 °C, followed by reheating at a rate of 5 °C/min up to 250 °C . Nitrogen was used as the sample purge gas at a flow rate of 50 mL/min. The data were collected using TA Instruments Explorer software (TA Instruments, Delaware, USA) and processed using Universal Analysis software (TA Instruments, Delaware, USA).

Stability analysis was performed at room temperature under accelerated conditions. The abiraterone acetate KSD and the abiraterone KSD samples were placed at room temperature and analyzed more than three months after manufacturing by XRPD and HPLC analysis using the method described above. An accelerated stability study was conducted using a stability chamber (Darwin Chambers Company, Missouri, USA) set at 40 °C and 75% RH. The abiraterone acetate KSD and the abiraterone KSD samples were loaded in appropriately labelled, open and closed, high-density polyethylene (HDPE) bottles and charged into the stability chamber. The sample bottles were removed at two weeks and four weeks after the day of charging, then analyzed using XRPD analysis and HPLC analysis as stated above.

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4.2.6.In Vitro Dissolution Study:

An in vitro, non-sink, gastric transfer dissolution method was developed to analyze the dissolution of neat abiraterone acetate API, neat abiraterone API, abiraterone acetate KSDs, and the abiraterone KSD. For dissolution analysis, samples equivalent to 44.6 mg abiraterone were loaded into an Erlenmeyer flask (dissolution vessel) containing 50 mL of 0.01N HCl (pH 2.0), then placed in an incubator-shaker-Excella E24 (New Brunswick Scientific, New Jersey, USA) set to 37 °C and agitated at a rotational speed of 180 rpm. After 30 min, 50 mL of FaSSIF (prepared in a 50 mMol phosphate buffer at pH 6.8) was added to the dissolution vessel. At predetermined time points, samples were drawn from the dissolution vessel and centrifuged using an ultracentrifuge (Eppendrof, Hamburg, Germany). The supernatants were further diluted using the HPLC diluent and analyzed using the HPLC method described above. The area under the drug dissolution curve (AUDC) was calculated using the linear trapezoidal method.

4.2.7.Supersaturation Study:

For the supersaturation study, abiraterone acetate KSD and abiraterone KSD samples equivalent to 44.6 mg, 89.2 mg, and 178.4 mg abiraterone were loaded into an Erlenmeyer flask (dissolution vessel) containing 50 mL of 0.01N HCl (pH 2.0), then placed in an incubator-shaker-Excella E24 (New Brunswick Scientific, New Jersey, USA) set to 37 °C and agitated at a rotational speed of 180 rpm. After 30 min, 50 mL of FaSSIF (prepared in a 50 mMol phosphate buffer at pH 6.8) was added to the dissolution vessel. At predetermined time points, samples were drawn from the dissolution vessel and centrifuged using an ultracentrifuge (Eppendorf, Hamburg, Germany). The supernatants were further diluted using the HPLC diluent and analyzed using the HPLC method described above. Volumes were scaled higher or lower based on sample availability. The area under the drug dissolution curve (AUDC) was calculated using the linear trapezoidal method.

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4.2.8.Tableting:

The KSD and tableting excipients Avicel PH-102, Pearlitol 200SD, Vivasol, Kleptose, AQOAT, Aerosil 200 P, and magnesium stearate were accurately weighed and dispensed. Aerosil 200 P was sieved through #40 mesh (420 µm) until all material passed through the sieve. The KSD and all tableting excipients, except magnesium stearate, were loaded into a vial and mixed using a vortex mixer (Thermo Scientific, Massachusetts, USA). Magnesium stearate was then added to the vial and blended using a spatula. The resultant tableting blend was then dispensed in aliquots equivalent to 56 mg abiraterone acetate or 50 mg abiraterone. Each aliquot was loaded into the tablet die and compressed using a single-station hand tablet press (BVA Hydraulics, Missouri, USA) with a target hardness of 8–12 kP.

4.2.9.In Vivo Pharmacokinetic Study in Beagle Dogs:

An in vivo pharmacokinetic study in fasted, non-naïve, male beagle dogs was conducted at Pharmaron (Ningbo, China). This animal study was conducted according to an approved Pharmaron IACUC protocol #PK-D-06012018. The 56.0 mg equivalent abiraterone acetate tablet, the 50.0 mg equivalent abiraterone tablet, and a 250 mg equivalent abiraterone acetate tablet (Zytiga®) were analyzed. Each study arm for each formulation consisted of 3–5 dogs.

The dogs were fasted overnight before dosing, and food was returned 4 h post dosing. Each dog was administered a single tablet of the respective formulation (per the study arm) along with a post-dose flush of 40 mL sterile water. At predefined time points of 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 16, 18, and 24 h post dose, 1 mL blood samples were drawn from each dog via the venipuncture of a peripheral vessel. These samples were then placed in tubes containing sodium heparin anticoagulant. The blood samples were centrifuged to isolate the plasma. The abiraterone content of the plasma samples were then evaluated using liquid chromatography with tandem mass spectrometry (LC- MS/MS). 189

4.2.10. Pharmacokinetic Analysis:

Pharmacokinetic parameters were estimated using Phoenix™ WinNonlin software, version 6.1 (Certara, New Jersey, USA), using a noncompartmental approach consistent with the oral route of administration. The area under the plasma concentration time curve (AUC) was calculated using the linear trapezoidal method. The relative bioavailability, i.e., the F value, was calculated using the following formula:

퐴푈퐶(0−24hr)(test abiraterone tablet) × 퐷표푠푒(abiraterone )(reference abiraterone acetate tablet) 퐹 = 퐴푈퐶(0−24hr)( reference abiraterone acetate tablet) × 퐷표푠푒(abiraterone ) (test abiraterone tablet)

5. RESULTS & DISCUSSION:

5.1.Development of abiraterone acetate KSDs:

Abiraterone acetate KSDs were developed in two stages. First, a suitable carrier was identified, then drug loading was optimized for each carrier.

5.1.1.Identification of a suitable carrier for abiraterone acetate KSDs:

Our previous study determined that the oligomer HPBCD was the optimum carrier for abiraterone [15]. Abiraterone acetate and abiraterone vary significantly, not only in their chemistry but also in the intermolecular interactions within their respective crystal structures, which corresponds to their disparate physical properties [7, 27, 28]. Thus, it would not be expected that the optimal ASD carrier for abiraterone would also be optimal for abiraterone acetate. Additionally, the KinetiSol processing parameters suitable for abiraterone do not necessarily apply directly to abiraterone acetate. Thus, in order to develop abiraterone acetate KSDs, we selected eight different polymers or oligomers that vary in their chemistries, chain lengths, and architectures to elicit the maximum possible noncovalent interactions between abiraterone acetate and the carrier to form stable abiraterone acetate KSDs (see Figure 4.2). 190

Figure 4.2. Polymer/oligomer space mapped for abiraterone acetate KSD development.

Table 4.1 lists abiraterone acetate KSD compositions, processing parameters, and their corresponding appearance. Lots 1 to 8 PMs were all processible using the KinetiSol technology. Since the melting point of abiraterone acetate is lower than that of abiraterone, the target processing temperature for these KSDs was kept at or below 150 °C [7]. The total processing time for all lots was less than 35 s. Lots 1 to 8 KSDs showed an opaque appearance owing to entrapped air as a result of the manual quenching process. Lots 1 to 6 showed slight discoloration, possibly due to the thermokinetic processing of long-chain polymers [29, 30]. Lot 7 KSD showed a marked speckled discoloration, indicating heterogeneity of the dispersion. The high mixing intensity of KinetiSol typically ensures that the mass ejected from the compounder chamber is homogenous. Jermain et al. demonstrated that KinetiSol technology can produce

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homogenous KSDs even at drug concentrations as low as 1% w/w [31]. Thus, the speckled appearance of the Lot 7 KSD is likely due to non-uniform quenching. No discoloration was observed in Lot 8 KSD, since it contained a short-chain oligomer. Lots 1 to 7 KSDs were more agglomerated, while the Lot 8 KSD was less agglomerated and highly brittle. All KSD masses were easily milled and sieved to yield KSD powders with a particle size of less than 250 µm.

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Table 4.1. Abiraterone acetate KSD compositions, processing parameters and their corresponding appearance.

Composition Shear KinetiSol® Batch Processing Total Polymer/ Stress Lot No. Drug (% Compound Size Temperatu Processing Appearance Oligomer (Rotational wt) er (g) re (°C) Time (s) (% wt) speed-rpm)

Abiraterone HPMC E5 1 Formulator 10 130 4,000, 5,000 13.9 Acetate (10) (90)

HPMCAS Abiraterone 2 126 G Formulator 10 130 4,000 9.5 Acetate (10) (90)

Abiraterone HPMCP 4,000, 3 Formulator 10 140 23.4 Acetate (10) (90) 5,000,6,000

Abiraterone PVP K30 5,000, 4 Formulator 10 130 32.4 Acetate (10) (90) 6,000,7,000

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Table 4.1 continued

Composition Shear KinetiSol® Batch Processing Total Stress Lot No. Compound Size Temperatu Processing Appearance Polymer/ (Rotational Drug (% er (g) re (°C) Time (s) Oligomer speed-rpm) wt) (% wt)

Abiraterone PVP-VA 3,000, 5 Formulator 10 130 28.3 Acetate (10) (90) 4,000,5,000

Abiraterone Soluplus® 4,000, 6 Formulator 10 140 24.5 Acetate (10) (90) 5,000,6,000

Eudragit® Abiraterone 5,000, 7 L 100-55 Formulator 10 120 27.7 Acetate (10) 6,000,7,000 (90)

Abiraterone HPBCD- 8 Formulator 10 130 5,000,6,000 14.5 Acetate (10) (90)

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Figure 4.3 illustrates X-ray diffractograms of neat abiraterone acetate API and the Lots 1–8 KSDs. Neat abiraterone acetate showed sharp diffraction peaks at 14.82°, 18.31°, 18.91°, and 21.66°, indicating its crystalline nature. The XRPD pattern of abiraterone acetate seen in our study did not match the pattern reported in the literature, most likely due to differences in polymorphs [7, 32]. However, it is unclear how many abiraterone acetate polymorphic forms exist [33, 34]. Zhou et al. reported the crystal structure of abiraterone acetate and found weak H-bonding between adjacent molecules. Wheatley et al. reported that the crystal structure of abiraterone acetate is dominated by van der Waals and electrostatic interactions [28, 32]. The characteristic peaks of abiraterone acetate were absent in the XRPD patterns of Lots 1–8 KSDs, indicating that abiraterone acetate was amorphous in these compositions. As seen in the previous study, the Lot 1 KSD with the HPMC E5 carrier showed a diffraction peak at 31.66° due to presence of sodium chloride in the bulk polymer [15, 35].

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Figure 4.3. X-ray diffractograms of neat abiraterone acetate API (top) and the Lots 1–8 KSDs (bottom). The dotted circle in the top figure indicates the characteristic peaks of abiraterone acetate API. The dotted lines in the bottom figure indicate the peak position region of these characteristic peaks, and the dotted rectangle in the bottom figure indicates the sharp diffraction peak seen in the Lot 1 KSD.

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Abiraterone acetate is prone to degradation under stress conditions, specifically under acidic and basic conditions [36, 37]. HPLC analysis showed that the Lots 1–8 KSDs contained total impurities of 0.66%, 0.33%, 3.23%, 0.69%, 0.47%, 0.72%, 1.03%, and 1.01%, respectively. Among these, the Lot 3 KSD contained the most impurities, likely due to the degradation of HPMCP and the subsequent degradation of abiraterone acetate [38].

Comparative evaluation of the various KSD carriers for abiraterone acetate, with respect to the dissolution enhancement of abiraterone acetate, was conducted via in vitro, two- stage, non-sink, gastric transfer dissolution testing (see Figure 4.4). Neat abiraterone acetate API showed poor dissolution throughout the study. On average, neat abiraterone acetate showed a concentration of 15.4 µg/mL in FaSSIF, which was slightly higher than the solubility value of ~11 µg/mL in FaSSIF reported by Solymosi et al. [7]. Stappaerts et al. reported abiraterone acetate solubility in FaSSIF to be 25.2 µg/mL [3]. The Lots 1, 2, 4, 5, and 6 KSDs showed enhanced abiraterone acetate dissolution in 0.01N HCl compared to neat abiraterone acetate API. The Lots 3 and 7 KSDs, which contained HPMCP and Eudragit® L-100 55, respectively, showed no abiraterone acetate release in 0.01N HCl. However, they exhibited enhanced dissolution in FaSSIF, owing to the enteric nature of the polymers [39]. Additionally, HPLC results suggested a loss of ionizable phthalate groups from the HPMCP backbone on processing, which caused a reduction in polymer ionization and poor drug release in FaSSIF media.

The Lot 8 KSD showed drastic dissolution enhancement of abiraterone acetate in 0.01N HCl. Although precipitation in FaSSIF was rapid and substantial, the concentration of abiraterone acetate in solution with this formulation was superior to the neat abiraterone acetate API. The total area under the drug dissolution curve (AUDC Total ) for the Lot 8 KSD was 1,037.6% relative to neat abiraterone acetate API. This was the highest value of

AUDCTotal achieved for all formulations, signifying that the HPBCD-based KSD significantly outperformed all other polymer-based KSDs. This can be attributed to strong positive drug–carrier interactions that enabled the complete amorphization of 197

abiraterone acetate on KinetiSol processing and a substantial drug concentration enhancement by HPBCD in solution [40].

Figure 4.4. In vitro, non-sink gastric transfer dissolution profiles of neat abiraterone acetate API and abiraterone acetate KSD. The red region represents 0.01N HCl, and the blue region represents FaSSIF. The inset represents the enlargement of the dissolution profile in FaSSIF.

Overall, the HPBCD-based Lot 8 KSD was processible using KinetSol and formed a completely amorphous composition. This KSD also exhibited minimal abiraterone acetate degradation and showed the greatest dissolution enhancement. These findings suggest that HPBCD is the optimal carrier not only for amorphous abiraterone but also for amorphous abiraterone acetate.

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5.1.2.Identification of an optimal drug loading for the abiraterone acetate KSD:

From the previous study, it was determined that 10% w/w drug loading was optimal for the abiraterone KSD [26]. As previously discussed, owing to differing physical and chemical properties, it could not be predicted a priori that the same would be true for KSDs of abiraterone acetate. Thus, a drug load range up to 20% w/w was explored for KSDs that contain abiraterone acetate and HPBCD (see Table 4.2). The Lot 9 KSD, with a 20% drug loading, was processible using KinetiSol and required a total processing time of less than 12 s. It was easily milled to form a KSD powder with a particle size of less than 250 µm.

Table 4.2. Abiraterone acetate KSD compositions with higher drug loading and their processing parameters.

Composition Shear Total Batch Processing Stress Lot KinetiSol® Processi Drug Oligomer Size Temperat (Rotationa No. Compounder ng Time (% wt) (% wt) (g) ure (°C) l speed- (s) rpm) Abirater 4,000, one HPBCD 9 Formulator 10 150 5,000, 11.9 Acetate (80) 6,000 (20) Abirater 2,400, one HPBCD Manufacturing 10 90 150 2,700, 23.0 Acetate (88.8) Compounder 3,000 (11.2)

The Lot 9 KSD showed a complete amorphous halo XRPD pattern and an absence of characteristic peaks of neat abiraterone acetate API, thereby indicating its amorphous nature (see Figure 4.5).

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Figure 4.5. X-ray diffractograms of the Lots 9 and 10 KSDs.

Upon HPLC analysis, the Lot 9 KSD showed a total impurities value of 0.70% and showed no individual unknown impurity at or above 0.5%, thus indicating minimal abiraterone acetate degradation during KinetiSol processing.

