Different Approaches Toward the Enhancement of Drug Solubility and Or Dissolution Rate

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Different Approaches Toward The Enhancement Of Drug Solubility And Or Dissolution Rate.

Princy Ann Abraham

A Dissertation submitted to the

Graduate School-New Brunswick

Rutgers, The State University of New Jersey

in partial fulfillment of the requirements

for the degree of Doctor of Philosophy

Graduate Program in Chemistry and Chemical Biology

written under the direction of

Professor Jing Li

and approved by:

______

New Brunswick, New Jersey

October, 2011

Princy Ann Abraham

ABSTRACT OF THE DISSERTATION

Different Approaches Toward The Enhancement Of Drug Solubility

And/Or Dissolution Rate.

By: Princy Ann Abraham Dissertation Director: Professor Jing Li

Nontraditional methods such as supercritical antisolvent processing and ultrasonic processor rely on physical alterations to enhance drug solubility and dissolution rate. They are advantageous because of their application to a wide variety of drugs and relatively short processing time. Our works show reduced particle size and complexation with these techniques results in the modification of dissolution rate and or solubility. Crystalline form of a drug is preferable because most drugs occur in this form and tend to be stable at this condition. Towards this effort new nanosized crystalline coordination drug polymers are synthesized to enhance drug solubility or dissolution rate.

γ-Indomethacin (IMC) is successfully processed with the supercritical antisolvent

(SAS) technique. Pure, acicular (needle-like) particles of the α-polymorph are consistently obtained with SAS as the solvent, concentration, temperature and pressures are varied. Controlled changes in process parameters yield significant changes in particle size. Enhanced dissolution profiles are observed with IMC processed with SAS as opposed to the unprocessed IMC. The reduced particle size, as well as the α- polymorphic form of IMC, contributes to the enhanced dissolution rate.

Hydroxypropyl-β- cyclodextrin complexation of γ-Indomethacin (IMC) was processed with the supercritical antisolvent (SAS) technique as well. SAS processing resulted in the highest initial dissolution rate of IMC complexed particles compared to ii

both spray drying and the physical mixture. This initial increase in dissolution rate is attributed to the micronization of particles. The addition of the water soluble polymer polyvinylpyrrolidone (PVP) to the IMC complex enhanced the dissolution rate further.

Finally, the synthesis of new nanosized crystalline coordination drug polymers is explored. These crystalline coordination drug polymers are composed of the drug molecules coordinated to zinc metal ion as a ligand. The dissolution is controlled mainly by the release of the bridging ligands. These new nanocrystalline CDPs serve as potential for enhanced dissolution.

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ACKNOWLEDGEMENT

I would like to express my heartfelt gratitude to Dr. Jing Li for her continued support, friendship, motherly concern, and advice throughout my whole graduate career.

I would like to thank Dr. Wayne Wang for his advice and for being a constant source of encouragement during my internship at Johnson and Johnson Pharmaceutical

Research and Development (PRD) and now as he is serving as an outside member of my committee.

I am grateful to Dr. Ralph Warmuth and Dr. KiBum Lee for being on my defense committee and for willing to take up another PhD defense. Thank you for your honest remarks and valuable comments.

I would also like to thank Dr. Gene Hall and the late Dr. Theodore Madey (may his soul rest in peace), for serving on my proposal committee.

I would like to thank IGERT for their support financially over the past few years and for all the meaningful discussions I was able to participate in. I would like to especially thank Prabhas Moghe for his useful critiques and Linda Anthony for her constant encouragement and support. I would also like to thank them for supporting my first internship at Johnson and Johnson PRD. Thank you to all the friends I made through this program.

I would like to recognize and thank my friends, mentors, and colleagues at

Johnson and Johnson Pharmaceutical Research and Development: Denita Winstead,

Pei Chu, Lian Liu, Nagy, Ronnie, Michelle, Brett, Teresa, Gary, Alexandra, Brigitte.

I would like to thank the chemistry graduate staff including Melissa, Ann, Eileen,

Dawn, John, Alexei, Tom Chapin and Tom Emge. From the department of cell biology and neuroscience I would like to thank Valentine Starovoytov for help with the use of the

SEM facilities. Many thanks to Bill (from the physics mechanics lab) and Dr. Olson

(chemistry department) for all their help in providing me with the resources to help me get my work to what it is now.

Group members past and present, ever since Dr. Li came to the New Brunswick campus; with a deep heart of gratitude I want to thank you all for your help, support, comments, jokes, and care that I have experienced from you. I want to thank Jungling

Sun and Longle Ma for providing all the H1NMR data. I also am very grateful to Yin

Wang for his work with me on the CDP systems, providing some supporting information on this and his friendship.

A special thanks to the Catholic center for their presence. Thanks to all the priests especially Fr. Tom, Fr. Peter, and Fr. Kevin for their example in holiness. I also want to thank Sister Jodie D Angiolillo (Director of evangelization of the diocese of

Metuchen), for being my spiritual guide and for her guidance and encouragement as well to finish this race.

To my Malankara ―Kara Krew‖ and all the friends I have met along my graduate life, I thank you for your kindness and for believing in me. A special thank you to Sandy for helping me access resources online to get much of my writing done.

I would like to thank my Jesus Youth family, especially for all their constant prayers and support from all over the world. In a special way I would like to thank

Sajichettan, Joseychi, Becky, Issac, Maria, and Joseph for opening up their home to me

to minimize my commute while I was pregnant. To Jottychettan and Jyothnichi, I thank them for bearing with me as faithful friends ever since my undergraduate days, and for always encouraging me even to the present day.

To my siblings and nephews and niece gifted to me by my husband;

Bobbychettan, Chechi, Liggy, Lukey, Mattymo and Lydiana. I thank you for your constant support, patience and for understanding when I could not spend the time very much meant for you, with you.

To my sisters, Priyachi, Preetichi, Penny, my brothers in-law, Bobychettan,

Shaunchettan and Szilvester, and my nephew and niece, Adrian and Kathryn: I thank you all for helping in more ways than one during these years of my graduate life. Thanks for believing in me, encouraging me and always knowing that if I put my heart into something that I will do a good job on it.

To daddy and mummy (my parents in law) thanks for bearing with me and for taking charge of the things that I should have been up to. One thing is for sure: I know that when God chose you all to become my new family, He knew exactly what He was doing.

To my parents, you have seen this from the very beginning and been with me through the toughest times of my graduate studies, the uncertain times, and the prosperous times. Now, in your much older age you have had to look after my Athu (who is less than 1 year old) for almost 24hrs on more than one occasion. First, thank you for bringing me into this world, even if things are not always easy. Thank you for always pushing and loving me regardless of my situation. Thank you for your unconditional love.

Thanks for teaching me about God from my childhood. He is the main reason that I have

gotten this far. Finally, thank you for your example of attending daily mass. It has become my daily source of my strength.

To my Athanasius (Athu), I thank God for allowing me to give birth to you. It was a life experience which taught me to sacrifice and persevere with the hope of a joy that awaits us. You have helped me relax during my stressful moments and to just be me.

To my husband Binu (Kuttan), my coach, my confidant, my love; your organization skills, intellectual capabilities, loving nature and most of all, your love for our

God has continued to bless me. You have helped me in a huge way to carry me through this huge milestone and you know it.

To the LORD who taught us that the last shall be first, I thank my God, the first in my life. You are the truth that I have felt, experienced, and can never deny the reality of, no matter how much I try. I thank You for being my hope, inner and outer strength, and guide especially through all these years of my graduate career. I thank God for placing all these amazing people in my life so I can become better in so many ways. These people include the ones I have mentioned, the ones who have prayed for me, and for any of those whom I have forgotten to mention here. Among the many Biblical verses there are two that have been the most encouraging throughout all these years. I have typed the verses in the way I have remembered it. The first is that God chooses the weak things of this world to do His greatest works through them (1 Corinthians 1:27) and the second verse is from (Philippians 1:6) He who began a good work in you will be faithful to complete it in you.

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TABLE OF CONTENTS

ABSTRACT OF THE DISSERTATION...... ii

ACKNOWLEDGEMENT...... iv

TABLE OF CONTENTS...... viii

LIST OF TABLES...... xi

LIST OF ILLUSTRATIONS...... xii

LIST OF ABBREVIATIONS...... xviii

LIST OF SYMBOLS...... xxi

CHAPTER 1……………………………………………………………………...... …… 1

Introduction 1…………………………………………………………………………. 1

Background……………………………………………………………………….…….. 3

1. Biological relevance of solubility...... 3

2. Solubility fundamentals...... 5

3. Kinetic and thermodynamic solubility...... 7

4. Formulation approaches...... 8

5. Supercritical fluids...... 11

a. Supercritical CO2...... 12

b. Fundamentals...... 18

6. Coordination drug polymers...... 22

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7. Size reduction...... 23

Objective………………………………………………………………………………… 26

References……………………………………………………………………………… 28

CHAPTER 2

Effects of solvent, concentration, pressure and temperature on SAS processed

-indomethacin for the purpose of -indomethacin solubility and dissolution rate enhancement……………………………………………………………………………. 38

2.1. Introduction...... 38

2.2 Background...... 39

2.3 Materials and methods...... 40

2.4 Results and discussion...... 45

2.5 Conclusion...... ……… 55

2.6 References...... 56

CHAPTER 3

Dissolution rate enhancement of indomethacin with one-step precipitation of indomethacin and cyclodextrin complexation……………………………………….. 60

3.1. Introduction...... 60

3.2 Background...... 62 ix

3.3 Materials and methods...... 66

3.4 Results and discussion...... 69

3.5 Summary...... 76

3.6 References...... 78

CHAPTER 4

Coordination drug polymers, a new approach to enhance drug dissolution...... 81

4.1. Introduction...... 81

4.2 Materials and methods...... 86

4.3 Results and discussion...... 92

4.4 Summary...... 104

4.5 References...... 105

CHAPTER 5

Closing remarks...... 107

APPENDIX...... 109

CURRICULUM VITAE…………………………………………………………………… 119

LIST OF TABLES

Table 1: United States Pharmacopeia descriptive terms for solubility

and their values………………………………………………………. 6

Table 1.1: Common formulation approaches and their marketed

products...... 10

Table 1.2: A) Compares physical and transport properties of a gas, SCF

and a liquid, B) Lists the critical temperature and pressures

of several supercritical fluids...... 13

Table 2: D10, D50, D90 of unprocessed and processed γ-IMC...... 47

Table 2.1: D10, D50, D90 of unprocessed and processed γ-IMC...... 49

Table 2.2: A summary of the process conditions and saturation

solubility of γ –IMC...... 53

Table 3: Properties of α, β, γ cyclodextrins…...... ……… 62

Table 4: BPY, BPE and BPEE ligands, their structure, basicity and solubility…………………………………………………………………………………… 87

Table 4.1: Unit cell parameters of CDPs…...... 90

Table A1: Additional solubility data…...... 115

LIST OF ILLUSTRATIONS

Figure 1: Passage of solid oral formulation into the bloodstream...... 4

Figure 1.1: Biopharmaceutics classification system……………...... 5

Figure 1.2: Phase diagram depicting the supercritical region……...... 11

Figure 1.3: Supercritical CO2 as a solvent (RESS) ……………...... 15

Figure 1.4: Supercritical CO2 as an antisolvent (SAS) ……………...... 17

Figure 1.5: Pressure vs. mole fraction of CO2 indicating the supercritical

phase with respect to the mixture critical point. ………...... 20

Figure 1.6: A) Supersaturation (s) vs. Time (t)

B) Average particle size vs. Supersaturation...... 21

Figure 1.7: Schematic of the breakdown of CDP in water…..……...... 23

Figure 1.8: Depiction of the diffusion layer…………………..……...... ……… 26

Figure 2: Structure of indomethacin……………………………...... ……….40

Figure 2.1: High pressure apparatus………………………………...... 43

Figure 2.2: SEM images of A) γ-IMC, IMC dissolved in DCM at solution

concentrations, B) 1.0%w/v, and C) IMC at 0.2% w/v…...... 48

Figure 2.3: Particle size distribution as A) concentration, B) pressure,

and C) temperature are varied……………………...... 50

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Figure 2.4: Powder x-ray diffraction of IMC in the A) -form, B) -form,

C) IMC as is, D) IMC processed with DCM (SAS) .…...... 51

Figure 2.5: Differential Scanning Calorimetry: Thermal properties of A) -IMC

and SAS processed IMC in B) Acetone and C) DCM……...... 52

Figure 2.6: Dissolution Profiles: A) γ -IMC, B) micronized γ -IMC,

C) α –IMC (recrystallized), D) γ -IMC (SAS-acetone), and

E) γ-IMC (SAS-DCM) …………………………………………………. 54

Figure 2.7: Particle size distribution of A) γ-IMC, B) micronized γ -IMC,

C) recrystallized IMC, and IMC processed in SAS with

D) acetone, and E) DCM as the solvent………………...... ……… 55

Figure 3: The molecular shape and chemical structure of

β-cyclodextrin……………………………………...... 63

Figure 3.1: Formation of an inclusion complex of salicylic acid with

β-cyclodextrin...... 64

Figure 3.2: Higuchi and Connors phase solubility diagram…….. ………………. 65

Figure 3.3: Phase-solubility plot of Indomethacin in increasing amount of

HP-β-CD...... 69

Figure 3.4: PXRD pattern of A) γ-IMC as is, B) HP-β-CD, IMC- HP-β-CD,

C) physical mixture, D) spray dried, E) SAS processed, and

F) SAS processed ternary mixture of IMC -HP-β-CD-PVP...... 70

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Figure 3.5: Observing the ibuprofen crystallization peak in the

differential scanning calorimeter of A) γ - Indomethacin

as is, B) IMC+ HP-β-CD physical mixture, C) Spray dried,

and D) SAS processed...... 71

Figure 3.6: Observing a shift of the benzoyl peak in the raman spectra of

A) γ - Indomethacin as is, B) IMC+ HPβCD physical mixture,

C) Spray dried, and D) SAS processed...... 72

Figure 3.7: Observing a shift of the benzoyl peak in the infrared spectra of

A) - Indomethacin as is, IMC- HPβCD B) physical mixture,

C) Spray dried, and D) SAS processed...... 73

Figure 3.8: Dissolution profile of A) γ - Indomethacin as is,

B) IMC- HPβCD physical mixture, C) Spray dried IMC- HPβCD,

D) SAS processed IMC- HPβCD, E) Spray dried IMC- HPβCD-PVP, and

F) SAS processed IMC- HPβCD –PVP...... 75

Figure 3.9: SEM images of γ - Indomethacin as is A), IMC processed with Spray

drying zoomed in view B) and zoomed out view C), IMC processed

with SAS zoomed in view D) and zoomed out view E) ...... 76

Figure 4: 1H NMR of BPY (top left), HIBP (bottom left) and CDP1 (top right) and CDP2

(bottom right) in deuterated water……………………...... 83

Figure 4.1: 1H NMR of BPE (left), and CDP3 (right) in deuterated water…………. 84

Figure 4.2: Structure of Ibuprofen…………………………………………….. ……….85

Figure 4.3: Absorbance peaks of HIBP (solid line), BPY (solid gray line) and BPE xiv