The dissolution testing of the Lot 9 KSD showed a marked decrease in in vitro performance after increasing its drug loading (see Figure 4.6). The relative AUDC Total for the Lot 9 KSD containing 20% drug loading was 67.2% compared to the Lot 8 KSD containing 10% drug loading. In our previous study on KSDs of abiraterone, we discovered that a molar ratio of at least 1.0:2.0 abiraterone:HPBCD was required for optimal performance [26]. The molar ratio of abiraterone acetate to HPBCD was 1.0:2.5 and 1.0:1.2 in the Lot 8 KSD and the Lot 9 KSD, respectively. This explains the decrease in dissolution enhancement after increasing the drug loading due to reduction in the number of molecules of HPBCD available for the complete inclusion of abiraterone acetate. Hence, a 10% w/w drug loading was determined to be optimal for the abiraterone acetate KSD, mirroring that of the abiraterone KSD.

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Figure 4.6. In vitro, non-sink gastric transfer dissolution profiles of the Lots 9 and 10 KSDs. The red region represents 0.01N HCl, and the blue region indicates FaSSIF.

In order to directly compare the abiraterone acetate KSD to the abiraterone KSD, it is necessary that the compositions are equivalent in terms of the percentage of active (i.e., abiraterone) drug loading. Thus, the composition of the Lot 8 KSD was adjusted for the scale-up and manufacturing of the Lot 10 KSD containing 11.2% abiraterone acetate, which is equivalent to 10% abiraterone (see Table 4.2). It is important to note that the abiraterone acetate:HPBCD ratio in the Lot 10 KSD was 1.0:2.2, i.e., at least two molecules of HPBCD were available to interact with one molecule of abiraterone acetate. The total processing time for the Lot 10 KSD was 23.0 s, yielding a fully amorphous formulation (see Figure 4.5).

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5.2.Development of abiraterone KSDs:

Scale-up of the abiraterone KSD was conducted using methods similar to those described in the previous study, with the exception that the manufacturing compounder was operated in semi-continuous mode rather than batch mode [26]. The average total processing time for the Lot 11 KSD, with a 1.0:2.2 (mol/mol) ratio of abiraterone to HPBCD, was 13.3 s. Its composition was fully amorphous (data not shown), and it contained total impurities of 0.41%. The in vitro dissolution profile of the Lot 11 KSD was deemed optimal, matching the profile of prior lots of the same composition [15, 26].

Table 4.3. Abiraterone KSD composition and its processing parameters.

Composition Batch Shear Processi Avg. Total Size Stress Lot KinetiSol® ng Processing Drug (% Per (Rotation No. Oligomer Compounder Tempera Time Per wt) Shot al speed- (% wt) ture (°C) Shot (s) (g) rpm)

Abiratero HPBCD Manufacturing 2,400, 11 90 180 13.3 ne (10) (90) Compounder 2,700

5.3.Comparison of the physicochemical stability of the abiraterone acetate KSD and abiraterone KSD:

Before investigating the physicochemical stability of KSDs, the physical and chemical stability of the neat APIs was first evaluated. Figure 4.7 shows heat–cool–heat DSC thermograms for neat abiraterone acetate and neat abiraterone. The neat abiraterone acetate API showed a melting endotherm at 147.59 °C in the first heating cycle and no thermal events during the cooling cycle. In the second heating cycle, the following thermal events were observed: a small glass transition event at 27.63 °C (midpoint), a broad recrystallization exotherm at 89.60 °C (peak maximum), and a small exotherm at

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98.75 °C (peak maximum), followed by a melting endotherm at 147.18 °C (peak minimum).

On the contrary, the neat abiraterone API showed a melting endotherm at 229.10 °C (peak minimum) in first heating cycle and a small exotherm at 139.70 °C (peak maximum), followed by a sharp significant recrystallization exotherm at 161.43 °C (peak maximum) in the cooling cycle. On the second heating cycle, the neat abiraterone showed a small endothermic event at 218.53 °C (peak minimum) and a melting endotherm at 229.13 °C (peak minimum).

From this thermal behavior, it can be concluded that, per the glass formation ability (GFA) categorization method proposed by Baird et al., abiraterone acetate is a Class II glass former since it showed recrystallization in the second heating cycle. However, abiraterone is a Class I glass former since it showed recrystallization during the cooling cycle [41, 42]. It is important to note that the heating and cooling rates employed in this study differed from those of Baird et al.; however, the results are considered valid, because the GFA classification system was shown to be robust for different heating and cooling rates [42]. Thus, abiraterone is likely to nucleate and crystallize faster than abiraterone acetate.

Additionally, the melting point of abiraterone (~229.0 °C) is much higher than that of abiraterone acetate (~147.0 °C). In a study by Ormes et al., it was shown that compounds with melting points higher than 180 °C have a stronger recrystallization tendency than compounds with melting points lower than 180 °C [43]. Thus, it would be expected that in its amorphous form, abiraterone acetate would exhibit greater physical stability than abiraterone.

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Figure 4.7. Heat–cool–heat DSC thermograms for neat abiraterone acetate API (top) and neat abiraterone API (bottom).

Upon forced degradation studies of neat abiraterone acetate API and neat abiraterone API, we found that abiraterone acetate underwent both acid- and base-catalyzed degradation, but abiraterone underwent only acid-catalyzed degradation (data not shown). The above results are consistent with other reports in the literature on the degradation 204

pathways of abiraterone acetate and abiraterone [36, 37, 44]. From this, it can be inferred that abiraterone acetate is likely to be chemically less stable in its amorphous form than abiraterone.

The physical stability of the abiraterone and abiraterone acetate KSDs was evaluated at both ambient and accelerated conditions (see Figure 4.8). Under all conditions, both the abiraterone acetate KSD and the abiraterone KSD remained amorphous in nature. This indicates that, despite the stronger recrystallization tendency of abiraterone, it remained physically stable in the KSD composition due to favorable interactions with HPBCD.

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Figure 4.8. X-ray diffractograms of the abiraterone acetate KSD (top) and the abiraterone KSD (bottom) at room temperature and under accelerated conditions.

Figure 4.9 shows total impurity profiles for the abiraterone acetate and abiraterone KSDs at ambient and accelerated conditions. After three months under ambient storage,

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abiraterone acetate showed extensive degradation, with total impurities increasing by ~345.2%. For the abiraterone KSD, the increase in total impurities was only ~17.1% after three months of ambient storage. Interestingly, after the initial increase in impurities in the abiraterone acetate KSD at room temperature, the further increase in total impurities under accelerated conditions was not pronounced.

Yonsa®, a micronized formulation of abiraterone acetate, contains the antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) [45]. On stability study, without the addition of BHA and BHT, these authors reported an increase in total impurities from 0.48% to 4.49% after three months of storage at 40 °C and 75% RH [46]. This further highlights the instability issues associated with abiraterone acetate. On the other hand, the abiraterone KSD formulation showed a minimal increase in impurities, and the total impurities remained below 1.0% at all time points under all tested conditions.

Figure 4.9. Total impurity profiles for abiraterone acetate KSD and abiraterone KSD at room temperature and under accelerated conditions.

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Thus, a comparison of the physicochemical stability of the abiraterone acetate KSD and the abiraterone KSD leads to the conclusion that both KSDs are physically stable, but the abiraterone KSD is significantly more chemically stable. The extent of the degradation of the abiraterone acetate KSD calls into question the viability of an amorphous formulation approach with this prodrug form of abiraterone.

5.4.Comparison of the in vitro performance and supersaturation ability of abiraterone acetate KSD and abiraterone KSD:

Figure 4.10 shows in vitro, non-sink, gastric transfer dissolution profiles of neat APIs and their corresponding KSDs. It can be seen that both the abiraterone acetate KSD and the abiraterone KSD show significantly enhanced dissolution of abiraterone acetate and abiraterone, respectively, in 0.01N HCl. Both KSDs showed similar precipitation behavior upon initiation of the second stage of the test, i.e., the addition of the FaSSIF medium. Thus, it was determined that the rate of precipitation of abiraterone from a supersaturated state was very similar to that of abiraterone acetate, despite abiraterone’s stronger tendency to recrystallize, as discussed previously. Hence, the potential advantage of the prodrug form (i.e., a reduced rate of precipitation) in a supersaturating delivery system was determined to be inconsequential.

Overall, the abiraterone acetate KSD (Lot 10 KSD) enhanced the dissolution of abiraterone acetate by 11.8-fold relative to neat abiraterone acetate API, and the abiraterone KSD (Lot 11 KSD) enhanced the dissolution of abiraterone by 15.8-fold relative to neat abiraterone API. The difference between their relative dissolution enhancement factors arises from the lower solubility of abiraterone compared to abiraterone acetate in FaSSIF.

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Figure 4.10. In vitro, non-sink gastric transfer dissolution profiles of neat abiraterone acetate API, neat abiraterone API, abiraterone acetate KSD, and abiraterone KSD. The red region indicates 0.01N HCl, and the blue region indicates FaSSIF. The inset represents the enlargement of the dissolution profile in FaSSIF.

To further evaluate the supersaturation ability of these KSDs, an in vitro, non-sink gastric transfer dissolution study was conducted using increased theoretical concentrations, i.e., an increased mass of KSD in an equivalent volume of dissolution media. Ester hydrolysis kinetics, along with dissolution enhancement, play a role in the supersaturation capacity of abiraterone acetate from the KSD formulation and, ultimately, its bioavailability. Therefore, a more biorelevant in vitro comparison of the abiraterone acetate KSD and the abiraterone KSD in media containing an esterase enzyme was considered. However, it was concluded that dissolution studies in media containing esterases was unnecessary, since the aim in this case was to understand the KSD formulations’ capacity for

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supersaturation, which arises strictly from the dissolution enhancement relative to the bulk crystalline drug substance.

Further, the subsequent in vivo performance comparison of these KSDs would account for ester hydrolysis kinetics. Moreover, the activity of externally added esterase enzymes in an in vitro study depends on the specific activity of the enzyme preparation as well as the somewhat arbitrary selection of enzyme concentration in the media, both of which may not be biorelevant.

Figure 4.11 shows the in vitro supersaturation ability of the abiraterone acetate KSD and the abiraterone KSD. The figure indicates that both the abiraterone acetate KSD and the abiraterone KSD linearly enhanced the dissolution of abiraterone acetate and abiraterone, respectively, with an increase in the theoretical concentration (i.e., sample loading). Surprisingly, even when the theoretical concentration was increased to ~3.7 mg/mL in 0.01N HCl, the KSD formulations continued to dissolve to completion (i.e., drive substantial supersaturation). Thus, it was concluded that the supersaturation potential of both KSDs was comparable, with achievable concentrations greater than ~3,700 µg/mL in 0.01N HCl and ~270 µg/mL in FaSSIF.

Figure 4.11. In vitro supersaturation ability of the abiraterone acetate KSD and abiraterone KSD. 210

Hence, based on a comparison of the in vitro performance and supersaturation ability of the abiraterone acetate KSD and the abiraterone KSD, it can be concluded that both KSDs provide similar performance enhancement and a similar potential for improved bioavailability.

5.5.Comparison of the in vivo performance of the abiraterone acetate KSD and the abiraterone KSD:

In order to compare the in vivo performance of the abiraterone and abiraterone acetate KSDs in a canine model, tablet formulations were developed and manufactured for both the Lot 10 KSD (the abiraterone acetate KSD tablet) and the Lot 11 KSD (the abiraterone KSD tablet) with strengths of 56 mg abiraterone acetate and 50mg abiraterone, respectively. The results of a single-dose pharmacokinetic study of these tablets and the Zytiga reference product (250 mg abiraterone acetate tablet) are listed in Table 4.4.

It can be seen that both KSD-based tablets achieved higher Cmax at one-fifth the dose compared to Zytiga. Among the KSD tablets, the abiraterone KSD tablet achieved greater

Cmax than the abiraterone acetate KSD tablet. This occurs possibly because abiraterone acetate must undergo esterase hydrolysis before in vivo absorption, while abiraterone is immediately absorbed. Thus, the absorption delay caused by esterase hydrolysis could lead to more precipitation of abiraterone acetate and reduced Cmax.

Zytiga showed a mean Tmax of 4.17 h, with high variability. Both the abiraterone acetate

KSD tablet and the abiraterone KSD tablet showed a mean Tmax of around 0.8 h. In a recent study, the oral absorption kinetics of a nano-amorphous abiraterone acetate formulation were studied, and a median Tmax in the range of 0.5–0.75 h was reported in both dogs and healthy humans [18, 19, 47]. It was concluded that the rapid absorption of this nano-amorphous abiraterone acetate formulation occurred not only because of its dissolution enhancement but also because of the enzymatic hydrolysis of abiraterone

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acetate [19]. On the contrary, since a rapid Tmax was observed for the abiraterone KSD tablet in this study, in which esterase activity has no role, it was concluded that the rapid absorption of abiraterone from the abiraterone acetate KSD tablet and the abiraterone KSD tablet is solely due to dissolution enhancement.

It was also observed that the KSD-based tablets reduced pharmacokinetic variability compared to Zytiga. The abiraterone acetate KSD tablet and the abiraterone KSD tablet were both able to enhance the oral bioavailability of abiraterone by 3.4-fold and 3.8-fold, respectively, compared to Zytiga. The total drug exposure (AUC) from both KSD-based tablets was comparable.

Table 4.4. Results from in vivo pharmacokinetic (PK) study in male beagle dogs.

Zytiga® (250 mg Lot 10 Tablet (56 Lot 11 Tablet (50 abiraterone mg abiraterone mg abiraterone) acetate) acetate) Average %CV Average %CV Average %CV

Cmax ng/mL 153.00 28.24 206.40 28.43 311.67 32.61

Tmax h 4.17 121.39 0.80 34.23 0.83 34.64

AUC(0–24 ng·h/mL 519.28 46.80 398.12 37.91 445.80 33.06 h) F Value (Dose unitless 1.0 3.4 3.8 Adjusted)

Thus, on comparison of the in vivo performance of abiraterone acetate KSD and abiraterone KSD, it can be concluded that both have similar performance and there is no additional bioavailability advantage associated with esterase hydrolysis of abiraterone acetate for this formulation approach.

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5.6.Consideration of other implications of abiraterone acetate use:

Bouhajib and Tayab developed a sensitive bioanalytical method that enabled the analysis of plasma concentrations of abiraterone and abiraterone acetate [48]. Following a single dose of 1,000 mg abiraterone acetate in humans, these authors were able to detect abiraterone acetate in plasma with a Cmax of 54.67 ± 68.30 pg/mL [48]. Haidar et al. evaluated the half maximal inhibitory concentration (IC50) values of abiraterone acetate and abiraterone for inhibiting 17α-hydroxylase/C17, 20-lyase (CYP17), a key enzyme involved in androgen production [49]. They found that the IC50 values of abiraterone acetate were much higher (110 nM and 1,600 nM) than that of abiraterone (73 nM and 220 nM) in human and rat testicular microsomes, respectively [49]. This suggests that abiraterone acetate has poor potency.

Also, it has been reported that the hydroxyl group of abiraterone (which is acetylated in abiraterone acetate) is involved in the binding of abiraterone to enzyme CYP17 binding sites 1 and 2 [50]. This further suggests reduced activity of the abiraterone acetate prodrug. The likelihood of the conversion of abiraterone acetate to abiraterone in plasma is minimal, since esterases in human plasma do not hydrolyze abiraterone acetate [6]. Thus, it would be preferable to deliver the active abiraterone moiety directly and thus eliminate the risk of circulating, poorly potent abiraterone acetate.

It has been reported that abiraterone has a minimal inhibitory effect on P-glycoproteins (P-gps), while abiraterone acetate significantly inhibits P-gps [6]. This presents a risk of negative drug–drug interactions when the prodrug abiraterone acetate is used. Additionally, the use of abiraterone acetate warrants the need for contraindication labeling for cholesterol esterase inhibiting drugs such as orlistat [19].