(dotted line)………………………………………………………………………………. 91

Figure 4.4: Absorbance peaks of HIMC (solid line), BPY (solid gray line) and BPE

(dotted line)………………………………………………………………………………. 91

Figure 4.5a: Structure of CDP1……………………………… ……………………….. 93

Figure 4.5b: Linear polymer backbone of CDP1…………………………………….. 93

Figure 4.5c: PXRD of CDP1…………………………………………………………… 93

Figure 4.6a: Structure of CDP2………………………………………………………… 94

Figure 4.6b: Zigzag chain of CDP2…………………………………………………… 94

Figure 4.6c: PXRD of CDP2. ………………………………………… ………………. 94

Figure 4.7a: Structure of CDP3. ………………………………………………………. 95

Figure 4.7b: Linear polymer backbone of CDP3. …………………………………… 95

Figure 4.7c: PXRD of CDP3. …………………………………………………. ……….95

Figure 4.8a: Structure of CDP5. ………………………………………………………. 96

Figure 4.8b: Linear polymer backbone of CDP5. …………………………………… 96

Figure 4.8c: PXRD of CDP5. ………………………………………… ………………. 96

Figure 4.9a: Structure of CDP6. ……………………………………………………… 97

Figure 4.9b: Linear polymer backbone of CDP6. ……………………………………97

Figure 4.9c: PXRD of CDP6 ………………………………………………………….. 97

Figure 4.10a: Time dependent concentration of CDP1 (black), 2 (red), 3 (blue) in distilled water. ……………………………………………………………………………. 99

Figure 4.10b: Time dependent concentration of CDP2 (black), and Ibuprofen as is (red) in distilled water. ………………………………………………………………………… 99

Figure 4.11: Time dependent concentration of CDP6 (blue) compared to

Indomethacin alone (red) in distilled water. ……………………………………………100

Figure 4.12: SEM images confirming particle size reduction: Drug as is (top left),

CDP2 (top right), ultrasonicated CDP2 (bottom).……...... 101

Figure 4.13: Powder x-ray pattern of CDP2 before and after

ultrasonication…………………………………………………………………………… 102

Figure 4.14: Powder x-ray pattern of the new CDP before and after ultrasonication……………………………………………………………………………. 102

Figure 4.15: Absorption vs, time of the ultrasonicated particles of the new CDP (red),

CDP2 (blue) and HIBP as is (black).………………………………………………. 103

Figure A1: Schematic of PXRD...... 109

Figure A2: Diffraction within atomic planes...... 110

Figure A3: Schematic of Ultraviolet Visible spectrophotometer...... 111

Figure A4: Illustration of the basic concept of an ATR…………………………….. 112

Figure A5: HP-β-CD (Hydroxypropyl-beta-cyclodextrin) is processed with the supercritical Antisolvent at 1%w/v Ethanol 35°C, 1500psi and varies from (A) 0.6 to (B)

1.0 in the degree of substitution. A significant difference in particle morphology and size is observed for (A) where there are irregular shaped nanoparticles and (B) is composed of microparticles that are spherical…………………………………………..……….. 113

Figure A6: DSC plot of HPCD……………………………………………………….. 114

FigureA7: Spray dried IMC-HPβCD in a 1:1 (solid line) and

1:4 (dashed line) ratio…………………………………………………………………… 116

Figure A8: Supporting raman spectra for chapter 3...... 117

Figure A9: A year old sample of IMC:HPβCD(SD) stored at room temperature

(DSC)……………………………………………………………………………………… 117

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Figure A10: Residual solvent analysis indicating that the SAS process meets the FDA requirements…………………………………………………………… 118

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LIST OF ABBREVIATIONS

AMCP Above the mixture critical point

API Active pharmaceutical ingredient

ASES Aerosol solvent extraction system

BCS Biopharmaceutics classification system

BMCP Below the mixture critical point

BPE 1,2-bis(4-pyridyl)-ethane

BPEE 1,2-bis(4-pyridyl)-ethylene

BPY 4,4’bipyridine

CCDC Cambridge crystallographic data center

CD Cyclodextrin

CDP Coordination drug polymer

DCM Dichloromethane

DMF N,N-dimethylformamide

DMSO Dimethylsulfoxide

DSC Differential scanning calorimetry

GAS Gas antisolvent recrystillization

xviii

HIBP Ibuprofen

HPβCD Hydroxypropyl beta cyclodextrin

HPLC High performance liquid chromatography

IR infrared

PM Physical mixture

PXRD Powder x-ray diffraction

NCE New chemical entity

NMP N-methyl-pyrolidone

NMR Nuclear magnetic resonance

NSAID non steroidal anti-inflammatory drug

RESS Rapid expansion of supercritical solution

RESS_SC Rapid expansion of supercritical solution with solid cosolvent

RESOLV Rapid expansion of supercritical solution into a liquid solvent

SAS Supercritical antisolvent

SEDS Solution enhanced dispersion with supercritical fluids

SEM Scanning electron microscopy

SCF Supercritical fluids

SC-CO2 Supercritical carbon dioxide

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SD Spray drying

PCA Particles by compressed antisolvent

PGSS Particles of gas saturated solution

PVP Polyvinylpyrrolidone

RDX Cyclotrimethylene trinitramine

USP United States pharmacopeia

LIST OF SYMBOLS

A surface area c real solute concentration

ceq equilibrium solute concentration

C concentration of bulk solution

Cs saturated concentration

D diffusion coefficient of the compound dC/dt dissolution rate

Gmix Gibbs free energy of mixture

Hmix Enthalpy of the mixture

Kc dissociation equilibrium constant g/mol gram per mole mg/ml milligram per milliliters h thickness of the diffusion boundary layer

γ activity coefficient

γ  activity coefficient at equilibrium concentrations

γi interfacial tension or surface free energy of drug

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ρ density of solid s supersaturation ratio s* supersaturation critical value

Smix Entropy of the mixture

MW molecular weight of drug

T temperature

Tc critical temperature

Tm mixture critical temperature t time

P pressure

Pc critical pressure

Pm mixture critical pressure r radius of the particle

R gas constant

µm micrometer nm nanometer w/v weight by volume

XCO2 mole fraction of carbon dioxide

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CHAPTER ONE

1. Introduction

New chemical entities (NCEs) are novel drugs or active pharmaceutical ingredients (APIs) entering the drug discovery pipeline due to technological innovation and pressure of competition1. NCEs were initiated in the mid-1990s by a combination of combinatorial chemistry and high throughput screening as opposed to wet chemistry1-4.

The resulting NCEs are mainly characterized as lipophilic with high molecular weight suitable for biological targets, which consequently exhibit poor water solubility. Both patients and the pharmaceutical industry are plagued with limited aqueous solubility of active pharmaceutical ingredients5,6. Poor solubility leads to poor absorption, bioavailability, and the need for high drug dosage to be administered, leading to increased side effects3. The result is the withdrawal of drugs from the market and/or the return of drugs to be reformulated at the minimum; costing time, money, and energy7.

Despite numerous efforts to alleviate these issues solubility remains the leading challenge in drug development8.

Various approaches are focused on particle design in drug development. Particle design in terms of particle size reduction, the formation of complex particles and of crystalline drug particles will be considered. The former two are among the most common approaches considered by pharmaceutics. Micronized particles with uniform particle size and narrow size distribution are desirable for optimum therapeutic efficacy.

For instance, oral delivery requires a particle size range between 1-5µm to cross the intestinal mucosa. Any particles larger than this remain in the gut lumen. Particles that are smaller than 0.1µm, exhibit higher gastrointestinal uptake and therapeutic efficacy 9,

10. Complex particles, specifically, cyclodextrin – based formulations, involve the encasing of lipophilic drugs inside its hydrophobic interior and attains water solubility through its hydrophilic exterior. Sporanox ® is an example of a marketed drug. It is the poorly water soluble drug Itraconazole complexed with hydroxypropyl β- cyclodextrin11.

The latter approach, the formation of crystalline drug particles is recently gaining more attention as alternative routes for enhancing dissolution rate limited solubility.

Pharmaceutical scientists investigate different crystalline forms, hydrates, solvates or amorphous forms because the arrangement of atoms and molecules in solids affects the different physicochemical properties the compound will possess12. Cocrystals are an example of crystalline drug particles that have been investigated with potential to provide greater solubility and stability. To this end, we have synthesized a new series of crystalline compounds called coordination drug polymers (CDPs). Coordination drug polymers, are composed of a drug substance as a ligand that is weakly coordinated to

Zinc metal. Zinc is an essential trace element that is found in our bodies in numerous zinc enzymes and proteins 13. It is commonly supplemented based on a person’s diet14.

The dissolution mechanism of these CDPs occurs by the replacement of the metal bridging amine ligands with water molecules. The potential of these CDPs, based on their structure, size and composition to enhance solubility and dissolution rate is evaluated as well.

Numerous methods exist to achieve the desired physical modifications to the

API. It is important to use the appropriate method to achieve the desired particle features, in order to save time, cost, obtain a narrow particle size distribution and have a stable product, among other attributes. From the existing methods, unconventional methods such as supercritical fluid processing continue to gain attention as a versatile, single step processing technique that uses carbon dioxide as a ―green‖ solvent15-19.

Supercritical antisolvent processing of 1-5 µm of insulin, trypsin and lysozyme proteins was also reported to maintain the biological activity after processing 20. Currently, no marketable drug products are reported for this technique and so much effort is directed

21 here . Supercritical antisolvent CO2 shows versatility with reduction in particle size and complexation all in one step at mild operating temperatures and pressures to enhance the solubility and dissolution rate of the model drug Indomethacin. Ultrasonic processor is another fast, easy to use, simple, unconventional method that uses acoustic sound to reduce particle size of CDPs by mechanical stress into micro and submicron or nanoparticles.

1.1 Background

1.1.1 Biological Relevance of Solubility

The most common routes to administer commercial APIs are the intravenous

(injection into vein), intraperitoneal (injection into abdomen cavity), subcutaneous

(beneath the skin), intramuscular (injection into muscle), and the oral route. Among all these, the oral route is the most safe, economic, noninvasive, and convenient route. This route, however, faces numerous challenges; mainly, poor drug absorption into the blood stream. Poor solubility limits absorption; that is the movement of a drug from its site of application into the bloodstream. Low absorption in turn inhibits sufficient bioavailability, or the measured amount of drug in the systemic circulation 3, 22.

In the oral route drugs must enter the systemic circulation to exert a therapeutic effect. Figure 1 illustrates the steps that a solid oral formulation passes through in order to get into the blood stream. First, the drug in its solid dosage form disintegrates. Then

4 the solid drug particles dissolve within an aqueous environment (gastrointestinal tract) into drug molecules. The extent and rate at which drug molecules go into solution is determined by the drug solubility and dissolution rate, respectively. This is then followed by permeation of the drug molecules into the bloodstream through the intestinal membrane23.

The biopharmaceutics classification system (BCS) correlates solubility and permeability to bioavailability24-27. According to the BCS classification system, APIs are grouped into categories I-IV as shown in Figure 1.1. Class I category drugs are defined as the drugs with the highest solubility and permeability, and therefore are readily absorbed when administered orally. The remaining classes II-IV suffer from poor solubility, permeability, or both and in turn affect the amount of absorption or bioavailability of the drug28. Solubility is a predetermined and rate limiting step for absorption, especially for class II drugs 29. According to Lipinksi, solubility is a much larger issue for drug discovery than permeability is 30.

Disintegration Drug in solution

Membrane Dissolution transfer c Blood stream v

Solubility Permeability

Figure 1: Passage of solid oral formulation into the bloodstream.

Class I Class II High solubility Low solubility High permeability High permeability

Class III Class IV Solubility High solubility Low solubility Low permeability Low permeability

Permeability

Figure 1.1: Biopharmaceutics classification system

1.1.2 Solubility Fundamentals

Solubility is an important physicochemical property of a drug that is pertinent in the pharmaceutical field because it helps determine the extent of absorption and oral bioavailability 31. It is defined as the maximum amount of solute that will dissolve the most stable crystalline form in a given volume of solvent at equilibrium conditions of temperature and pressure32. More specifically solubility is defined as buffered, unbuffered and intrinsic solubility 33. Unbuffered solubility is usually measured in water and is the saturated solubility of the compound at the final pH of the solution. Buffered solubility is the solubility at a given pH. It neglects the possibility of salt formation with the counterions of the buffering system. Finally intrinsic solubility is the solubility of the neutral form of an ionizable drug.

According to the United States Pharmacopeia (USP), solubility is described as the number of milliliters needed to dissolve 1 gram of solute. The varying degrees of

6 solubility from very soluble to practically insoluble are defined and listed in Table 134.

High solubility according to BCS is 85% within 30 minutes from pH 1 to 7.5 3.

Descriptive solubility term Parts of solvent required Solubility cutoff (mg/ml) for 1 part per solute Very soluble Less than 1 >1000 Freely soluble From 1 to 10 >100 soluble From 10 to 30 >33.3 Sparingly soluble From 30 to 100 >10 Slightly soluble From 100 to 1000 >1 Very slightly soluble From 1000 to 10,000 >0.1 Practically insoluble or Greater than or equal to >0.01 insoluble 10,000

Table 1: United states pharmacopeia descriptive terms for solubility and their values.

On the molecular level, solubility involves dissolution; the breaking of intermolecular attractions between solute-solute, solvent-solvent, and the formation of new interactions between solute-solvent22. It is these interactions, which are identified as ionic, van der Waals, and hydrogen bonding, which govern solubility. The first step is to free a solute molecule from its cavity. Next is to create a cavity in the solvent. Finally, the solvent cavity is occupied by the free solute molecule. The extent to which this mixing between solute and solvent will occur is determined by the gibbs free energy equation

(1).

Drug + Solvent  Solvated Drug,

Equation (1)

Mixing will occur when the free energy is negative. Favorable mixing occurs when Hmix is negative or close to zero. This implies that the reaction will continue until all available solid is dissolved. If on the other hand, the net enthalpy change is positive,

7 the reaction will progress until a saturated solution or equilibrium is reached and G = 0.

Hmix takes into account the differences between water-solute adhesive interaction with the sum of the solute-solute and water-water adhesive interactions. In general, molecules have more freedom in a mixture. Smix is a measure of the randomness or disorder of the molecules of the mixture that is formed. It is usually positive with the formation of a mixture. An increase in water solubility of the drug would mean an increase in the entropy of mixing 31. Maximum solubility is attained when the dissolution process has reached saturation 35.

Several factors play a role in determining the solubility of a compound. These include compound structure, pH, temperature, physical state of the compound when placed in solution either solid or liquid, composition and physical conditions of solvent.

Solubility is determined by the conditions it is measured in.

1.1.3 Solubility Measurements

Kinetic solubility is a strongly time dependent method that is relevant in the early discovery stage for high throughput measurements that involve compounds greater than

600 per week and with limited compound supply. It differs from thermodynamic solubility in that the desired compound is dissolved in an organic solvent such as DMSO and it is a measurement of the dynamic state rather than the equilibrium state 3.

Typically a stock solution of known concentration is made of the desired compound dissolved in dimethylsulfoxide (because it is a strong organic solvent). This stock is then gradually added to the aqueous solvent of interest until the anti-solvent properties of the water drive the compound out of the solution. The resulting precipitation is detected optically, and the kinetic solubility is defined at this point, at which the aqueous component can no longer solvate the drug.

Risks are associated when using this solubilization method, since the measurements are often carried out on compounds that have not been purified or crystallized. The results are irreproducible between different kinetic methods. This is because there is no time for equilibration of the compound and the compound is already in a dissolved state so the precipitation can occur either through an amorphous, metastable or stable form. Kinetic measurements are dependent on the physical form of the initial precipitate. In addition the energy required to break the crystal lattice of the compound by DMSO is unaccounted for in the solubility measurements. Kinetic solubility measurements are intended to evaluate feasibility for biological assays such as structure-solubility relationships36. Thermodynamic solubility, on the other hand, is an equilibration method that is independent of time and dependent on the initial physical form of the compound. It determines the solubility of the stable crystalline form of the compound. The results obtained for thermodynamic solubility are more reproducible and this allows a more reliable assessment of solubility issues of the compound 25.

Commonly measurements are taken by the traditional shake- flask method.

Excess drug is added to solvent at desired temperature and shaken for 24 hours a day for 7 days a week. The excess drug is removed from filtration, and the dissolved amount is detected by high pressure liquid chromatography or ultra-violet spectroscopy or mass spectrometry detection37.