For ester prodrugs, the interindividual variation in esterase expression can cause variability in drug exposure [51]. The specific esterase involved in abiraterone acetate hydrolysis is yet to be identified, and the effect of its variability on drug exposure is yet

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to be studied. The direct use of the active form, abiraterone, precludes the risks associated with variable esterase expression.

6. CONCLUSION:

From this study, it was concluded that formulating amorphous solid dispersions of poorly water-soluble active drugs can eliminate the need for prodrug synthesis to improve solubility. After comparing the prodrug-based abiraterone acetate KSD to the active moiety-based abiraterone KSD, it was concluded that both are physically stable and exhibit comparable in vitro and in vivo performance. However, the use of the acetate prodrug in the KSD formulation presents risks of chemical instability and negative drug– drug interactions. Thus, formulating amorphous solid dispersions of the active abiraterone moiety is preferable, and it shows a greater potential to provide commercially viable pharmacokinetic and pharmacodynamic benefits.

7. ACKNOWLEDGEMENTS AND DISCLOSURE:

Gala acknowledges and thanks Pharmaron, China, for their support with the animal study. Williams acknowledges financial support for Gala from DisperSol Technologies, LLC. Gala and Miller are coinventors of intellectual property related to this work.

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26. Gala, U., Chapter 3- The Effect Of Drug Loading On The Properties Of Kinetisol® Processed Abiraterone-Hydroxypropyl Β Cyclodextrin Solid Dispersions. 2019. 27. Burke, D.F., et al., Active-site conformation of 17-(3-pyridyl)androsta-5,16-dien- 3β-ol, a potent inhibitor of the P450 enzyme C17α-hydroxylase/C17-20 lyase. Bioorganic & Medicinal Chemistry Letters, 1995. 5(11): p. 1125-1130. 28. Wheatley, A.M., et al., Crystal structure of abiraterone acetate (Zytiga), C26H33NO2. Powder Diffraction, 2018. 33(1): p. 72-72. 29. Hughey, J.R., et al., Preparation and characterization of fusion processed solid dispersions containing a viscous thermally labile polymeric carrier. International Journal of Pharmaceutics, 2012. 438(1): p. 11-19. 30. LaFountaine, J.S., et al., Thermal Processing of PVP- and HPMC-Based Amorphous Solid Dispersions. AAPS PharmSciTech, 2016. 17(1): p. 120-132. 31. Jermain, S.V., et al., Homogeneity of amorphous solid dispersions – an example with KinetiSol®. Drug Development and Industrial Pharmacy, 2019. 45(5): p. 724- 735. 32. Zhou, S., H. Huang, and R. Huang, Crystal structure of (3S)-3-acet-oxy-17- (pyridin-3-yl)androsta-5,16-diene. Acta crystallographica. Section E, Crystallographic communications, 2015. 71(Pt 3): p. o146-o147. 33. Solymosi, T., Development, preclinical evaluation and first-in-human clinical trial of a nano-amorphous abiraterone acetate formulation. 2019. 34. Łaszcz, M., K. Trzcińska, and E.U. Stolarczyk, Physicochemical characteristics of abiraterone acetate used for the treatment of prostate cancer. 2016. 35. Lee, Y.-E., et al., Influence of NaCl Concentration on Food-Waste Biochar Structure and Templating Effects. Energies, 2018. 11: p. 2341. 36. Gadhave, R.V., et al., Stability indicating RP-HPLC-PDA method for determination of abiraterone acetate and characterization of its base catalyzed degradation product by LC-MS. 2016. 8: p. 76-81.

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37. Chandra Reddy, B.J. and N.C. Sarada, Development and validation of a novel RP- HPLC method for stability-indicating assay of Abiraterone acetate. Journal of Liquid Chromatography & Related Technologies, 2016. 39(7): p. 354-363. 38. Karandikar, H., et al., Systematic identification of thermal degradation products of HPMCP during hot melt extrusion process. International Journal of Pharmaceutics, 2015. 486(1): p. 252-258. 39. Raymond C. Rowe, et al., Handbook of Pharmaceutical Excipients – 7th Edition. Pharmaceutical Development and Technology, 2013. 18(2): p. 544-544. 40. Saokham, P., et al., Solubility of Cyclodextrins and Drug/Cyclodextrin Complexes. Molecules, 2018. 23(5). 41. Wyttenbach, N. and M. Kuentz, Glass-forming ability of compounds in marketed amorphous drug products. European Journal of Pharmaceutics and Biopharmaceutics, 2017. 112: p. 204-208. 42. Baird, J.A., B. Van Eerdenbrugh, and L.S. Taylor, A Classification System to Assess the Crystallization Tendency of Organic Molecules from Undercooled Melts. Journal of Pharmaceutical Sciences, 2010. 99(9): p. 3787-3806. 43. Ormes, J.D., Predicting the Risk of Crystallization for Suspensions of Amorphous Spray Dried Dispersions from Structural, Thermal and Hydrophilicity Properties. 2014, University of Kansas. 44. Khedr, A., I. Darwish, and F. Bamane, Analysis of abiraterone stress degradation behavior using liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry. J Pharm Biomed Anal, 2013. 74: p. 77-82. 45. FDA, U., Highlights of prescribing information- Yonsa®. 2018. 46. Maura Murphy, P.N., William Bosch, Matthew CALLAHAN, Satya Bhamidipati, Jason Coleman, Christopher Hill, Marck Norret, Abiraterone Acetate Formulation and Methods of Use. 2016.

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47. Solymosi, T., et al., Novel formulation of abiraterone acetate might allow significant dose reduction and eliminates substantial positive food effect. 2017. 80(4): p. 723-728. 48. Bouhajib, M. and Z. Tayab, Evaluation of the Pharmacokinetics of Abiraterone Acetate and Abiraterone Following Single-Dose Administration of Abiraterone Acetate to Healthy Subjects. Clin Drug Investig, 2019. 39(3): p. 309-317. 49. Haidar, S., et al., Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo. J Steroid Biochem Mol Biol, 2003. 84(5): p. 555-62. 50. Haider, S.M., et al., Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy. Journal of Molecular Biology, 2010. 400(5): p. 1078-1098. 51. Williams, F.M., Clinical Significance of Esterases in Man. Clinical Pharmacokinetics, 1985. 10(5): p. 392-403.

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Chapter Five: Improved Apparent Solubility Of Abiraterone Leads To Enhanced Therapeutics In A Preclinical Model

1. ABSTRACT:

Abiraterone is primarily a CYP17A1 inhibitor used in treatment of metastatic castration resistant prostate cancer. In clinical studies, it has been observed that increased abiraterone exposure leads to increased probability of overall survival. In preclinical studies, it has been shown that increased abiraterone exposure leads to inhibition of secondary steroidogenic enzymes and formation of more potent Δ4 abiraterone metabolite, leading to higher androgen suppression and slower disease progression. Due to poor pharmacokinetics of the commercial abiraterone acetate formulation, the desired abiraterone exposure has not been achieved. In our previous studies, we reported the development of KinetiSol® processed solid dispersion (KSD) of abiraterone that improved abiraterone dissolution and pharmacokinetics in beagle dogs. The aim of this study was to further investigate the apparent solubility and pharmacokinetic enhancement by abiraterone KSD, and evaluate potential pharmacodynamic enhancement in a prostate cancer xenograft model. It was found that the KSD improved the apparent solubility of abiraterone by 25-96 fold at biorelevant pH. The results of a pharmacokinetic study in SCID mice demonstrated that the abiraterone KSD improved drug exposure linearly with increased dose. A tumor growth inhibition study in a 22Rv1 prostate cancer xenograft model revealed that the abiraterone KSD statistically significantly inhibited the tumor growth in a dose dependent manner as compared to control. Whereas, similar doses of neat abiraterone acetate API were unable to significantly inhibit tumor growth. Thus, this study demonstrated that the solubility and pharmacokinetic improvements enabled by the abiraterone KSD resulted in enhanced therapeutic outcomes in a preclinical prostate cancer model.

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2. GRAPHICAL ABSTRACT:

3. INTRODUCTION:

Prostate cancer is one the leading causes of cancer related death in men worldwide [1]. According to statistics presented by prostate cancer foundation, in 2019 nearly 175,000 men will be diagnosed with prostate cancer and about 32,000 men would die from the diseases in United States [2]. This equates to a new case every 3 minutes and a death every 17 minutes [2]. Such a high rate of mortality in prostate cancer patients is primarily due to metastasis of the tumor [3]. Metastatic prostate cancer (mPC) patients have a five year survival rate of merely 29.3% [4]. The first line of treatment for mPC involves androgen deprivation therapy (ADT), with or without chemotherapy [5]. In spite of a positive initial response to ADT, almost all cases of mPC stop responding to ADT and the disease progresses to a stage called castration resistant prostate cancer (CRPC) [5].

A decade ago, docetaxel was the only approved drug available for treatment in CRPC patients [6, 7]. In April 2011, abiraterone acetate, a prodrug of active drug abiraterone, was approved for treatment of metastatic castration resistant prostate cancer (mCRPC) under the commercial name Zytiga® [8]. Abiraterone is a selective and irreversible 221

inhibitor of enzyme 17α-hydroxylase/C17, 20-lyase (CYP17A1), which is involved in production of androgens [9]. It acts primarily by reducing androgen production in the testicular, adrenal, and prostatic tumor tissues, thereby leading to slower disease progression [10, 11]. A recent report suggests, that abiraterone can exhibit competitive androgen receptor antagonism in tumor xenografts expressing the mutation AR-F876L [12].

While a complete understanding of the relationship between abiraterone exposure to its therapeutic effect is yet to be developed, several reports have shown promising efficacy results with increased abiraterone exposure [6]. An exploratory analysis was performed on data from the pivotal study COU-AA-301, to find a relationship between abiraterone pharmacokinetics and pharmacodynamics [13]. Patients were subdivided into two groups with abiraterone trough concentration at steady state (Cmin_ss) ≤ 11.0 ng/mL and Cmin_ss >11.0 ng/mL. These researchers reported better overall survival (OS) for patients with higher Cmin_ss ; however, since there was no statistically significant separation between two groups, they concluded no further effect with higher Cmin_ss [13]. It should be noted that there were only a few data points (i.e. pharmacokinetic data was only available for 15% of patients) and the median value of 11.0 ng/mL selected for this analysis represented <10% of the highest value seen for C min_ss in the study. A predicted probability of OS v/s Cmin plot was developed based on a tumor growth inhibition (TGI) model to encompass the entire range of observed Cmin values, and a clear separation was seen between groups, i.e., predicted probability of OS increased with increased Cmin [14, 15].

Prostate specific antigen (PSA) is used as a surrogate biomarker for abiraterone treatment, and a decline in PSA level is indicative of effective treatment [16]. Upon analyzing the data from pivotal studies COU-AA-301 and COU-AA-302, Xu et al. showed that all patients with Cmin_ss greater than or equal to 40.0 ng/mL had high PSA decay rate [17]. In a prospective observational study in mCRPC patients treated with abiraterone acetate, Carton et al. observed that patients with high abiraterone exposure 222

had a median progression free survival (PFS) value of 12.2 months; whereas, those with lower abiraterone exposure had a median PFS value of only 7.4 months [18]. Similar observations were made by Friedlander et al., wherein the patients responding to abiraterone treatment (i.e. having PSA decline) had higher Cmin_ss as compared to those patients which were non-responders and had become resistant to abiraterone [19]. In a case study reported by Woei-A-Jin et al., they saw a rapid increase in PSA levels, radiographic progression and worsening of disease, when they decreased the dose of abiraterone acetate which led to lower Cmin [20]. They were able to stabilize the patient only when the dose was increased, leading to a higher Cmin of 73.3 µg/L [20]. All these studies clearly indicate that higher abiraterone exposure leads to slower disease progression.

There are only a few studies exploring the effect of higher abiraterone exposure, and thus the complete mechanism of action is yet to be explored. With current abiraterone acetate treatment, i.e., Zytiga at a 1,000 mg daily dose, urinary androgen metabolites are detectable [21]. This suggests that the inhibition of CYP17A1 is partial, with current treatment leading to the formation of residual androgens and disease progression [22]. Thus, it is likely that complete inhibition CYP17A1 can be achieved by higher abiraterone exposure. Additionally, it has been suggested that resistance to abiraterone is developed through induction of steroidogenic enzymes, androgen receptors and androgen receptor splice variants [23]. Li et al. suggested that an increase in abiraterone exposure can lead to inhibition of steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (3βHSD) and can thus reverse the resistance developed at current abiraterone exposures achieved by Zytiga [22]. In recent studies, it has been discovered that 3βHSD isozyme converts abiraterone to more potent metabolite Δ4 abiraterone (D4A), which not only blocks more steroidogenic enzymes, but also directly antagonizes androgen receptors [24, 25]. Thus, all these studies suggest that increased abiraterone exposure leads to better androgen suppression and slower disease progression. Figure 5.1, derived from available literature, illustrates the effect of increased abiraterone exposure on its

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pharmacodynamics. Primarily, increased abiraterone exposure leads to complete inhibition of CYP17A1, but it also inhibits 3βHSD and gets converted to more potent metabolite D4A. D4A inhibits CYP17A1, 3βHSD and the androgen receptor.

Figure 5.1. Effect of increased abiraterone exposure on its pharmacodynamics [22, 24- 26]. [Blue - action of abiraterone at its standard exposure, Red and Green- action of abiraterone and its potent metabolite D4A at elevated exposure]; [CYP17A1 - 17α-hydroxylase/C17, 20-lyase; 3βHSD - 3β-hydroxysteroid dehydrogenase; AKR31C - Aldo-keto reductase family 1 member C3; PSA - Prostate specific antigen]

While increasing abiraterone exposure increases its efficacy, it can also impact its safety. Friedlander et al. suggested that higher doses of abiraterone acetate, i.e., Zytiga at 1,000 mg twice daily, was well tolerated [19]. Also, the adverse events noted for lower abiraterone exposure (mean Cmin_ss = 6.2 ng/mL) groups, are similar and acceptable,

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when compared to higher abiraterone exposure (mean Cmin_ss = 35.2 ng/mL) groups [13]. Thus, abiraterone is likely to be safe at higher exposure, but studies are needed to find its maximum tolerable dose (MTD) in humans.

The intuitive way to increase abiraterone exposure would be to increase Zytiga’s dose. However, Zytiga has poor oral bioavailability, high pharmacokinetic variability and high food effect [10, 13, 27]. When Zytiga’s dose was doubled from 1,000 mg to 2,000 mg, an increase of only 8% in the mean area under plasma concentration-time curve (AUC) was observed [10]. Another way to increase abiraterone exposure, is to administer Zytiga with low to high fat meals [28]. However, this approach is inherently risky, since diet of entire patient population cannot be controlled and there is increased chance of unpredictable and variable drug exposure [29]. Thus, due to formulation limitations, Zytiga is unable to deliver higher abiraterone exposure.

In our previous studies, we described the development of a KinetiSol® processed amorphous solid dispersion (KSD) of abiraterone with hydroxypropyl β cyclodextrin (HPBCD) [30-32]. We demonstrated that the abiraterone KSD increased the oral bioavailability of abiraterone and improved its overall pharmacokinetics in a healthy canine model [30-32].

Based on these results, it was hypothesized that if the abiraterone KSD could also enhance the drug’s solubility and bioavailability in a preclinical xenograft model then a concurrent improvement in pharmacodynamics, i.e., greater tumor response, would also be observed. Thus, the objective of the current study is to investigate the apparent solubility enhancement by abiraterone KSD and study its impact on abiraterone pharmacokinetics and pharmacodynamics in a preclinical xenograft model.

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4. MATERIAL AND METHODS:

4.1.Materials:

Abiraterone active pharmaceutical ingredient (API) was purchased from MSN Laboratories Pvt. Ltd. (Telangana, India). Abiraterone acetate active pharmaceutical ingredient (API) was purchased from Attix Pharma (Ontario, Canada). Hydroxypropyl β cyclodextrin i.e. Kleptose® HPB was purchased from Roquette America (USA). The solvents used for HPLC analysis were of HPLC grade. All other chemicals and reagents used for solubility study and HPLC analysis were of ACS grade.

4.2.Methods:

4.2.1. Abiraterone KSD Preparation:

Abiraterone KSD was prepared according to the method mentioned in our previous study [31, 32]. Briefly, prior to compounding, the 10% w/w abiraterone and 90% w/w HPBCD were accurately weighed and thoroughly mixed to prepare the physical mixture. The physical mixture was then divided into sub-lots and charged into the KinetiSol manufacturing compounder chamber, operated in semi-continuous mode. Inside the chamber, a shaft with protruding blades was rotated at varying incremental speeds ranging from 2400 rpm to 2700 rpm, without external heat addition, to impart frictional and shear forces to the sample material. The temperature of the mass was monitored using an infrared probe. When molten mass temperature reached a value of 180 °C, the mass was rapidly ejected, collected, and pressed between two stainless steel plates to rapidly quench the sample. The resultant sample was milled and sieved through a #60 mesh screen to yield KSD powder of particle size ≤ 250 μm.

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4.2.2. Solubility Study:

The solubility study was carried out at Ceutix Labs (California, USA). Four media varying in pH, i.e., 0.1 N HCl (pH= 1.03), 0.03 N HCl (pH= 1.65), 0.01 N HCl (pH= 2.08) and 50mM phosphate buffer (pH= 6.8) were prepared. 10 to 15 mL of each medium was added into a scintillation vial containing two ¼ inch Teflon balls and an excess of neat abiraterone API or abiraterone KSD were added to respective vials. The vials were placed in an incubator shaker i.e. New Brunswick™ I24R shaker (New Brunswick Scientific, New Jersey, USA), and agitated at 37°C and 160 rpm for 24 hrs. At 24 hr, 1000 µL aliquots were pulled from the vials and centrifuged. The clear supernatant was then diluted using methanol : water (9:1 v/v) diluent. These samples were then analyzed for dissolved abiraterone content by high-performance liquid chromatography (HPLC).

4.2.3. Pharmacokinetic Study:

The pharmacokinetic study was carried out at Charles River Laboratories (Massachusetts, USA). This animal study was conducted according to an approved Charles River Laboratories IACUC protocol (#20111396). Male CB.17 severe combined immunodeficient (SCID) mice (~ 5 – 8 weeks of age) were used for the study. The mice (n=3) were randomly assigned to test arms based on test articles, dose levels and time points (Table 5.1). A freshly prepared stock solution of 0.5% methylcellulose aqueous solution, adjusted to pH 2, was used as a vehicle. The mice were fasted overnight. Prior to dose administration the body weights of mice were recorded. The test articles (neat abiraterone acetate API and abiraterone KSD in vehicle) were prepared as per the concentrations mentioned in Table 5.1 and administered to mice via oral gavage. At defined whole blood collection time points, the mice were euthanized and maximum obtainable blood volume was drawn via cardiac puncture. The blood was placed into tubes containing sodium heparin anticoagulant. The blood samples were centrifuged to isolate the plasma. The plasma samples were then analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for abiraterone content.

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4.2.4. Pharmacokinetic Analysis:

4.2.5. Xenograft Model Development:

The 22Rv1 human prostate tumor xenograft model was developed at Translational Drug Development (TD2) (Arizona, USA). The protocol # TD4198 was reviewed and approved by TD2 IACUC, Arizona. Male CB.17 SCID mice were sourced from Charles River Laboratories (Massachusetts, USA). 22Rv1 tumor cells were sourced from American Type Culture Collection i.e. ATCC (Virginia, USA). The mice were inoculated in the subcutaneous right flank with 0.1 mL of a 50% Media / 50% Matrigel® mixture containing a suspension of 5x106 cells/mouse of live 22Rv1 tumor cells. At time of inoculation, the mice were 6 weeks old. Tumor bearing animals were monitored and tumors were measured periodically until they reached designated start size. The tumor dimensions of 22Rv1 xenograft model i.e. tumor bearing mice was measured using digital calipers and recorded in study management software called Study Director version 3 (Studylog, California, USA). The tumor volume was calculated utilizing the following formula: 푎 × 푏2 푇푢푚표푟 푉표푙푢푚푒 (푚푚3) = 2 where ‘b’ is the smallest diameter and ‘a’ is the largest diameter [33]. After 16th day following inoculation, fifty mice with tumor sizes of 93-186 mm3 were randomized into five groups of ten mice, each with a mean tumor volume of 132 mm3, by random equilibration method using the Study Director software.

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4.2.6. Tumor Growth Inhibition Study:

The tumor growth inhibition study was carried out at TD2 (Arizona, USA). The protocol # TD4198 was reviewed and approved by TD2 IACUC, Arizona. The study design is shown in table 5.2. The study contained a total of five groups (n=10 animals/group) which were a vehicle control group and four test article groups. Freshly prepared 0.5% methylcellulose aqueous solution, adjusted to pH 2 was used as vehicle. The test articles were freshly prepared daily, just prior to dosing. To prepare the test articles, the neat abiraterone acetate API and abiraterone KSD were mixed in vehicle using a magnetic stirrer, to prepare uniform suspension/ solution. The dosing was performed via oral gavage over 3-5 seconds for all groups beginning on Day 1, and continued daily to study end, as described in table 5.2. Food was withdrawn from the mice two hours prior to dosing and returned at four hours post dose. Nutritional supplementation was provided starting on Day 8 to all mice due to observed body weight loss. Tumor volumes and body weights were recorded when the mice were randomized and were taken twice weekly thereafter. Clinical observations were made daily. The study was ended on Day 26 when the vehicle control group reached endpoint of mean tumor volume exceeding 1500 mm3.

4.2.7. Tumor Growth Inhibition Analysis:

The mean tumor growth inhibition (TGI) was calculated for Day 26 (the final day of the study) utilizing the following formula:

(푋̅푇푟푒푎푡푒푑(퐷푎푦 푓𝑖푛푎푙) − 푋̅푇푟푒푎푡푒푑(퐷푎푦 1)) 푇퐺퐼 = [1 − ] × 100 (푋̅푉푒ℎ𝑖푐푙푒 퐶표푛푡푟표푙(퐷푎푦 푓𝑖푛푎푙) − 푋̅푉푒ℎ𝑖푐푙푒 퐶표푛푡푟표푙(퐷푎푦 1)) where 푋̅ is the mean tumor volume. All statistical analyses in the xenograft study were performed with Prism GraphPad® software version 6 (GraphPad®, California, USA). Differences in Day 26 tumor volumes of each test article group and the vehicle control group were confirmed using a two-tailed Student’s t-test with Welch’s correction.

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5. RESULTS AND DISCUSSION:

5.1.Apparent solubility of neat abiraterone API and abiraterone KSD:

Abiraterone is weakly basic in nature and has pKa of 4.81 [34]. Thus, it ionizes at lower pH and hence has higher solubility in acidic media. Figure 5.2 illustrates the apparent solubility of neat abiraterone API and abiraterone KSD across different pH. It can be seen that across entire range of pH tested, the neat abiraterone API, being crystalline in nature, has lower solubility as compared to the abiraterone KSD which is amorphous. At lower pH (≤ 2), abiraterone KSD enhanced apparent solubility by ~96 fold, and at higher pH of 6.8, the abiraterone KSD enhanced apparent solubility by ~25 fold. Under fasting conditions, the pH of the gastrointestinal tract ranges from 1.0 to 6.5 [35]. Thus, the abiraterone KSD enhances the apparent solubility of abiraterone across entire biorelevant pH range, and thus is likely to cause higher flux of abiraterone across the gastrointestinal membrane, leading to higher oral bioavailability.

Figure 5.2. Apparent solubility of neat abiraterone API and abiraterone KSD across different pH. 230

5.2.Pharmacokinetic performance of neat abiraterone acetate API and abiraterone KSD:

Since the overall goal of this study was to investigate the effect of improved pharmacokinetics on pharmacodynamics, a male SCID mouse model, which was to be used to develop the xenograft model, was chosen for a pharmacokinetic study. This was done to eliminate the impact of species variation on study results and assist in appropriate dose selection for pharmacodynamic study. The pharmacokinetic study design is shown in table 5.1. Three dose levels were chosen to cover a wide range of drug exposure and to evaluate dose-exposure relationship.

Table 5.1. Pharmacokinetic study design.

Whole (Abiraterone acetate / Abiraterone) Blood Group Test No. of Test Dose Dose Dose collectio No. Arm animals Article Level Concentration Volume n Time (mg/kg) (mg/mL) (mL/kg) point 1.1 5 min 3 1.2 30 min 3 1.3 1hr 3 Neat 1.4 2 hr 3 1 abiraterone 10 1 10 1.5 4 hr 3 acetate API 1.6 6 hr 3 1.7 8 hr 3 1.8 12 hr 3 2.1 5 min 3 2.2 30 min 3 Neat 2 2.3 1hr 3 abiraterone 50 5 10 2.4 2 hr 3 acetate API 2.5 4 hr 3

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Table 5.1 continued Whole (Abiraterone acetate / Abiraterone) Blood Group Test No. of Test Dose Dose Dose collectio No. Arm animals Article Level Concentration Volume n Time (mg/kg) (mg/mL) (mL/kg) point 2.6 6 hr 3 2.7 8 hr 3 2.8 12 hr 3 3.1 5 min 3 3.2 30 min 3 3.3 1hr 3 Neat 3.4 2 hr 3 3 abiraterone 100 10 10 3.5 4 hr 3 acetate API 3.6 6 hr 3 3.7 8 hr 3 3.8 12 hr 3 4.1 5 min 3 4.2 30 min 3 4.3 1hr 3 4.4 2 hr 3 Abiraterone 4 10 1 10 4.5 4 hr 3 KSD 4.6 6 hr 3 4.7 8 hr 3 4.8 12 hr 3 5.1 5 min 3 5.2 30 min 3 5.3 1hr 3 Abiraterone 5 50 5 10 5.4 2 hr 3 KSD 5.5 4 hr 3 5.6 6 hr 3

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5.8 12 hr 3 6.1 5 min 3 6.2 30 min 3 6.3 1hr 3 6.4 2 hr 3 Abiraterone 6 100 10 10 6.5 4 hr 3 KSD 6.6 6 hr 3 6.7 8 hr 3 6.8 12 hr 3

Figure 5.3 shows the pharmacokinetic performance and Figure 5.4 shows the dose- exposure relationship of neat abiraterone acetate API and abiraterone KSD. From figure 5.3 it can be seen that all three groups of abiraterone KSD had higher overall abiraterone exposure as compared to corresponding groups of neat abiraterone acetate API.

Interestingly, the Tmax for all three groups of abiraterone KSD and two lower dose groups of neat abiraterone acetate API was 0.5 hr. Only the high dose group of neat abiraterone acetate API had a Tmax of 1.0 hr. For the abiraterone KSD groups, an earlier

Tmax is expected due to its rapid dissolution rate and supersaturation ability. The abiraterone acetate API, must undergo esterase catalyzed hydrolysis prior to absorption, and thus this could have caused a delayed Tmax for the high dose group of neat abiraterone acetate API [36]. In non-clinical pharmacokinetic studies of neat abiraterone

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acetate API in mice, rats and monkeys the Tmax was seen within 1 to 2 hr after dosing [14].

Most pharmacokinetic-pharmacodynamic models developed from patient data utilize Cmin or Cmin_ss as the metric for pharmacokinetics/drug exposure because it is logistically easier to obtain trough plasma concentrations of patients [37]. However, it is inaccurate to assume that any single pharmacokinetic parameter governs the pharmacodynamics without extensive pharmacokinetic-pharmacodynamic studies [38].

Also, it is important to note that if a drug has a perfectly linear dose-exposure relationship, then merely changing the doses will not be indicative of the pharmacokinetic driver, since in this case the Cmax, Cmin and AUC will change proportionately [38]. Thus, more complex study designs such as changing dose regimen along with dose levels would be needed.

Hence, in this study we did not aim to identify a particular pharmacokinetic driver, but instead investigated the impact of abiraterone KSD on all key pharmacokinetic parameters. From figure 5.4, it can be seen that the abiraterone KSD groups had higher

AUC0-12hr, Cmax and Cmin/C12hr as compared to corresponding dose levels for the neat abiraterone acetate API groups. For both sets of groups, the AUC0-12hr, Cmax and

Cmin/C12hr increased with increase in dose. However, the abiraterone KSD groups showed better dose- exposure linearity for AUC0-12hr as compared to neat abiraterone acetate API groups. Similar observations were reported for neat abiraterone acetate API pharmacokinetics in animals such as mice, rats and monkeys, wherein the plasma concentration of abiraterone increased with abiraterone acetate dose, but less than dose proportionally [14]. Overall, the apparent solubility enhancement by abiraterone KSD led to improved pharmacokinetics of abiraterone in the mouse model.

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Figure 5.3. Pharmacokinetic performance of neat abiraterone acetate API and abiraterone KSD in male SCID mice.

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Figure 5.4. Dose- exposure relationship of neat abiraterone acetate API and abiraterone KSD in male SCID mice.

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5.3.Pharmacodynamic performance of neat abiraterone acetate API and abiraterone KSD:

The pharmacodynamic performance was evaluated in a 22Rv1 xenograft model developed in male SCID mice, and the study design is shown in table 5.2. The 22Rv1 tumor cell line was chosen since it is representative of castration resistant prostate cancer [39, 40]. From the dose-exposure plot for AUC (see Figure 5.4 a) it can be seen that a 22.4 mg/kg dose of neat abiraterone acetate API confers a drug exposure equivalent to the standard Zytiga dose in humans [10]. Thus, 22.4mg/kg was chosen as the low dose for neat abiraterone acetate API group. A high dose of 100 mg/kg was chosen for neat abiraterone acetate API group, since it was found to be tolerable in the pharmacokinetic study mentioned above. Corresponding abiraterone equivalent doses were selected for abiraterone KSD groups.

The results of the pharmacodynamic investigation of neat abiraterone acetate API and abiraterone KSD are shown in Figure 5.5 and Table 5.3. From figure 5.5, it can be seen that significant separation between the vehicle control group and some treatment groups started from Day 19. On Day 22, the mean tumor volumes of all treatment groups were lower than that of vehicle control group. On Day 26, the vehicle control group reached a mean tumor volume of 1500mm3, and hence the study was ended.

On Day 26, the low dose neat abiraterone acetate API group had reduced mean tumor volume as compared to control group, however high variability rendered the result statistically insignificant. Contrastingly, the high dose neat abiraterone acetate API group had higher mean tumor volume as compared to the control group, yet it too showed high variability. Both abiraterone KSD dose groups showed lower mean tumor volume as compared to the control group with less variability. The lower variability seen from the abiraterone KSD groups can be attributed to consistent drug exposure enabled by the KSD formulation. Neither of the neat abiraterone acetate API groups were able to statistically significantly inhibit the tumor growth as compared to control group.

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Whereas, both abiraterone KSD groups were able to statistically significantly inhibit the tumor growth as compared to control group.

The tumor growth inhibition for the high dose neat abiraterone acetate API group was non reportable since it showed higher tumor volume as compared to, not only the control group, but also the low dose neat abiraterone acetate API group. The reason for this conflicting observation could be that on repeated high doses of poorly soluble neat abiraterone acetate API, it would accumulate in gastrointestinal tract of the mice and lead to precipitation of subsequent doses. This ultimately culminates into lower drug exposure at steady state. Motwani et al. reported a TGI of -16% (mean tumor volume of treatment group greater than that of control group) in 22Rv1 xenograft model, after 200 mg/kg of abiraterone acetate dose, once a day, for 40 days [41]. The highest tumor growth inhibition of 33.1% was seen for high dose abiraterone KSD group. This suggests that increased drug exposure enabled by a high dose of abiraterone KSD, led to increased pharmacodynamic performance.