1.1.4 Formulation Approaches

Approaches to enhance solubility are commonly based on chemical or physical modifications. Chemical modifications such as salt and prodrug formation involve structure modification by modifying the lipophilicity, charge, and crystal energy (the arrangement causes a variation in solubility). These modifications are limited to select drugs and are time consuming38. For instance, a limited amount of APIs, have desired

9 functional groups such as ionizable or polar groups with potential for salt formation. In addition, changing one property can adversely affect the other. This is observed when structural modifications that enhance solubility of APIs often also increase the toxicity and decrease permeability 39, 40.

Physical modifications on the other hand are applicable for a wider group of

APIs, are easier to prepare, and in industry are considered more often over chemical modifications. Formulation approaches by physical modifications involve particle size reduction, modification of crystal form, and formulation as a liquid. These formulation approaches along with the most common techniques are listed in Table 1.1. A number of listed companies that are still developing marketable products involve supercritical fluids. Supercritical fluids have the potential to reduce particle size and form composite particles or liquid formulations all in one step.

Formulation Description Marketed Advantages Disadvantages approach (techniques) products

Rely on mechanical Particle Top-down attrition to denaturing, Micronization reduce particle Avoids use electrostatic Rapamune 43, size (milling, of harsh charges, rough Emend 44, (nanoparticle) high pressure solvents surfaces, wide Triglide 45 homogenization, particle size microfluidization) distribution 41, 42

Generation of unstable Under Direct particle polymorphs, Useful for development formation solvates, Bottom-up thermolabile by companies hydrates, Micronization (supercritical drugs, such as Ferro solvent based (nanoparticle) fluids, narrow Corp. and needle shaped precipitation, particle size Thar Tech, particles result Dow, Niro 48, 49 spray –drying ) distribution 46, 47 from rapid growth in one direction.

Modify Gris-Peg, crystalline form Higher Physical Nabilone- of drug (hot melt PVP(Cesamet) Composite particles 50 entropic instability and extrusion) and Rezulin 51, or Solid dispersion crystalline lack of 52 form reproducibility

High cost, large Norvir applied dose (ritonovir), Liquid-like Drug in liquid and high Ease of Fortovase formulations dispersion amounts of processing, (Squauinavir) (emulsions, excipients, enhances Vfend complexation) 32, toxicity of stability (voriconazole), 53, 54 surfactants and Sporanox even at low 55, 56 levels.

Table 1.1: Common formulation approaches and their marketed products

1.1.5 Supercritical Fluids

As early as the 1970s, supercritical fluids (SCFs) were realized for use in saving energy. They have found extensive applications from extraction, to polymerization, to the purification of organic and inorganic substrates, and to process micro- and nanoparticles.

Any substance can achieve a supercritical state above its critical point, which is defined as the highest pressure (critical pressure, Pc) and temperature (critical temperature, Tc). At the critical point a substance can exist in equilibrium with its vapor and liquid state (figure 1.2). Beyond the critical point a homogeneous fluid exists in a supercritical state exhibiting both liquid and gas like properties. Its low viscosity, low conductivity, near zero surface tension and high diffusivities resemble gas-like properties, whereas its density is similar to liquids. SCFs’ gas-like properties enhance mass transfer and by slight changes in pressure or temperature near its critical point its

Solid

Supercritical

Liquid

Pressure Pressure

Gas

Temperature Figure 1.2: Phase diagram depicting the supercritical region density or solvent power can be tuned as well (table 1.2A).

1.1.6 Supercritical CO2

Supercritical CO2 (SC-CO2), is the most suitable and widely used SCF to process pharmaceutical materials. It is abundant, relatively inexpensive, non-toxic, non- flammable, recyclable, relatively safe and environmentally benign. Its’ near ambient critical temperature of 31.3°C and pressure of 73.8 bars are highly desirable, especially for processing thermally sensitive materials, when compared to other SCFs such as water, and ethanol, to name a few (table 1.2B).

Numerous methods to process particles using supercritical CO2 have been developed and these are broadly classified into three categories based on whether the supercritical fluid is used: 1) as a solvent for drug and excipients 2) as an antisolvent when the drug and excipients are dissolved in an organic solvent that is miscible with

SC-CO2 and 3) as a vehicle or medium for other fluids techniques such as expansion in particle gas saturated solution (PGSS), milling, as supercritical fluid extraction of

57 emulsions, supercritical CO2 assisted spray-drying . Major works have been reported for the use of supercritical CO2 as a solvent as in RESS and as an antisolvent following section serves to provide a brief description of these methods. Excellent review articles are available15, 17, 58-60.

A Property Gas SCF Liquid

Density -103 0.2-0.9 0.8-1.0 (g/cm3) Viscosity 0.5-3.5e-4 0.2-1.0e-3 0.3-2.4e-2 (Poise) Diffusivity 0.01-1.0 0.1-3.3e-4 0.5-2.0e-5 (cm2/s) B

Fluid Tc(C) Pc(bar) Ethylene 9.3 50.4 Carbon Dioxide 31.1 73.8 Nitrous Oxide 31.3 73.8 Ethane 32.3 48.8 Chlorodifluoromethane 96.0 51.9 Propylene 91.8 46.2 Propane 96.7 42.5 Ethanol 240.8 61.4 Water 374.1 217.7

Table 1.2:

A) Compares density, viscosity, and diffusivity of a gas, SCF and a liquid.

B) Lists the critical temperature and pressures of several supercritical fluids.

1.1.7 SC-CO2 as Solvent

RESS, first applied by Krukonis in 1984, is where the non-polar SC-CO2 is used as a solvent as depicted in Figure 1.3. In this process the solute is dissolved in the supercritical fluid and then expanded through rapid depressurization into atmospheric

14 conditions through an orifice or a nozzle. The pressure drop from a supercritical fluid to a gas can be as fast as 10-5 seconds. The lowered vapor pressure decreases the density of the solvent and causes it to evaporate which results in the precipitation of solute particles 19, 61, 62. The main advantage of RESS is that this process is free of organic solvents. Many small non-polar molecules and low molecular weight drugs, such as benzoic acid, salicylic acid, aspirin , ibuprofen are soluble in SC-CO2 and have been processed using this method 63, 64. Nanoparticles within the size range of 60nm-1000 nm have been obtained with RESS.

Several modifications were made where, instead of the rapid expansion into air, which results in agglomeration, a nonsolvent medium was used such as water, which decreased agglomeration and produced nano-particles (2.5-200 nm) 65 . The acronym

RESOLV (rapid expansion into a liquid solvent) was coined for this modified technique.

RESS_SC (rapid expansion of supercritical solution with solid cosolvent) is another modification of the conventional RESS method where Griseofulvin nanoparticles were obtained from 50-250nm with no agglomeration. Solubility enhancement was reported to be 28-fold greater than the conventional RESS process without the addition of the cosolvent. RESS-SC processed Phenytoin nanoparticles had a 400-fold increase in solubility. In both these cases menthol was used as a solid co-solvent 66, 67.

The solubility of numerous polar molecules, pharmaceutics and high molecular weight polymers, however, is limited in SC-CO2 which results in high operating pressures and temperatures. For instance microparticles (20-90 m) of L-PLA (polylactic acid) with naproxen, an anti-inflammatory agent, were processed at a pressure and temperature of

190 bars and 114°C, respectively. The use of supercritical carbon dioxide as an antisolvent allows a wider range of drug substances and its excipients to be processed at mild temperature and pressures.

CO2 Nozzle

P1 P1> P2 P2 Solid c c ccccccc Particles

CO2

Figure 1.3: Supercritical CO2 as a solvent (RESS)

1.1.8 SC-CO2 as Anti-solvent The antisolvent processes were developed since most drugs and high molecular weight polymers are insoluble in SC-CO2. Several variations within the antisolvent method have been developed, for example, GAS, SAS, PCA, ASES and SEDS. All are based on the antisolvent process, where the desired solute is dissolved in an organic solvent that is miscible with the Sc-CO2 and the solute is insoluble in SC-CO2. The selected organic solvent must both dissolve the solute and be totally miscible with SC-

CO2. The drug solution is brought in contact with the SC-CO2 where a solvent-induced phase separation, marked by precipitation, is observed.