Giatromanolaki et al. reported that CY17A1 expression is significantly reduced in 22Rv1 cells after abiraterone exposure suggesting that the primary mode of action of abiraterone is CY17A1 inhibition in 22Rv1 cells [42]. While we saw appreciable and statistically significant tumor growth inhibition in the high dose abiraterone KSD group, the result was far from a complete response (>90% tumor reduction). This could be because of abiraterone-resistance developed in 22Rv1 cells. It has been reported that 22Rv1 cells can express androgen receptor splice variant ARV-7, that can make 22Rv1 tumor cells resistant to abiraterone [43-45].

From in-life observations, mild to moderate hunched posture and emaciation was reported in conjunction with body weight loss in some animals. Tumor necrosis was seen throughout the groups, which is typical for this cell line, but non-impactful on study results. No major treatment related toxicities were observed, thus suggesting that the experimental doses were below toxicity limits.

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Overall, it can be concluded that increased abiraterone exposure enabled by the KSD formulation leads to greater tumor growth inhibition in a 22Rv1 mouse xenograft model.

Table 5.2. Tumor growth inhibition study design.

Dose (mg/kg) Group No. of [Abiraterone Test Article Dosing Regimen No. animals acetate/ Abiraterone] None (Vehicle 1 10 Not applicable QD to end control)

Neat abiraterone 2 10 22.4 QD to end acetate API

Neat abiraterone 3 10 100.0 QD to end acetate API

4 10 Abiraterone KSD 20.0 QD to end

5 10 Abiraterone KSD 89.2 QD to end

QD= Once a day

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Figure 5.5. Pharmacodynamic performance of neat abiraterone acetate API and abiraterone KSD in 22Rv1 xenograft model.

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Table 5.3. Results of tumor growth inhibition study.

Dose (mg/kg) Day 26 Mean Student's t Group [Abiraterone Tumor Volumes Day 26 % Test Article test - No. acetate/ (mm3) ± TGI (n) p value Abiraterone] Standard Error None (Vehicle 1 Not applicable 1502.6 ± 75.0 - control) Neat abiraterone 2 22.4 1264.2 ± 129.9 17.4 (10) 0.165 acetate API Neat abiraterone 3 100.0 1549.4 ± 149.6 NR 0.757 acetate API 4 Abiraterone KSD 20.0 1203.8 ± 67.5 21.8 (10) 0.014 5 Abiraterone KSD 89.2 1048.4 ± 41.2 33.1 (10) <0.001 NR-not reportable

6. CONCLUSION:

From this study, it was concluded that increased apparent solubility of abiraterone enabled by the KSD formulation led to enhanced pharmacokinetics in SCID mice. Improved abiraterone pharmacokinetics ensured higher drug exposure resulting in significant tumor growth inhibition in the 22Rv1 xenograft model. Thus, this study suggests the potential for the abiraterone KSD formulation to improve therapeutic outcomes for mCRPC patients.

7. ACKNOWLEDGEMENTS AND DISCLOSURE:

Gala acknowledges and thanks Ceutix Labs, California, Charles River Laboratories, Massachusetts and Translational Drug Development, Arizona for their support with the

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study. Williams acknowledges financial support for Gala from DisperSol Technologies, LLC.

Parts of this manuscript were presented in a symposium at the Annual Meeting of the American Association of Pharmaceutical Scientists (AAPS), 2018, Washington DC and in a poster at AAPS PharmSci 360 Meeting, 2019, San Antonio. Gala and Miller are coinventors on intellectual property related to this work.

8. REFERENCES:

1. Rawla, P., Epidemiology of Prostate Cancer. World journal of oncology, 2019. 10(2): p. 63-89. 2. Foundation, P.C. Top 10 Things You Should Know About Prostate Cancer. 2019; Available from: https://www.pcf.org/c/top-10-things-you-should-know-about- prostate-cancer/. 3. Nandana, S. and L.W. Chung, Prostate cancer progression and metastasis: potential regulatory pathways for therapeutic targeting. American journal of clinical and experimental urology, 2014. 2(2): p. 92-101. 4. Damodaran, S., C.E. Kyriakopoulos, and D.F. Jarrard, Newly Diagnosed Metastatic Prostate Cancer: Has the Paradigm Changed? The Urologic clinics of North America, 2017. 44(4): p. 611-621. 5. Dawson, N.A., C.J. Ryan, and J.P. Richie, Castration-resistant prostate cancer: Treatments targeting the androgen pathway. 2013. 6. Benoist, G.E., et al., Pharmacokinetic Aspects of the Two Novel Oral Drugs Used for Metastatic Castration-Resistant Prostate Cancer: Abiraterone Acetate and Enzalutamide. Clin Pharmacokinet, 2016. 55(11): p. 1369-1380. 7. Dong, L., et al., Metastatic prostate cancer remains incurable, why? Asian J Urol, 2019. 6(1): p. 26-41.

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8. Belfort, G.M.H., Boyd L. and Botella, GabrielMartinez Abiraterone Acetate (Zytiga®): An Inhibitor of CYP17 as a Therapeutic for Castration-Resistant Prostate Cancer, in Successful Drug Discovery. 2016. p. 115-135. 9. Attard, G., et al., Phase I clinical trial of a selective inhibitor of CYP17, abiraterone acetate, confirms that castration-resistant prostate cancer commonly remains hormone driven. J Clin Oncol, 2008. 26(28): p. 4563-71. 10. FDA, U. Highlights of prescribing information- Zytiga®. 2011-19. 11. Rehman, Y. and J.E. Rosenberg, Abiraterone acetate: oral androgen biosynthesis inhibitor for treatment of castration-resistant prostate cancer. Drug Des Devel Ther, 2012. 6: p. 13-8. 12. Norris, J.D., et al., Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer. The Journal of clinical investigation, 2017. 127(6): p. 2326-2338. 13. FDA, U., Clinical pharmacology and biopharmaceutics review(s)- Zytiga®. 2010. 14. EMA, Assessment Report For Zytiga. 2011. 15. TGA, Australian Public Assessment Report for abiraterone acetate. 2012. 16. Xu, X.S., et al., Correlation between Prostate-Specific Antigen Kinetics and Overall Survival in Abiraterone Acetate-Treated Castration-Resistant Prostate Cancer Patients. Clin Cancer Res, 2015. 21(14): p. 3170-7. 17. Xu, X.S., et al., Modeling the Relationship Between Exposure to Abiraterone and Prostate-Specific Antigen Dynamics in Patients with Metastatic Castration- Resistant Prostate Cancer. Clin Pharmacokinet, 2017. 56(1): p. 55-63. 18. Carton, E., et al., Relation between plasma trough concentration of abiraterone and prostate-specific antigen response in metastatic castration-resistant prostate cancer patients. Eur J Cancer, 2017. 72: p. 54-61. 19. Friedlander, T.W., et al., High-Dose Abiraterone Acetate in Men With Castration Resistant Prostate Cancer. Clin Genitourin Cancer, 2017. 15(6): p. 733-741.e1.

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20. Woei-A-Jin, F.J.S.H., et al., Dose Reduction May Jeopardize Efficacy of Abiraterone Acetate. Journal of Clinical Oncology, 2018. 36(30): p. 3062-3064. 21. Attard, G., et al., Clinical and biochemical consequences of CYP17A1 inhibition with abiraterone given with and without exogenous glucocorticoids in castrate men with advanced prostate cancer. J Clin Endocrinol Metab, 2012. 97(2): p. 507-16. 22. Li, R., et al., Abiraterone inhibits 3beta-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer. Clin Cancer Res, 2012. 18(13): p. 3571-9. 23. Mostaghel, E.A., et al., Resistance to CYP17A1 inhibition with abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants. Clin Cancer Res, 2011. 17(18): p. 5913-25. 24. Li, Z., et al., Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature, 2015. 523(7560): p. 347-51. 25. Li, Z., et al., Redirecting abiraterone metabolism to fine-tune prostate cancer anti-androgen therapy. Nature, 2016. 533(7604): p. 547-51. 26. Crona, D.J. and Y.E. Whang, Androgen Receptor-Dependent and -Independent Mechanisms Involved in Prostate Cancer Therapy Resistance. Cancers, 2017. 9(6): p. 67. 27. Chi, K.N., et al., Food effects on abiraterone pharmacokinetics in healthy subjects and patients with metastatic castration-resistant prostate cancer. J Clin Pharmacol, 2015. 55(12): p. 1406-14. 28. Szmulewitz, R.Z., et al., Prospective International Randomized Phase II Study of Low-Dose Abiraterone With Food Versus Standard Dose Abiraterone In Castration-Resistant Prostate Cancer. J Clin Oncol, 2018. 36(14): p. 1389-1395. 29. Todd, M., et al., Fast and flawed or scientifically sound: the argument for administering oral oncology drugs during fasting. J Clin Oncol, 2012. 30(8): p. 888-9; author reply 889.

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30. Gala, U., Chapter 2- Improved Dissolution And Pharmacokinetics Of Abiraterone Through KinetiSol® Enabled Amorphous Solid Dispersion 2019. 31. Gala, U., Chapter 3- The Effect Of Drug Loading On The Properties Of Kinetisol® Processed Abiraterone-Hydroxypropyl Β Cyclodextrin Solid Dispersions. 2019. 32. Gala, U., Chapter 4- Comparing the KinetiSol® processed amorphous solid dispersions of Abiraterone acetate and Abiraterone: Is the Prodrug necessary? 2019. 33. Euhus, D.M., et al., Tumor measurement in the nude mouse. J Surg Oncol, 1986. 31(4): p. 229-34. 34. Drugbank. Abiraterone. 2007 09/05/2019]; Available from: https://www.drugbank.ca/drugs/DB05812. 35. Gala, U.H., D.A. Miller, and R.O. Williams, Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2020. 1873(1): p. 188319. 36. Stappaerts, J., et al., Rapid conversion of the ester prodrug abiraterone acetate results in intestinal supersaturation and enhanced absorption of abiraterone: In vitro, rat in situ and human in vivo studies. European Journal of Pharmaceutics and Biopharmaceutics, 2015. 90: p. 1-7. 37. Giles, F.J., et al., Nilotinib population pharmacokinetics and exposure-response analysis in patients with imatinib-resistant or -intolerant chronic myeloid leukemia. European Journal of Clinical Pharmacology, 2013. 69(4): p. 813-823. 38. Tuntland, T., et al., Implementation of pharmacokinetic and pharmacodynamic strategies in early research phases of drug discovery and development at Novartis Institute of Biomedical Research. Frontiers in pharmacology, 2014. 5: p. 174-174. 39. Sramkoski, R.M., et al., A new human prostate carcinoma cell line, 22Rv1. In Vitro Cell Dev Biol Anim, 1999. 35(7): p. 403-9. 40. Cunningham, D. and Z. You, In vitro and in vivo model systems used in prostate cancer research. J Biol Methods, 2015. 2(1). 245

41. Motwani, V.B., Dorothy; Huang, Liyue; Pantano, Chloe; Estanek,Vania; Keats,Jeffrey A.; Rodrigues,Lindsey U.; Stickland, Kimberly; Dransfield, Daniel T. ; Raimondi , Alejandra Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion. 2019, Epizyme. 42. Giatromanolaki, A., et al., CYP17A1 and Androgen-Receptor Expression in Prostate Carcinoma Tissues and Cancer Cell Lines. Current Urology, 2019. 13(3): p. 157-165. 43. Bovinder Ylitalo, E. and P. Wikström, Mechanisms behind tumor relapse in 22Rv1 xenografts after treatment with abiraterone or cabazitaxel. Endocrine Abstracts, 2016. 44. He, Y., et al., Androgen receptor splice variants bind to constitutively open chromatin and promote abiraterone-resistant growth of prostate cancer. Nucleic acids research, 2018. 46(4): p. 1895-1911. 45. Pham, S., et al., Next-generation steroidogenesis inhibitors, dutasteride and abiraterone, attenuate but still do not eliminate androgen biosynthesis in 22RV1 cells in vitro. The Journal of Steroid Biochemistry and Molecular Biology, 2014. 144: p. 436-444.

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Chapter Six: Concluding Remarks And Future Direction

1. DISSERTATION CONCLUSION:

A majority of anticancer drugs have inherent poor water solubility that imparts suboptimal pharmacokinetics and limits their therapeutic potential. Amorphous solid dispersion (ASD) is a promising formulation technology that can not only improve the pharmacokinetics of poorly water soluble anticancer drugs but can also enhance their pharmacodynamics. One such anticancer drug is abiraterone, whose complete therapeutic potential in the treatment of prostate cancer has not been realized, due to its oral delivery limitations. This study, for the first time, demonstrated development of an abiraterone ASD through implementation of the KinetiSol® Technology. Also, this study revealed that short chain oligomers like hydroxypropyl β cyclodextrin (HPBCD), can be processed by KinetiSol. This study presented a new solvent free platform to make drug- cyclodextrin complexes. This study also showed that KinetiSol processed ASDs (i.e. KSD) can eliminate the need for synthesis of a prodrug for solubility enhancement. The abiraterone KSD developed in this study showed significant improvement in abiraterone pharmacokinetics and pharmacodynamics in a preclinical prostate cancer model.

The abiraterone KSD developed in this study was investigated in pharmacokinetic studies in healthy human subjects by DisperSol Technologies LLC, Georgetown, Texas [1]. The pharmacokinetic studies in fasted healthy human subjects demonstrated that the abiraterone KSD based tablet improved drug exposure by an impressive 4 to 6 fold as compared to Zytiga (commercial abiraterone acetate tablet) [1]. Also the abiraterone KSD based tablet eliminated the undesirable food effect seen with Zytiga [1].

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2. FUTURE DIRECTION:

DisperSol Technologies will be investigating the abiraterone KSD based formulation in metastatic castration resistant prostate cancer (mCRPC) patients [1]. It is hypothesized that the abiraterone KSD based formulation will have a high tolerable dose and would lead to slower disease progression. Thus, the abiraterone KSD developed in this study is expected to improve overall survival for prostate cancer patients.

Also, several reports have indicated that abiraterone may benefit treatment of androgen receptor positive breast cancer and ovarian cancer [2-6]. Hence, the abiraterone KSD formulation may also have potential application in breast cancer and ovarian cancer treatment. Overall, the abiraterone KSD product developed in this study has the potential to improve lives of millions of cancer patients worldwide (Figure 6.1).

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Figure 6.1. Illustrative summary of the dissertation. 249

3. REFERENCES: 1. Keen, J.M. Case studies in human bioavailability enhancement with KinetiSol®. in AAPS Pharm Sci 360. 2019. San Antonio. 2. Grellety, T., et al., Enhancing Abiraterone Acetate Efficacy in Androgen Receptor-positive Triple-negative Breast Cancer: Chk1 as a Potential Target. Clin Cancer Res, 2019. 25(2): p. 856-867. 3. Grellety, T., et al., Long-Term Complete Response of an Androgen Receptor– Positive Triple-Negative Metastatic Breast Cancer to Abiraterone Acetate. JCO Precision Oncology, 2018(2): p. 0-0. 4. Schweizer, M.T. and E.Y. Yu, AR-Signaling in Human Malignancies: Prostate Cancer and Beyond. Cancers, 2017. 9(1): p. 7. 5. Simigdala, N., et al., Abiraterone shows alternate activity in models of endocrine resistant and sensitive disease. British Journal of Cancer, 2018. 119(3): p. 313- 322. 6. Banerjee, S., et al., Principal results of the cancer of the ovary abiraterone trial (CORAL): A phase II study of abiraterone in patients with recurrent epithelial ovarian cancer (CRUKE/12/052). Annals of Oncology, 2016. 27(suppl_6).

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Appendix

Oncology drug products approved by the US FDA between January 2000 and September 2018:

Route of No. Oncology Drug Product administration Erleada (apalutamide); Janssen Oncology; For the treatment 1 ORAL of prostate cancer, Approved February 2018 Lutathera (lutetium Lu 177 dotatate); Advanced Accelerator 2 Applications; For the treatment of gastroenteropancreatic IV neuroendocrine tumors, Approved January 2018 Aliqopa (copanlisib); Bayer; For the treatment of follicular 3 IV lymphoma , Approved September 2017 Alunbrig (brigatinib); Ariad Pharmaceuticals; For the 4 treatment of advanced ALK-positive metastatic non-small ORAL cell lung cancer, Approved April 2017 Bavencio (avelumab) ; EMD Serono/Pfizer; For the treatment 5 IV of Merkel cell carcinoma , Approved March 2017 Besponsa (inotuzumab ozogamicin); Pfizer; For the treatment 6 of adults with relapsed or refractory B-cell precursor acute IV lymphoblastic leukemia, Approved August 2017 Calquence (acalabrutinib); Acerta Pharmaceuticals; For the 7 treatment of mantle cell lymphoma , Approved November ORAL 2017 IDHIFA (enasidenib); Celgene; For the treatment of relapsed 8 or refractory acute myeloid leukemia with IDH2 mutation , ORAL

Approved August 2017 Imfinzi (durvalumab); AstraZeneca; For the treatment of 9 advanced or metastatic urothelial carcinoma and Stage III IV non-small cell lung cancer, Initially approved May 2017

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Route of No. Oncology Drug Product administration Kisqali (ribociclib); Novartis; For the treatment of breast 10 ORAL cancer, Approved March 2017 Kymriah (tisagenlecleucel); Novartis; For the treatment of 11 refractory B-cell precursor acute lymphoblastic leukemia , IV Approved August 2017 Nerlynx (neratinib); Puma Biotech; For the treatment of 12 ORAL HER2 breast cancer, Approved July 2017 Rydapt (midostaurin); Novartis; For the treatment of FLT3 13 positive acute myeloid leukemia and mastocytosis , Approved ORAL April 2017 Verzenio (abemaciclib); Eli Lilly; For the treatment of HR+, 14 ORAL HER2- breast cancer, Approved September 2017 Vyxeos (daunorubicin and cytarabine) ; Jazz Pharma; For the 15 treatment of newly-diagnosed therapy-related AML or AML IV with myelodysplasia-related changes, Approved August 2017 Xermelo (telotristat ethyl); Lexicon Pharmaceuticals; For the 16 treatment of carcinoid syndrome diarrhea, Approved ORAL February 2017 Yescarta (axicabtagene ciloleucel); Kite Pharmaceuticals; For 17 the treatment of relapsed or refractory large B-cell IV lymphomas, Approved October 2017 Zejula (niraparib); Tesaro; For the treatment of recurrent 18 epithelial ovarian, fallopian tube, or primary peritoneal cancer ORAL , Approved March 2017 Cabometyx (cabozantinib); Exelixis; For the treatment of 19 ORAL advanced renal cell carcinoma, Approved April 2016 Keytruda (pembrolizumab); Merck; For the treatment of head 20 IV and neck squamous cell cancer , Approved August 2016 21 Lartruvo (olaratumab) ; Eli Lilly; For the treatment of soft IV

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Route of No. Oncology Drug Product administration tissue sarcoma, Approved October 2016 Lenvima (lenvatinib); Eisai; For the treatment of advanced 22 ORAL

renal cell carcinoma, Approved May 2016 Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment 23 IV of classical Hodgkin lymphoma, Approved May 2016 Opdivo (nivolumab)-rep.; Bristol-Myers Squibb; For the 24 treatment of recurrent or metastatic squamous cell carcinoma IV of the head and neck, Approved November 2016 Rubraca (rucaparib); Clovis Oncology; For the treatment of 25 advanced ovarian cancer in women with deleterious germline ORAL or somatic BRCA mutation, Approved December 2016 Sustol (granisetron); Heron Therapeutics; For the prevention 26 of chemotherapy-induced nausea and vomiting, Approved SC August 2016 Syndros (dronabinol oral solution); Insys Therapeutics; For the treatment of anorexia associated with AIDS and nausea 27 ORAL and vomiting associated with cancer chemotherapy, Approved July 2016 Tecentriq (atezolizumab); Genentech; For the treatment of 28 urothelial carcinoma and metastatic non-small cell lung IV cancer, Approved May 2016 Venclexta (venetoclax); AbbVie; For the treatment of chronic 29 lymphocytic leukemia with 17p deletion, Approved April ORAL 2016 Alecensa (alectinib); Roche; For the treatment of ALK- 30 positive, metastatic non-small cell lung cancer , Approved ORAL December 2015 Cotellic (cobimetinib) ; Genentech; For the treatment of 31 ORAL BRAF V600E or V600K melanoma, Approved November

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Route of No. Oncology Drug Product administration 2015

Darzalex (daratumumab); Janssen Biotech; For the treatment 32 IV of multiple myeloma, Approved November 2015 Empliciti (elotuzumab); Bristol-Myers Squibb; For the 33 treatment of patients with multiple myeloma who have IV received prior therapies, Approved November 2015 Farydak (panobinostat); Novartis; For the treatment of 34 ORAL multiple myeloma, Approved February 2015 Ibrance (palbociclib); Pfizer; For the treatment of ER- 35 positive, HER2-negative breast cancer, Approved February ORAL

2015 Imlygic (talimogene laherparepvec) ; Amgen; For the 36 treatment of unresectable recurrent melanoma, Approved LESIONAL

October 2015 Keytruda (pembrolizumab); Merck; For the treatment of PD- 37 L1 positive advanced non-small cell lung cancer, Approved IV October 2015 Lenvima (lenvatinib); Eisai; For the treatment of thyroid 38 ORAL cancer, Approved February 2015 Lonsurf (trifluridine and tipiracil); Taiho Oncology; For the 39 treatment of metastatic colorectal cancer , Approved ORAL September 2015 Ninlaro (ixazomib); Millennium Pharmaceuticals; For the 40 ORAL treatment of multiple myeloma, Approved November 2015 Odomzo (sonidegib); Novartis; For the treatment of locally 41 ORAL advanced basal cell carcinoma, July 2015 Onivyde (irinotecan liposome injection); Merrimack; For the 42 IV treatment of metastatic pancreatic cancer following

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Route of No. Oncology Drug Product administration gemcitabine-based therapy, Approved October 2015

Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment 43 IV of advanced renal cell carcinoma, Approved November 2015 Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment 44 of metastatic squamous non-small cell lung cancer, Approved IV March 2015 Portrazza (necitumumab) ; Eli Lilly; For the treatment of 45 metastatic squamous non-small cell lung cancer, Approved IV November 2015 Tagrisso (osimertinib); AstraZeneca; For the treatment of 46 EGFR T790M mutation positive non-small cell lung cancer , ORAL Approved November 2015 Unituxin (dinutuximab); United Therapeutics; For the 47 treatment of pediatrics with high-risk neuroblastoma, IV Approved March 2015 Varubi (rolapitant); Tesaro; For the prevention of delayed 48 nausea and vomiting associated with chemotherapy, ORAL Approved September 2015 Vistogard (uridine triacetate); Wellstat Therapeutics; For the 49 emergency treatment of patients with a fluorouracil or ORAL capecitabine overdose, Approved December 2015 Yondelis (trabectedin); Janssen; For the treatment of 50 IV liposarcoma or leiomyosarcoma, Approved October 2015 Akynzeo (netupitant and palonosetron); Helsinn; For the 51 prevention of chemotherapy-induced nausea and vomiting, ORAL Approved October 2014 Beleodaq (belinostat); Spectrum Pharmaceuticals; For the 52 IV treatment of relapsed or refractory peripheral T-cell

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Route of No. Oncology Drug Product administration lymphoma, Approved July 2014

Blincyto (blinatumomab); Amgen; For the treatment of Philadelphia chromosome-negative relapsed /refractory B cell 53 IV precursor acute lymphoblastic leukemia, Approved December 2014 Cyramza (ramucirumab); Eli Lilly; For the treatment of 54 IV gastric cancer, Approved April 2014 Imbruvica (ibrutinib); Pharmacyclics; For the treatment of 55 chronic lymphocytic leukemia and Waldenstrom ORAL macroglobulinemia, Approved February 2014 Keytruda (pembrolizumab); Merck; For the treatment of 56 unresectable or metastatic melanoma, Approved September IV 2014 Lynparza (olaparib); AstraZeneca; For the treatment of 57 previously treated BRCA mutated advanced ovarian cancer, ORAL Approved December 2014 Opdivo (nivolumab); Bristol-Myers Squibb; For the treatment 58 of unresectable or metastatic melanoma, Approved December IV 2014 Zydelig (idelalisib); Gilead; For the treatment of relapsed 59 CLL, follicular B-cell NHL and small lymphocytic ORAL lymphoma, Approved July 2014 Zykadia (ceritinib); Novartis; For the treatment of ALK+ 60 ORAL metastatic non-small cell lung cancer, Approved April 2014 Gazyva (obinutuzumab); Genentech; For the treatment of 61 previously untreated chronic lymphocytic leukemia, IV Approved October of 2013

62 Gilotrif (afatinib); Boehringer Ingelheim; For the treatment of ORAL

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Route of No. Oncology Drug Product administration metastatic non-small cell lung cancer with EGFR mutations, Approved July 2013 Imbruvica (ibrutinib); Pharmacyclics; For the treatment of 63 ORAL mantle cell lymphoma, Approved November of 2013 Kadcyla (ado-trastuzumab emtansine); Genentech; For the 64 treatment of HER2-positive metastatic breast cancer, IV Approved February 2013 Mekinist (trametinib); GlaxoSmithKline; For the treatment of 65 unresectable or metastatic melanoma with BRAF V600E or ORAL V600K mutations, Approved May of 2013 Pomalyst (pomalidomide); Celgene; For the treatment of 66 relapsed and refractory multiple myeloma, Approved ORAL February 2013 Revlimid (lenalidomide); Celgene; For the treatment of 67 ORAL mantle cell lymphoma, Approved June 2013 Stivarga (regorafenib); Bayer; For the treatment of 68 ORAL gastrointestinal stromal tumor, Approved February 2013 Tafinlar (dabrafenib); GlaxoSmithKline; For the treatment of 69 unresectable or metastatic melanoma with BRAF V600E ORAL mutation, Approved May 2013 Valchlor (mechlorethamine) gel; Ceptaris Therapeutics; For 70 the treatment of Stage IA/IB mycosisfungoides-type TOP cutaneous T-cell lymphoma, Approved August 2013 Xgeva (denosumab); Amgen; For the treatment of giant cell 71 SC tumor of bone, Approved June 2013 Xofigo (radium Ra 223 dichloride); Bayer Healthcare 72 Pharmaceuticals; For the treatment of prostate cancer with IV bone metastases, Approved May 2013

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Route of No. Oncology Drug Product administration Abraxane (paclitaxel protein-bound particles for injectable 73 suspension); Celgene; For the treatment of non-small cell IV lung cancer, Approved October 2012 Afinitor (everolimus); Novartis; For the treatment of renal 74 angiomyolipoma associated with tuberous sclerosis complex, ORAL Approved April 2012 Afinitor (everolimus); Novartis; For the treatment of hormone 75 receptor-positive, HER2-negative breast cancer, Approved ORAL July 2012 Bosulif (bosutinib); Pfizer; For the treatment of Ph+ chronic 76 ORAL myelogenous leukemia, Approved September 2012 Cometriq (cabozantinib); Exelixis; For the treatment of 77 metastatic medullary thyroid cancer, Approved November ORAL 2012 Erivedge (vismodegib); Genentech; For the treatment of basal 78 ORAL cell carcinoma, Approved January 2012 Iclusig (ponatinib); Ariad Pharmaceuticals; For the treatment of chronic myeloid leukemia and Philadelphia chromosome 79 ORAL positive acute lymphoblastic leukemia, Approved December 2012 Inlyta (axitinib); Pfizer; For the treatment of advanced renal 80 ORAL cell carcinoma, Approved January 2012 Kyprolis (carfilzomib); Onyx Pharmaceuticals; For the 81 IV treatment of multiple myeloma, Approved July 2012 Marqibo (vinCRIStine sulfate LIPOSOME injection); Talon 82 Therapeutics; For the treatment of Ph- acute lymphoblastic IV leukemia, Approved August 2012 Neutroval (tbo-filgrastim); Teva Pharmaceutical; For the 83 SC reduction in the duration of severe chemotherapy-induced

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Route of No. Oncology Drug Product administration neutropenia, Approved August 2012

Perjeta (pertuzumab); Genentech; For the first-line treatment 84 IV of HER2+ metastatic breast cancer, Approved June 2012 Picato (ingenol mebutate) gel; LEO Pharma; For the 85 TOP treatment of actinic keratosis, Approved January 2012 Stivarga (regorafenib); Bayer HealthCare Pharmaceuticals; 86 For the treatment of previously treated patients with ORAL metastatic colorectal cancer, Approved September 2012 Subsys (fentanyl sublingual spray); Insys Therapeutics; For 87 the treatment of breakthrough cancer pain, Approved January SUBLINGUAL of 2012 Synribo (omacetaxine mepesuccinate); Teva Pharmaceutical; 88 For the treatment of chronic or accelerated phase chronic SUBLINGUAL myeloid leukemia, Approved October 2012 Votrient (pazopanib); GlaxoSmithKline; For the treatment of 89 ORAL soft tissue sarcoma, Approved April 2012 Xtandi (enzalutamide); Medivation; For the treatment of 90 metastatic castration-resistant prostate cancer, Approved ORAL August 2012 Zaltrap (ziv-aflibercept); Sanofi-aventis; For the treatment of 91 IV metastatic colorectal cancer, Approved August 2012 Abstral (fentanyl sublingual tablets); ProStrakan; For the 92 treatment of breakthrough cancer pain in opioid-tolerant SUBLINGUAL patients, Approved January 2011 Adcetris (brentuximab vedotin); Seattle Genetics; For the 93 treatment of Hodgkin lymphoma and anaplastic large cell IV lymphoma, Approved August 2011

94 Afinitor (everolimus); Novartis; For the treatment of ORAL

259

Route of No. Oncology Drug Product administration advanced pancreatic neuroendocrine tumors, Approved May 2011 Erwinaze (asparaginase Erwinia chrysanthemi); Eusa 95 Pharma; For the treatment of acute lymphoblastic leukemia, MUS Approved November of 2011 Lazanda (fentanyl citrate) nasal spray; Archimedes; For the 96 management of breakthrough cancer pain, Approved June NASAL 2011 Sutent (sunitinib malate); Pfizer; For the treatment of 97 ORAL pancreatic neuroendocrine tumors, Approved May 2011 Sylatron (peginterferon alfa-2b); Merck; For the treatment of 98 IV melanoma, Approved April 2011 Vandetanib (vandetanib); AstraZeneca; For the treatment of 99 ORAL thyroid cancer, Approved April 2011 Xalkori (crizotinib); Pfizer; For the treatment of ALK+ non- 100 ORAL small cell lung cancer, Approved August of 2011 Yervoy (ipilimumab); Bristol-Myers Squibb; For the 101 IV treatment of metastatic melanoma, Approved March 2011 Zelboraf (vemurafenib); Roche; For the treatment of BRAF + 102 ORAL melanoma, Approved August of 2011 Zytiga (abiraterone acetate); Centocor Ortho Biotech; For the 103 ORAL treatment of prostate cancer, Approved May 2011 Halaven (eribulin mesylate); Eisai; For the treatment of 104 IV metastatic breast cancer, Approved November 2010 Herceptin (trastuzumab); Genentech; For the treatment of 105 IV gastric cancer, Approved October 2010 Jevtana (cabazitaxel); sanofi aventis; For the treatment of 106 IV prostate cancer, Approved June 2010