GAS technique differs from the other supercritical antisolvent techniques because it is a batch process. The particle production in GAS occurs in the liquid phase as the compressed gas is gradually added to the organic solution. This causes a volumetric expansion of the solvent which reduces the solvent power of the liquid solvent

and solid particles precipitate. The rate of addition of supercritical CO2 determines the size and morphology of the particles. First proposed by Gallagher in 1989, this technique has been used to micronize explosives such as RDX (Cyclotrimethylene trinitramine) 19, pharmaceuticals, and several biodegradable polymers such as the composite of insulin in PEG/PLA within a size range of 0.4 to 0.6m 68. This technique offers the advantage of processing a wider range of materials than RESS and PGSS at lower pressures and milder temperatures. However, it is limited in that it uses an organic solvent and it provides little control over the particle size distribution because of low supersaturation

19, 69 rates as compared to other SC-CO2. anti-solvent methods

In the SAS68, 69, PCA70, 71 and ASES72 techniques the solution is injected into the precipitating chamber (Figure 1.4), filled with SC-CO2. In these techniques particle size and morphology are mainly dependent on solvent extraction and mass transfer. PCA was originated by Dixon, where a capillary is used to precipitate particles in a semi- continuous mode into subcritical or supercritical compressed gas. SAS was a modification of PCA, described by Lim, who suggested a continuous mode where the organic solution and SC-CO2 will flow continuously. ASES was first described by Bleich, although not fundamentally different from SAS it uses a nozzle rather than a capillary tube to disperse its solution. However, at high atomization (conversion of liquid into very fine droplets) conditions in the capillary no distinction between SAS and ASES exists.

Overall PCA, SAS and ASES are used interchangeably, ―in current publications almost no distinction is made between SAS, PCA, ASES.‖19. For these semi-continuous antisolvent processes the smallest particle size was obtained Tetracycline dissolved in

NMP (N-methyl-pyrolidone) was precipitated with an average size of 150nm69. The main difference between these techniques and SEDS, the Solution enhanced dispersion method is that SEDS uses a coaxial nozzle. The supercritical fluid is injected through the

17 inner tube whereas the outer tube is filled with the organic solution. The coaxial nozzle incorporates a premixing chamber within its nozzle which enhances mixing, uniform and continuous particle formation73. The lowest size at 0.05 m was obtained with Albumin19.

A modification of the semicontinuous antisolvent processes was reported where an enhanced mass transfer technique is employed. A vibrating surface is used to increase the mass transfer and formation of small droplets. This showed that the particles formed were about 10 times smaller than the simple SAS method with a significant decrease in agglomeration L-PLA with Tetracosactide was precipitated with a mean volume diameter of about 10.1 m74.

SAS will be used in this thesis because this clearly describes the antisolvent process that was used.

Solution

CO2

Nozzle

Solvent + CO2

cccc Dry particles ccccc

Solvent + CO2

Figure 1.4: Supercritical CO2 as an antisolvent (SAS).

1.1.9 Solvent Selection

In the antisolvent process, the organic solution (organic solvent and solute) is added to the compressed gas; the antisolvent (SC-CO2). At the appropriate temperature and pressure the dissolution of the SC-CO2 in the liquid solvent is accompanied by the evaporation and reduction in the solvent powers of the liquid solvent. A supersaturated solution results; which leads to the precipitation of solute particles into dry particles75.

Unlike liquid antisolvent processes, precipitation in compressed CO2 occurs at a much faster rate.

Solvent selection, in the antisolvent process, requires that the solvent dissolve the desired solute and that the solvent be partially or completely miscible with the antisolvent. The latter, miscibility criteria, is directly related to the extent of the volume expansion. CO2 is a non-polar molecule but can be miscible with polar solvents such as acetonitrile and dimethylsulfoxide because of the interaction between the quadrupole

75 moment of CO2 and the dipole moment of the polar solvents . A comparison of the volumetric expansions ( a measure of the reduction in solvent power) of DMSO and

DCM (dichloromethane or methylene chloride) showed that DCM (which has a lower polarity than DMSO) exhibited a larger volume expansion at a lower pressure because

76 of its higher miscibility in SC-CO2 than DMSO . Based on the concept of ―like dissolves like‖ a higher miscibility for DCM in SC-CO2 is observed than for DMSO because of the higher polarity of the latter solvent. This implies that a faster supersaturated solution, therefore faster precipitation with smaller particles can be achieved with DCM than with

DMSO at a given temperature and pressure.

1.1.10 Mass Transfer Proper solvent selection is paralled with an understanding of the solute-solvent and solvent-antisolvent interactions. Different particle morphologies and sizes result when operating at either subcritical or supercritical points77, 78. The mixture critical point

(MCP) is defined as the point at and above which the binary mixture (solvent and SCF) is completely miscible and there exists one phase or the supercritical phase. Below the mixture critical point (BMCP) or at the subcritical point, both the vapor and liquid phases coexist. Figure 1.5 plots out that the mixture critical point will change depending on the material, but is ignored at low solute concentrations. Supersaturation both AMCP (above the mixture critical point) and BMCP (Below the mixture critical point) is governed by two different mechanisms and will be described below.

AMCP or P>Pm and TTm, is where two-phases coexist. The interfacial tension results in droplet dispersion and mass transfer that govern supersaturation and particle formation. Here the nucleation and particle growth occur within the solvent droplet phase.

1.1.11 Supersaturation

Supersaturation is the driving force for precipitation. It occurs when the amount of solute that can be dissolved in a solvent exceeds the maximum level at the equilibrium conditions. Supersaturation is defined by the difference between the real solute concentration c and the equilibrium solute concentration ceq at a given pressure and temperature with their corresponding activity coefficients, see equation (2). It is a metastable state that leads to precipitation 19.

Supercritical Phase

Mixture Critical Point

Pressure Pressure Liquid Vapor Liquid-Vapor

X CO2

Figure 1.5: Pressure vs. mole fraction of CO2 (XCO2) indicating the supercritical phase with respect to the mixture critical point.

Precipitation or crystallization is a two step process of nucleation and particle growth which drives the metastable solution to equilibrium. The formation of a nucleus is termed nucleation. Particle growth is the addition of more molecules to the nucleus. At high supersaturation rates nucleation is dominant over particle growth and smaller particles are obtained 76.

c s = Equation (2)  c  eq

where s= supersaturation ratio, c, ceq are solute and solute equilibrium concentrations,

and and γ and γ  are activity coefficient and activity coefficient at equilibrium concentrations, respectively.

Figure 1.6A is a plot of supersaturation (s) vs. time (t). There are three zones depicted. In zone I particles remain dissolved in solution until they reach saturation at s=1. Zone II is an area below the supersaturation critical value (s*) whereas zone III depicts areas of high supersaturation. Trace A and B in the anti-solvent precipitation represent the rate of anti-solvent addition. According to trace A, a fast supersaturation rate will lead to solely nucleation and thus monodispersed particles. Trace B shows a lower supersaturation rate, which would lead to crystal growth and a wide particle size distribution when compared to trace A. Figure 1.6B relates supersaturation to particle size. At low supersaturation rates (i) large particles would be obtained. At higher supersaturation rates (ii) smaller particles would be obtained and a further increase in supersaturation (iii) would lead to an increase in particle size due to agglomeration.

Agglomerated particles are particles that are strongly bonded whereas aggregates are bound by weak van der Waals forces.