260

Route of No. Oncology Drug Product administration Provenge (sipuleucel-T); Dendreon; For the treatment of 107 IV hormone refractory prostate cancer, Approved May 2010 Xgeva (denosumab); Amgen; For the prevention of skeletal- 108 related events in patients with bone metastases from solid SC tumors, Approved November 2010 Zuplenz (ondansetron oral soluble film); Strativa Pharmaceuticals; For the prevention of post-operative, 109 ORAL chemotherapy and radiotherapy induced nausea and vomiting, Approved July 2010 Afinitor (everolimus); Novartis; For the treatment of renal 110 ORAL cell carcinoma, Approved March 2009 Arzerra (ofatumumab); GlaxoSmithKline; For the treatment 111 IV of chronic lymphocytic leukemia, Approved October 2009 Avastin (bevacizumab); Genentech; For the treatment of renal 112 IV cell carcinoma, Approved July 2009 Cervarix [Human Papillomavirus Bivalent (Types 16 and 18) Vaccine, Recombinant; GlaxoSmithKline; For the prevention 113 MUS of cervical cancer and cervical intraepithelial neoplasia caused by HPV types 16 and 18, Approved October 2009 Elitek (rasburicase); sanofi-aventis; For the management of 114 plasma uric acid levels in adults with malignancies, Approved IV October 2009 Folotyn (pralatrexate injection); Allos Therapeutics; For the 115 treatment of peripheral T-cell lymphoma, Approved IV September 2009 Istodax (romidepsin); Gloucester Pharmaceuticals; For the 116 treatment of cutaneous T-cell lymphoma, Approved IV November 2009

261

Route of No. Oncology Drug Product administration Onsolis (fentanyl buccal); BioDelivery Sciences; For the 117 management of breakthrough cancer pain, Approved July BUCCAL 2009 Votrient (pazopanib); GlaxoSmithKline; For the treatment of 118 ORAL renal cell carcinoma, Approved October of 2009 Degarelix (degarelix for injection); Ferring Pharmaceuticals; 119 For the treatment of prostate cancer, Approved December of SC 2008 Fusilev (levoleucovorin); Spectrum Pharmaceuticals; For rescue after high-dose methotrexate therapy in osteosarcoma 120 IV and to reduce the toxicity of methotrexate, Approved March of 2008 Mozobil (plerixafor injection); Genzyme; For the treatment of 121 non-Hodgkin’s lymphoma and multiple myeloma, Approved SC December 2008 Sancuso (granisetron); ProStrakan; For the treatment of 122 chemotherapy-induced nausea and vomiting, Approved TRANSDERMAL September 2008 Treanda (bendamustine hydrochloride); Cephalon; For the 123 treatment of Chronic lymphocytic leukemia and B-cell non- IV Hodgkin’s lymphoma, Approved October 2008 Evista (raloxifene hydrochloride); Eli Lilly; For the treatment/prevention of osteoporosis and reduction of breast 124 ORAL cancer risk in postmenopausal women, Approved September 2007 Hycamtin (topotecan hydrochloride); GlaxoSmithKline; For 125 the treatment of small cell lung cancer, Approved October ORAL 2007 126 Ixempra (ixabepilone); Bristol-Myers Squibb; For the IV

262

Route of No. Oncology Drug Product administration treatment of breast cancer, Approved October 2007 Tasigna (nilotinib hydrochloride monohydrate); Novartis; For 127 the treatment of chronic myelogenous leukemia, Approved ORAL October 2007 Torisel (temsirolimus); Wyeth; For the treatment of renal cell 128 IV carcinoma, Approved May 2007 Tykerb (lapatinib); GlaxoSmithKline; For the treatment of 129 ORAL breast cancer, Approved March 2007 Gardasil (quadrivalent human papillomavirus (types 6, 11, 16, 18) recombinant vaccine); Merck; For the prevention of 130 MUS cervical cancer associated with human papillomavirus, Approved June 2006 Sprycel (dasatinib); Bristol-Myers Squibb; For the treatment 131 of imatinib-resistant chronic myeloid leukemia, Approved ORAL June 2006 Sutent (sunitinib); Pfizer; For the treatment of kidney cancer 132 ORAL and gastrointestinal stromal tumors, Approved January 2006 Vectibix (panitumumab); Amgen; For the treatment of 133 IV colorectal cancer, Approved September 2006 Arranon (nelarabine); GlaxoSmithKline; For the treatment of 134 T-cell acute lymphoblastic leukemia and T-cell lymphoblastic IV lymphoma, Approved October 2005 Nexavar (sorafenib); Bayer/Onyx; For the Treatment of Renal 135 ORAL Cell Carcinoma, Approved December 2005 Alimta (pemetrexed for injection); Eli Lilly; For the treatment 136 IV of malignant pleural mesothelioma, Approved February 2004 Avastin (bevacizumab); Genentech; For the treatment of 137 metastatic carcinoma of the colon or rectum, Approved IV February 2004

263

Route of No. Oncology Drug Product administration Clolar (clofarabine); Genzyme; For the treatment of acute 138 lymphoblastic leukemia in pediatric patients, Approved IV December, 2004 Erbitux (cetuximab); Imclone, Bristol-Myers Squibb; For the 139 treatment of EGFR-expressing, metastatic colorectal cancer, IV Approved February 2004 Sensipar (cinacalcet); Amgen; For the treatment of secondary 140 hyperparathyroidism and hypercalcemia in parathyroid ORAL carcinoma patients, Approved March 2004 Tarceva (erlotinib, OSI 774); Genentech, OSI Pharmaceuticals; For the treatment of advanced refractory 141 ORAL metastatic non-small cell lung cancer, Approved November, 2004 Aloxi (palonosetron); MGI Pharma, Helsinn Healthcare; For 142 the prevention of nausea and vomiting associated with IV emetogenic cancer chemotherapy, Approved August 2003 Bexxar; Corixa; For the treatment of patients with CD20 143 positive, follicular, non-Hodgkin's lymphoma following IV chemotherapy relapse, Approved June 2003 Emend (aprepitant); Merck; For the treatment of nausea and 144 vomiting associated with chemotherapy, Approved March ORAL 2003 Iressa (gefitinib); AstraZeneca; For the second-line treatment 145 ORAL of non-small-cell lung cancer, Approved May 2003 Plenaxis (abarelix for injectable suspension); Praecis 146 Pharmaceuticals; For treatment of advanced prostate cancer, MUS Approved December 2003 Premarin (conjugated estrogens); Wyeth; For the prevention 147 ORAL of postmenopausal osteoporosis and treatment of vasomotor

264

Route of No. Oncology Drug Product administration menopause symptoms, Approved July of 2003

UroXatral (alfuzosin HCl extended-release tablets); Sanofi- 148 aventis; For the treatment of of the signs and symptoms of ORAL benign prostatic hyperplasia, Approved June 2003 Velcade (bortezomib); Millennium Pharmaceuticals; Injectable agent for the treatment of multiple myeloma 149 IV patients who have received at least two prior therapies., Approved May 2003 Eligard (leuprolide acetate); Atrix Laboratories; For the 150 palliative treatment of advanced prostate cancer, Approved IV January 2002 Faslodex (fulvestrant); AstraZeneca; For the treatment of 151 hormone receptor positive metastatic breast cancer, Approved IV April 2002 Gleevec (imatinib mesylate); Novartis; For the treatment of 152 gastrointestinal stromal tumors (GISTs), Approved February ORAL 2002 Neulasta; Amgen; Treatment to decrease the chance of 153 infection by febrile neutropenia in patients receiving IV chemotherapy, Approved January 2002 SecreFlo (secretin); Repligen; To aid in the diagnosis of 154 IV pancreatic dysfunction and gastrinoma, Approved April 2002 Zevalin (ibritumomab tiuxetan); Biogen IDEC; For the 155 treatment of non-Hodgkin's lymphoma, Approved February IV 2002 Zometa (zoledronic acid); Novartis; For the treatment of 156 multiple myeloma and bone metastases from solid tumors, IV Approved February 2002

265

Route of No. Oncology Drug Product administration Campath; Berlex Laboratories; Injectable treatment of B-cell 157 IV chronic lymphocytic leukemia, Approved May 2001 Femara (letrozole); Novartis; First-line treatment of 158 postmenopausal women with locally advanced or metastatic ORAL breast cancer, Approved January 2001 Gleevec (imatinib mesylate); Novartis; Oral therapy for the 159 ORAL treatment of chronic myeloid leukemia, Approved May 2001 Kytril (granisetron) solution; Roche; For the prevention of 160 nausea and vomiting associated with cancer therapy, BOTH Approved June 2001 Trelstar LA (triptorelin pamoate); Debiopharm; Intramuscular 161 injection for the treatment of advanced stage prostate cancer, IV Approved June 2001 Xeloda; Roche; Oral chemotherapy for the treatment of 162 ORAL metastatic colorectal cancer, Approved May 2001 Zometa (zoledronic acid); Novartis; For the treatment of 163 IV hypercalcemia of malignancy, Approved August 2001 Mylotarg (gemtuzumab ozogamicin); Wyeth; For the 164 treatment of CD33 positive acute myeloid leukemia (AML), IV Approved May 2000 Trelstar Depot (triptorelin pamoate); Debio Rechereche Pharmaceutique, Target Research Associates; For the 165 MUS palliative treatment of advanced prostate cancer, Approved June 2000 Trisenox (arsenic trioxide); Cell Therapeutics; For the induction of remission and consolidation in patients with 166 IV acute promyelocytic leukemia (APL), Approved September 2000

266

Route of No. Oncology Drug Product administration Viadur (leuprolide acetate implant); Alza; For pain relief in 167 IMPLANT men with advanced prostate cancer, Approved March 2000

Categorization based on route of administration:

Route No. Percent Oral 74.5 44.6% Intravenous 71.5 42.8% Intramuscular 5 3.0% Subcutaneous 6 3.6% Topical 2 1.2% Intralesional 1 0.6% Others 7 4.2%

267

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Chapter Two:

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Chapter Three:

3. Chi, K.N., et al., Food effects on abiraterone pharmacokinetics in healthy subjects and patients with metastatic castration-resistant prostate cancer. J Clin Pharmacol, 2015. 55(12): p. 1406-14. 4. FDA, U., Clinical pharmacology and biopharmaceutics review(s)- Zytiga®. 2010. 5. FDA, U., Highlights of prescribing information- Yonsa®. 2018. 6. Goldwater, R., et al., Comparison of a Novel Formulation of Abiraterone Acetate vs. the Originator Formulation in Healthy Male Subjects: Two Randomized, Open-Label, Crossover Studies. Clin Pharmacokinet, 2017. 56(7): p. 803-813. 7. Li, R., et al., Abiraterone inhibits 3beta-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer. Clin Cancer Res, 2012. 18(13): p. 3571-9. 8. Xu, X.S., et al., Modeling the Relationship Between Exposure to Abiraterone and Prostate-Specific Antigen Dynamics in Patients with Metastatic Castration- Resistant Prostate Cancer. Clin Pharmacokinet, 2017. 56(1): p. 55-63. 9. Xu, X.S., et al., Correlation between Prostate-Specific Antigen Kinetics and Overall Survival in Abiraterone Acetate-Treated Castration-Resistant Prostate Cancer Patients. Clin Cancer Res, 2015. 21(14): p. 3170-7. 10. Davis, M.E. and M.E. Brewster, Cyclodextrin-based pharmaceutics: past, present and future. Nature Reviews Drug Discovery, 2004. 3(12): p. 1023-1035. 11. Loftsson, T., et al., Cyclodextrins in drug delivery. Expert Opin Drug Deliv, 2005. 2(2): p. 335-51. 12. Sharma, N. and A. Baldi, Exploring versatile applications of cyclodextrins: an overview. Drug Delivery, 2016. 23(3): p. 729-747. 13. Saokham, P., et al., Solubility of Cyclodextrins and Drug/Cyclodextrin Complexes. Molecules, 2018. 23(5). 14. Varma, M.M. and P.S. Kumar, Formulation and Evaluation of GLZ Tablets Containing PVP K30 and Hydroxyl Propyl Beta Cyclodextrin Solid Dispersion.

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Chapter Four: 1. Rautio, J., et al., The expanding role of prodrugs in contemporary drug design and development. Nature Reviews Drug Discovery, 2018. 17: p. 559. 2. Najjar, A. and R. Karaman, The prodrug approach in the era of drug design. Expert Opinion on Drug Delivery, 2019. 16(1): p. 1-5. 308

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14. Van den Mooter, G., The use of amorphous solid dispersions: A formulation strategy to overcome poor solubility and dissolution rate. Drug Discovery Today: Technologies, 2012. 9(2): p. e79-e85. 15. Gala, U., Chapter 2- Improved Dissolution And Pharmacokinetics Of Abiraterone Through KinetiSol® Enabled Amorphous Solid Dispersion 2019. 16. Gala, U.H., D.A. Miller, and R.O. Williams, Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2020. 1873(1): p. 188319. 17. Hywel Williams, M.M., David Vodak, Eduardo Jule, Hassan Benameur, Improving Oral Drug Absorption–WhichTechnology to Select? A Spray- DriedDispersionand Lipid-Based Formulation Case Study Using Abiraterone Acetate, in AAPS- Pharm Sci 360 2019. 2019: San Antonio. 18. Solymosi, T., et al., Development of an abiraterone acetate formulation with improved oral bioavailability guided by absorption modeling based on in vitro dissolution and permeability measurements. Int J Pharm, 2017. 532(1): p. 427- 434. 19. Basa-Denes, O., et al., Investigations of the mechanism behind the rapid absorption of nano-amorphous abiraterone acetate. Eur J Pharm Sci, 2019. 129: p. 79-86. 20. Rumondor, A.C.F., S.S. Dhareshwar, and F. Kesisoglou, Amorphous Solid Dispersions or Prodrugs: Complementary Strategies to Increase Drug Absorption. Journal of Pharmaceutical Sciences, 2016. 105(9): p. 2498-2508. 21. Newman, A., J.E. Hastedt, and M. Yazdanian, New directions in pharmaceutical amorphous materials and amorphous solid dispersions, a tribute to Professor George Zografi – Proceedings of the June 2016 Land O’Lakes Conference. AAPS Open, 2017. 3(1): p. 7. 22. EMA, Harvoni-Assesment Report. 2014. 23. EMA, Epclusa-Assesment Report. 2016.

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24. Hajnal, K., et al., Prodrug Strategy in Drug Development. Acta Medica Marisiensis, 2016. 62. 25. Strickley, R.G. and R. Oliyai, Formulation Challenges of Prodrugs, in Prodrugs: Challenges and Rewards Part 1, V.J. Stella, et al., Editors. 2007, Springer New York: New York, NY. p. 1083-1110. 26. Gala, U., Chapter 3- The Effect Of Drug Loading On The Properties Of Kinetisol® Processed Abiraterone-Hydroxypropyl Β Cyclodextrin Solid Dispersions. 2019. 27. Burke, D.F., et al., Active-site conformation of 17-(3-pyridyl)androsta-5,16-dien- 3β-ol, a potent inhibitor of the P450 enzyme C17α-hydroxylase/C17-20 lyase. Bioorganic & Medicinal Chemistry Letters, 1995. 5(11): p. 1125-1130. 28. Wheatley, A.M., et al., Crystal structure of abiraterone acetate (Zytiga), C26H33NO2. Powder Diffraction, 2018. 33(1): p. 72-72. 29. Hughey, J.R., et al., Preparation and characterization of fusion processed solid dispersions containing a viscous thermally labile polymeric carrier. International Journal of Pharmaceutics, 2012. 438(1): p. 11-19. 30. LaFountaine, J.S., et al., Thermal Processing of PVP- and HPMC-Based Amorphous Solid Dispersions. AAPS PharmSciTech, 2016. 17(1): p. 120-132. 31. Jermain, S.V., et al., Homogeneity of amorphous solid dispersions – an example with KinetiSol®. Drug Development and Industrial Pharmacy, 2019. 45(5): p. 724-735. 32. Zhou, S., H. Huang, and R. Huang, Crystal structure of (3S)-3-acet-oxy-17- (pyridin-3-yl)androsta-5,16-diene. Acta crystallographica. Section E, Crystallographic communications, 2015. 71(Pt 3): p. o146-o147. 33. Solymosi, T., Development, preclinical evaluation and first-in-human clinical trial of a nano-amorphous abiraterone acetate formulation. 2019. 34. Łaszcz, M., K. Trzcińska, and E.U. Stolarczyk, Physicochemical characteristics of abiraterone acetate used for the treatment of prostate cancer. 2016.

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35. Lee, Y.-E., et al., Influence of NaCl Concentration on Food-Waste Biochar Structure and Templating Effects. Energies, 2018. 11: p. 2341. 36. Gadhave, R.V., et al., Stability indicating RP-HPLC-PDA method for determination of abiraterone acetate and characterization of its base catalyzed degradation product by LC-MS. 2016. 8: p. 76-81. 37. Chandra Reddy, B.J. and N.C. Sarada, Development and validation of a novel RP- HPLC method for stability-indicating assay of Abiraterone acetate. Journal of Liquid Chromatography & Related Technologies, 2016. 39(7): p. 354-363. 38. Karandikar, H., et al., Systematic identification of thermal degradation products of HPMCP during hot melt extrusion process. International Journal of Pharmaceutics, 2015. 486(1): p. 252-258. 39. Raymond C. Rowe, et al., Handbook of Pharmaceutical Excipients – 7th Edition. Pharmaceutical Development and Technology, 2013. 18(2): p. 544-544. 40. Saokham, P., et al., Solubility of Cyclodextrins and Drug/Cyclodextrin Complexes. Molecules, 2018. 23(5). 41. Wyttenbach, N. and M. Kuentz, Glass-forming ability of compounds in marketed amorphous drug products. European Journal of Pharmaceutics and Biopharmaceutics, 2017. 112: p. 204-208. 42. Baird, J.A., B. Van Eerdenbrugh, and L.S. Taylor, A Classification System to Assess the Crystallization Tendency of Organic Molecules from Undercooled Melts. Journal of Pharmaceutical Sciences, 2010. 99(9): p. 3787-3806. 43. Ormes, J.D., Predicting the Risk of Crystallization for Suspensions of Amorphous Spray Dried Dispersions from Structural, Thermal and Hydrophilicity Properties. 2014, University of Kansas. 44. Khedr, A., I. Darwish, and F. Bamane, Analysis of abiraterone stress degradation behavior using liquid chromatography coupled to ultraviolet detection and electrospray ionization mass spectrometry. J Pharm Biomed Anal, 2013. 74: p. 77-82. 45. FDA, U., Highlights of prescribing information- Yonsa®. 2018. 312

46. Maura Murphy, P.N., William Bosch, Matthew CALLAHAN, Satya Bhamidipati, Jason Coleman, Christopher Hill, Marck Norret, Abiraterone Acetate Formulation and Methods of Use. 2016. 47. Solymosi, T., et al., Novel formulation of abiraterone acetate might allow significant dose reduction and eliminates substantial positive food effect. 2017. 80(4): p. 723-728. 48. Bouhajib, M. and Z. Tayab, Evaluation of the Pharmacokinetics of Abiraterone Acetate and Abiraterone Following Single-Dose Administration of Abiraterone Acetate to Healthy Subjects. Clin Drug Investig, 2019. 39(3): p. 309-317. 49. Haidar, S., et al., Effects of novel 17alpha-hydroxylase/C17, 20-lyase (P450 17, CYP 17) inhibitors on androgen biosynthesis in vitro and in vivo. J Steroid Biochem Mol Biol, 2003. 84(5): p. 555-62. 50. Haider, S.M., et al., Molecular Modeling on Inhibitor Complexes and Active-Site Dynamics of Cytochrome P450 C17, a Target for Prostate Cancer Therapy. Journal of Molecular Biology, 2010. 400(5): p. 1078-1098. 51. Williams, F.M., Clinical Significance of Esterases in Man. Clinical Pharmacokinetics, 1985. 10(5): p. 392-403.

Chapter Five:

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4. Damodaran, S., C.E. Kyriakopoulos, and D.F. Jarrard, Newly Diagnosed Metastatic Prostate Cancer: Has the Paradigm Changed? The Urologic clinics of North America, 2017. 44(4): p. 611-621. 5. Dawson, N.A., C.J. Ryan, and J.P. Richie, Castration-resistant prostate cancer: Treatments targeting the androgen pathway. 2013. 6. Benoist, G.E., et al., Pharmacokinetic Aspects of the Two Novel Oral Drugs Used for Metastatic Castration-Resistant Prostate Cancer: Abiraterone Acetate and Enzalutamide. Clin Pharmacokinet, 2016. 55(11): p. 1369-1380. 7. Dong, L., et al., Metastatic prostate cancer remains incurable, why? Asian J Urol, 2019. 6(1): p. 26-41. 8. Belfort, G.M.H., Boyd L. and Botella, GabrielMartinez Abiraterone Acetate (Zytiga®): An Inhibitor of CYP17 as a Therapeutic for Castration-Resistant Prostate Cancer, in Successful Drug Discovery. 2016. p. 115-135. 9. Attard, G., et al., Phase I clinical trial of a selective inhibitor of CYP17, abiraterone acetate, confirms that castration-resistant prostate cancer commonly remains hormone driven. J Clin Oncol, 2008. 26(28): p. 4563-71. 10. FDA, U. Highlights of prescribing information- Zytiga®. 2011-19. 11. Rehman, Y. and J.E. Rosenberg, Abiraterone acetate: oral androgen biosynthesis inhibitor for treatment of castration-resistant prostate cancer. Drug Des Devel Ther, 2012. 6: p. 13-8. 12. Norris, J.D., et al., Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer. The Journal of clinical investigation, 2017. 127(6): p. 2326-2338. 13. FDA, U., Clinical pharmacology and biopharmaceutics review(s)- Zytiga®. 2010. 14. EMA, Assessment Report For Zytiga. 2011. 15. TGA, Australian Public Assessment Report for abiraterone acetate. 2012.

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16. Xu, X.S., et al., Correlation between Prostate-Specific Antigen Kinetics and Overall Survival in Abiraterone Acetate-Treated Castration-Resistant Prostate Cancer Patients. Clin Cancer Res, 2015. 21(14): p. 3170-7. 17. Xu, X.S., et al., Modeling the Relationship Between Exposure to Abiraterone and Prostate-Specific Antigen Dynamics in Patients with Metastatic Castration- Resistant Prostate Cancer. Clin Pharmacokinet, 2017. 56(1): p. 55-63. 18. Carton, E., et al., Relation between plasma trough concentration of abiraterone and prostate-specific antigen response in metastatic castration-resistant prostate cancer patients. Eur J Cancer, 2017. 72: p. 54-61. 19. Friedlander, T.W., et al., High-Dose Abiraterone Acetate in Men With Castration Resistant Prostate Cancer. Clin Genitourin Cancer, 2017. 15(6): p. 733-741.e1. 20. Woei-A-Jin, F.J.S.H., et al., Dose Reduction May Jeopardize Efficacy of Abiraterone Acetate. Journal of Clinical Oncology, 2018. 36(30): p. 3062-3064. 21. Attard, G., et al., Clinical and biochemical consequences of CYP17A1 inhibition with abiraterone given with and without exogenous glucocorticoids in castrate men with advanced prostate cancer. J Clin Endocrinol Metab, 2012. 97(2): p. 507-16. 22. Li, R., et al., Abiraterone inhibits 3beta-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer. Clin Cancer Res, 2012. 18(13): p. 3571-9. 23. Mostaghel, E.A., et al., Resistance to CYP17A1 inhibition with abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants. Clin Cancer Res, 2011. 17(18): p. 5913-25. 24. Li, Z., et al., Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature, 2015. 523(7560): p. 347-51. 25. Li, Z., et al., Redirecting abiraterone metabolism to fine-tune prostate cancer anti-androgen therapy. Nature, 2016. 533(7604): p. 547-51.

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26. Crona, D.J. and Y.E. Whang, Androgen Receptor-Dependent and -Independent Mechanisms Involved in Prostate Cancer Therapy Resistance. Cancers, 2017. 9(6): p. 67. 27. Chi, K.N., et al., Food effects on abiraterone pharmacokinetics in healthy subjects and patients with metastatic castration-resistant prostate cancer. J Clin Pharmacol, 2015. 55(12): p. 1406-14. 28. Szmulewitz, R.Z., et al., Prospective International Randomized Phase II Study of Low-Dose Abiraterone With Food Versus Standard Dose Abiraterone In Castration-Resistant Prostate Cancer. J Clin Oncol, 2018. 36(14): p. 1389-1395. 29. Todd, M., et al., Fast and flawed or scientifically sound: the argument for administering oral oncology drugs during fasting. J Clin Oncol, 2012. 30(8): p. 888-9; author reply 889. 30. Gala, U., Chapter 2- Improved Dissolution And Pharmacokinetics Of Abiraterone Through KinetiSol® Enabled Amorphous Solid Dispersion 2019. 31. Gala, U., Chapter 3- The Effect Of Drug Loading On The Properties Of Kinetisol® Processed Abiraterone-Hydroxypropyl Β Cyclodextrin Solid Dispersions. 2019. 32. Gala, U., Chapter 4- Comparing the KinetiSol® processed amorphous solid dispersions of Abiraterone acetate and Abiraterone: Is the Prodrug necessary? 2019. 33. Euhus, D.M., et al., Tumor measurement in the nude mouse. J Surg Oncol, 1986. 31(4): p. 229-34. 34. Drugbank. Abiraterone. 2007 09/05/2019]; Available from: https://www.drugbank.ca/drugs/DB05812. 35. Gala, U.H., D.A. Miller, and R.O. Williams, Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer, 2020. 1873(1): p. 188319. 36. Stappaerts, J., et al., Rapid conversion of the ester prodrug abiraterone acetate results in intestinal supersaturation and enhanced absorption of abiraterone: In 316

vitro, rat in situ and human in vivo studies. European Journal of Pharmaceutics and Biopharmaceutics, 2015. 90: p. 1-7. 37. Giles, F.J., et al., Nilotinib population pharmacokinetics and exposure-response analysis in patients with imatinib-resistant or -intolerant chronic myeloid leukemia. European Journal of Clinical Pharmacology, 2013. 69(4): p. 813-823. 38. Tuntland, T., et al., Implementation of pharmacokinetic and pharmacodynamic strategies in early research phases of drug discovery and development at Novartis Institute of Biomedical Research. Frontiers in pharmacology, 2014. 5: p. 174-174. 39. Sramkoski, R.M., et al., A new human prostate carcinoma cell line, 22Rv1. In Vitro Cell Dev Biol Anim, 1999. 35(7): p. 403-9. 40. Cunningham, D. and Z. You, In vitro and in vivo model systems used in prostate cancer research. J Biol Methods, 2015. 2(1). 41. Motwani, V.B., Dorothy; Huang, Liyue; Pantano, Chloe; Estanek,Vania; Keats,Jeffrey A.; Rodrigues,Lindsey U.; Stickland, Kimberly; Dransfield, Daniel T. ; Raimondi , Alejandra Synergistic activity of tazemetostat in combination with androgen signaling inhibitors in preclinical models of prostate cancer demonstrates potential for clinical expansion. 2019, Epizyme. 42. Giatromanolaki, A., et al., CYP17A1 and Androgen-Receptor Expression in Prostate Carcinoma Tissues and Cancer Cell Lines. Current Urology, 2019. 13(3): p. 157-165. 43. Bovinder Ylitalo, E. and P. Wikström, Mechanisms behind tumor relapse in 22Rv1 xenografts after treatment with abiraterone or cabazitaxel. Endocrine Abstracts, 2016. 44. He, Y., et al., Androgen receptor splice variants bind to constitutively open chromatin and promote abiraterone-resistant growth of prostate cancer. Nucleic acids research, 2018. 46(4): p. 1895-1911. 45. Pham, S., et al., Next-generation steroidogenesis inhibitors, dutasteride and abiraterone, attenuate but still do not eliminate androgen biosynthesis in 22RV1

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cells in vitro. The Journal of Steroid Biochemistry and Molecular Biology, 2014. 144: p. 436-444.

Chapter Six:

1. Keen, J.M. Case studies in human bioavailability enhancement with KinetiSol®. in AAPS Pharm Sci 360. 2019. San Antonio. 2. Grellety, T., et al., Enhancing Abiraterone Acetate Efficacy in Androgen Receptor-positive Triple-negative Breast Cancer: Chk1 as a Potential Target. Clin Cancer Res, 2019. 25(2): p. 856-867. 3. Grellety, T., et al., Long-Term Complete Response of an Androgen Receptor– Positive Triple-Negative Metastatic Breast Cancer to Abiraterone Acetate. JCO Precision Oncology, 2018(2): p. 0-0. 4. Schweizer, M.T. and E.Y. Yu, AR-Signaling in Human Malignancies: Prostate Cancer and Beyond. Cancers, 2017. 9(1): p. 7. 5. Simigdala, N., et al., Abiraterone shows alternate activity in models of endocrine resistant and sensitive disease. British Journal of Cancer, 2018. 119(3): p. 313- 322. 6. Banerjee, S., et al., Principal results of the cancer of the ovary abiraterone trial (CORAL): A phase II study of abiraterone in patients with recurrent epithelial ovarian cancer (CRUKE/12/052). Annals of Oncology, 2016. 27(suppl_6).

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Vita

Urvi Hasmukhlal Gala was born in Mumbai, India. Urvi pursued her undergraduate studies in Pharmacy at Institute of Chemical Technology, Mumbai, India. During her undergraduate studies, Urvi developed keen interest in the subject of Pharmaceutics. During Summer of 2011, she interned at Pfizer Animal Health Pharmaceutical Sciences, Mumbai, India. Urvi graduated with a Bachelor’s degree in Pharmacy in 2012 and began working as a Research Associate in formulation development department at Rubicon Research Pvt. Ltd., Mumbai, India. At Rubicon, Urvi worked on development of oral osmotic drug delivery system, gastro-retentive drug delivery system, fast melt granules and chewable tablets for small molecules. In 2013, Urvi was admitted to graduate program at Creighton University, Omaha Nebraska. She worked in Dr. Harsh Chauhan’s lab and developed ternary amorphous solid dispersions for polyphenolic drugs. During her graduate studies at Creighton, Urvi published several review articles and a research paper in high impact pharmaceutical journals. Urvi also presented her work at conferences in Omaha, San Diego and Orlando. Urvi graduated from Creighton with Master’s in Pharmaceutical Sciences in 2015. Later Urvi volunteered in Dr. Hugh Smyth’s Lab at the University of Texas at Austin for five months, wherein she assisted in controlled release coating of materials, thermal characterization of exothermic reactions and development of protein dry powder inhaler system. In 2016, Urvi began working as a Scientific Consultant and later joined as an Associate Principal Scientist at DisperSol Technologies, LLC, Georgetown, Texas. At DisperSol, Urvi worked on development of amorphous solid dispersion for poorly water soluble drugs using KinetiSol® technology. In 2017, Urvi joined the doctoral program at the University of Texas at Austin, under the supervision of Dr. Robert. O. Williams III. Urvi has presented her doctoral research at conferences in Washington D.C and San Antonio. Urvi has published her first chapter in Biochimica et Biophysica Acta - Reviews on Cancer and other chapters will be submitted to high impact pharmaceutical journals. 319

Urvi is an inventor on one published patent application and has two other applications pending publication.

Email address : [email protected] This dissertation was typed by the author.

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