III (nucleation and crystal growth)

) )

s s ( ( B s * i ii iii II (predominantly crystal growth) s=1

I (unsaturated solution)

Supersaturation Supersaturation

Average size of particles particles size size of of Average Average

Time(t) Supersaturation (s) Figure 1.6: A) Supersaturation (s) vs. Time (t). B) Average particle size vs. supersaturation

1.1.12 Coordination Drug Polymers

Coordination Drug Polymers (CDPs) are crystalline and stable material. CDPS were designed with a similar dissolution mechanism as that reported by Dr. Uhrich and her coworkers’s on polyanhydride esters. Polyanhydride esters are polymers that breakdown in water to release the drug component salicylic acid which is part of the polymer backbone. The releasing behavior of these polymes can be modified by adjusting the polymer backbone80, 81.

With a similar idea, coordination drug polymers breakdown in water is illustrated as follows (Figure 1.7).Coordination polymers are composed of weak bonds to the metal ion .When CDP polymers are placed inside water, the affinity of amines and water molecules to Zinc metal is similar. The higher concentration of water molecules causes a shift in equilibrium by replacing all the BPY ligands with water molecules. By varying the bridging ligand depending on the degree of basicity, the dissolution rate of CDPs can be tuned.

Figure 1.7: Schematic of the breakdown of CDP in water.

1.1.13 Particle Size

Particle size control remains to be one of the most desirable aspects in particle design by pharmaceutics. It is a critical variable that plays a role in determining solubility and dissolution rate. The Ostwald-Freundlich equation relates solubility to particle size equation (3). Based on this equation the intrinsic solubility or saturation solubility has an indirect relation to particle radius. A decrease in particle size exponentially increases saturation solubility. A significant increase in solubility is observed when particle size is reduced below 0.1μm 82, 83. Solubility is greater for fine particles than large particles84.

 Ln Cs = Equation (3) r

2M   RT

where, M= molecular weight of Drug,

γ = interfacial tension or surface free energy of drug,

ρ = density of solid,

T = temperature,

R = gas constant,

r = radius of the particle

Cs is the solubility of micronized particles.

Dissolution is often the rate limiting step in solubility. The Noyes-Whitney equation, equation (4), relates saturation solubility to dissolution rate. According to equation (4), a decrease in particle size leads to an increase in surface area which leads to a direct increase in dissolution rate. Dissolution rate is the time required for a substance to dissolve at the absorption site. Dissolution describes the process when a solid dissolves in a liquid and dissolution rate is the time it takes for this to occur, a dynamic process. Solubility implies that dissolution is complete and there is a saturated solution, an equilibrium state 3, 85.

The Noyes –Whitney equation is based on the diffusion layer model. The diffusion layer or stagnant layer theory explains the different factors that affect dissolution. According to the diffusion layer theory, the diffusion layer is adjacent to the surface of the dissolving particle. See Figure 1.8 for an illustration of this concept. The diffusion of drug through this layer determines the rate of dissolution of the particle.

Solution of the solute forms a thin layer at the solid/liquid interface, a boundary layer known as the stagnant or diffusion layer. This is then followed by the transfer of the solute molecules from the boundary layer to the bulk solution, with a concentration of C under the influence of diffusion or convection. A decrease in h increases Cs (saturation solubility) and leads to an increase in the gradient (Cs-C)/h and thus to an increase in dissolution velocity. Particle size reduction increases surface area therefore more of the solid is exposed to solution. An increase in surface area then leads to an increase in dissolution rate.

dC ADCs C  Equation (4) dt h

where, dC/dt= dissolution rate,

A = surface area,

D = diffusion coefficient of the compound,

Cs is the solubility of the compound in the dissolution medium it is the solution that is in contact with the surface of the solid and is therefore the saturated concentration,

C or Cb is the concentration of the bulk solutions the concentration of drug in the medium at time t and

h is the thickness of the diffusion boundary layer adjacent to the surface of the dissolving compound.

Solid dosage Stagnant Bulk form film solution Bulk solution Cs

Stagnant film Solid Matrix Drug

X= 0 X= h

Figure 1.8: Depiction of the diffusion layer.

1.2 Objective

Overall this thesis is directed to: 1) explore non-traditional methods to enhance

API solubility and dissolution rate, 2) provide systematic studies with select drug model systems to reduce size with or without the addition of excipients using non-traditional methods, and 3) to explore new routes to enhance solubility and dissolution rates by an understanding of solvent-solvent, solute-solute, and solvent-solute interactions.

In Chapter 2, a fundamental study involving the precipitation of a poorly water soluble model drug, indomethacin, by the supercritical antisolvent (SAS) process is discussed. A systematic approach to reduce particle size is conducted by varying temperature, pressure, and solution concentration. Characterization that involves solubility and dissolution studies of the resulting precipitates are conducted as well.

Based on the dissolution properties of SAS processed indomethacin shows enhanced solubility and dissolution rate.

Dissolution enhancement for Indomethacin is explored further in Chapter 3 with the addition of cyclodextrin, a well-known complexing agent. The resulting particles are compared with spray drying- another competing precipitation method. The analysis is accompanied by appropriate characterizations. SAS versatility is confirmed by the formation of indomethacin-complex with enhanced dissolution properties.

The synthesis of new nano-crystalline coordination drug polymers (CDPs) to enhance solubility is described in Chapter 4. CDPs were synthesized with two model drugs, Ibuprofen and Indomethacin. These drugs were ligands with coordination bonds to Zinc metal. A series of novel CDPs are reported along with their respective dissolution information by varying the bridging ligand structure. Modifications in size to the CDP that displayed the highest dissolution resulted in further enhancement of the dissolution rate.

Formation of nanoparticles by the ultrasonic processor assures faster dissolution properties for this crystalline compound.

Finally, the conclusion summarizes the studies that have been conducted by particle size reduction, complexation and formation of a new crystalline compound in order to enhance solubility and dissolution rate. Future steps toward a better understanding of particle design by SAS and by ultrasonication are suggested, followed by steps left to confirm bioavailability.

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CHAPTER TWO

Effects of solvent, concentration, pressure and temperature on SAS processed - indomethacin for the purpose of -indomethacin solubility and dissolution rate enhancement.

2.1 Introduction:

In developmental pharmacy nearly 40% of the active pharmaceutical ingredients

(APIs) exhibit poor water-solubility1. For oral delivery, these APIs exhibit dissolution limited poor bioavailability. Numerous approaches to enhance the solubility and dissolution rates of these APIs include: increasing surface area by a reduction in particle size, and altering the crystalline form of the drug2, 3. To achieve physical and chemical property modifications, several conventional processing techniques such as kneading, freeze-drying, solvent evaporation and spray drying are employed4. Spray drying and the relatively new supercritical processing techniques are the only two methods that are fast, one-step processes. Supercritical processing, unlike the conventional spray drying technique, results in much higher solubility and dissolution rate because of very fast precipitation which result in smaller particles with a narrow particle size range5-8.

Previously supercritical CO2 (Sc-CO2) have been reported to use CO2 as a solvent in

RESS (rapid expansion of supercritical solutions), an antisolvent in SAS (supercritical anti-solvent), GAS (gaseous anti-solvent), or as the solute as in PGSS (particles from gas saturated solution), to name a few. Several thorough reviews of these processes can be found in review articles9, 10. The semi-continuous antisolvent (SAS) process allows a wide range of materials to be processed since the process is not limited to the solubility of the solute in CO2. This allows a vast variety of solvents and drugs to be used.

Supercritical antisolvent processing on the well studied API, Indomethacin (IMC) alone is limited11. Studies with copper-indomethacin and the addition of polymers to IMC

7,12-14 has been conducted with the use of SC-CO2 as antisolvent . IMC alone processed with SC-CO2 as a solvent was reported to yield insignificant effects on the crystalline form and the particle reduction, even at high pressures15. A systematic study on IMC alone, however, is lacking. Poor results with IMC in the RESS process provide the motivation to understand the behavior of IMC alone in a fundamental study with the SAS process.

Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) that serves as an antipyretic, and an analgesic agent in alleviating ailments such as rheumatoid arthritis and gout16,17. According to the Biopharmaceutics Classification System (BCS),

Indomethacin is categorized as a Class II API with poor water solubility and high permeability18. This study, investigates the possibility of enhancing the dissolution rate of

IMC by means of the supercritical antisolvent technique. Micronization and modification of Indomethacin crystal form contribute to enhanced dissolution rate with SAS.

Experimental parameters are explored by variation in the solvent (Acetone,

Dichloromethane, and Dimethylsulfoxide), the concentration (0.2% to 1.5% w/v), the pressure (83 to 117 bar) and the temperature (35C to 55C).

2.2 Background

Indomethacin (figure 2), 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3- acetic acid, has a molecular weight of 357.79 g/mol. It exhibits a reported poor water solubility of 0.02mg/ml in water19 and a high permeability greater than 85%20.

Indomethacin has a pH dependent saturation solubility which decreases with increasing pH18. The dissolution rate for an approximately 20mg sample of IMC is about 60%

40 dissolution within 60minutes21. Indomethacin is reported to have two common in addition to its amorphous form22. The - phase is metastable, monoclinic, and acicular appearance. The monoclinic form has 6 molecules of IMC with three different conformations packed in a unit cell. - phase IMC is the most stable form of

Indomethacin with a triclinic crystal structure and plate-like morphology. The unit cell of the triclinic form has two molecules of the same conformation, where a molecular dimer is formed by hydrogen bonding between carboxylic acids23.

Cl O

HO N

Figure 2: Structure of indomethacin

2.3 Materials And Methods

2.3.1 Materials

99% pure Indomethacin was purchased from Sigma Aldrich (Saint Louis, MO).

99.9% pure CO2 was purchased from GTS (Allentown, PA). Acetone and dichloromethane, are reagent grade chemicals that were purchased from VWR (West

Chester, PA). Reagent grade DMSO was purchased from Alfa Aesar (Ward Hill, MA).

200 proof Ethyl alcohol was purchased from Pharmaco (Brookfield, CT). Poly

(dimethylsiloxane) with a molecular weight of 4000, was purchased from Alfa Aesar

(Ward Hill, MA). Phosphate buffer solutions were prepared in distilled water by dissolving potassium phosphate monohydrate (0.5M) and sodium hydroxide pellets. This solution was then adjusted to a final pH of 7.2.

2.3.2 Methods

2.3.2.1 Recrystallization

The recrystallization process was conducted as described previously24. Basically, about 2 grams of -IMC was heated in approximately 7mls of ethyl alcohol (before reaching a boiling temperature of 78.29C) until the IMC was completely dissolved. The solution was cooled to room temperature followed by the addition of distilled water

(~14ml). Filtration was used to remove the precipitated crystals. The precipitated crystals were dried in a vacuum oven overnight. The whole recrystallization procedure was repeated twice.

2.3.2.2 High Pressure Apparatus

The supercritical high pressure experimental apparatus (figure 2.1) was operated

25 in a semi-continuous mode . Basically, the precipitation chamber (A) is filled with CO2, is heated, and pressurized to supercritical conditions. The desired drug solution with/without excipients is fed through the high pressure HPLC syringe pump (B). The drug substance solution is injected from the high pressure syringe pump (Teledyne,

Thousand Oaks, CA) into the injection nozzle (C_1 and inset at C_2) into the precipitation chamber. The coaxial nozzle (Sonotek, Milton, NY) has inner and outer diameters of 152 and 800µm, respectively. The solution flows through the inner nozzle and the CO2 passes through the outer nozzle. Particle precipitation occurs in the precipitation chamber. The precipitation vessel is comprised of a Jerguson gauge

(Northeast Controls, Upper Saddle River, NJ). The visual monitoring of the precipitation process is enabled through the transparent borosilicate windows. As precipitation occurs, the fluid mixture passes through the filter (D). The stainless steel filter unit

(Swagelok, Mountainside, NJ) has a porosity of 0.5µm where particles are collected. The fluid mixture is then separated with a cyclone (E) into the solvent and the CO2 gas which is vented into atmospheric pressure at a designated hood.

The back-pressure regulator (F) maintains the chamber pressure, and the rotameter measures the CO2 flow rate. The thermocouple and pressure gauges are represented by the letter T and P, respectively. An air driven pump (G) from Haskel,

Costa Mesa, CA pressurized liquid carbon dioxide.

C_2 Organic solution CO2 CO2

Figure 2.1: High pressure apparatus

2.3.2.3 Micro-Mill Grinder (Scienceware L929_2)

For 5 minutes approximately 3 grams of -IMC was placed under dry-milling.

Samples were processed twice.

2.3.2.4 Powder X-ray Diffraction (Rigaku D-Max/2200T)

Characterization of compound structure was carried out on a powder X-ray diffractometer. Data was measured between a 2 of 5-50, a step size of 0.01 and at a scanning speed of 8/min.

2.3.2.5 Differential Scanning Calorimetry (DSC Q100)

Hermetically sealed aluminum pans were used to measure at a scanning rate of

10°C/min using approximately 5 mg samples.

2.3.2.6 Scanning Electron Microscopy (SEM AMRAY 1830I)

A clean adhesive tape attached to a metal stub was used to place the sample.

Sputter coated samples with Au-Pd mixture under an Argon atmosphere were placed in the SEM microscope and viewed under a voltage of 15KV.

2.3.2.7 Particle Size Distribution (Malvern Mastersizer 2000, Hydro S)

Poly (dimethylsiloxane) was used to disperse the powder samples. The refractive index for the powder samples and poly (dimethylsiloxane) was set to 1.52, and 1.40, respectively. Measurements were run thrice with about 1000 particles being measured per run. Data is reported in % volume distribution because it is pharmaceutically relevant. The mean standard deviation represented by error bars was obtained after measurements were conducted three times.

2.3.2.8 Solubility Determined By HPLC

The powders were rotated in dissolution medium for 24 hours and then filtered.

The solubility of saturated aqueous solutions of the powders was determined by HPLC.

Indomethacin was detected at a wavelength of 318 nm. The mobile phase composition consisted of acetonitrile: triethylamine: glacial acetic acid in a ratio of 505: 0.65: 495. The column dimensions were 150 × 4.6mm, 3.5m, Eclipse XDB-C. It was maintained at a constant temperature of 30C and a flow rate of 1ml/min.

2.3.2.9 Dissolution (Vankel VK7010/ VK 8000)

The dissolution medium was made of 900ml of phosphate buffer and set at a constant temperature of 37.0°C +/- 0.5°C. IMC equivalent to 50 mg was placed into 0

45 inch gray capsules. These capsules were then encased within four-ring sinkers. The sinkers were then dropped into the dissolution medium which had been previously degassed with helium. The USP apparatus (II) paddle dissolution method26 was followed with continuous stirring set at 50 rpm for the duration of sixty minutes. Data points were collected by taking aliquots every 5 minutes for the first 30 minutes and then by every 10 minutes for the next 30 minutes. The samples were processed at least three times with

SAS and dissolution was conducted on 3 separate batches as well. Error bars represent the mean standard deviation on these samples. Two dissolution runs were conducted for

-IMC.

2.4 Results and Discussion

2.4.1 Effect of Solvents

Particle size and/or morphology of precipitates in the SAS process are well known to be affected by the choice of solvent 27-29. For this study the solvents selected are as follows: acetone, dimethylsulfoxide (DMSO), and methylene chloride (DCM). Both acetone and DMSO are pharmaceutically accepted solvents. Dichloromethane is a common choice for SAS experiments.

DCM is able to dissolve a wide number of compounds or materials and obtain good miscibility with Sc-CO2 at low temperatures and pressures.

IMC with a solution concentration of 1% w/v, was processed at a constant temperature of 35.0°C and the pressures for each solvent were selected above the mixture critical point of the binary supercritical CO2 – solvent systems. The following were the mixture critical points for the selected solvents at the set operating temperature of 35.0C: DMSO, acetone, and DCM are below 80 bar,30 around 72, and 78 bar29,

46 respectively. Above or near the mixture critical point, solvents are miscible with

31 supercritical CO2 and exhibit a single, supercritical phase .

When DMSO was the selected solvent the precipitation chamber was observed to be cloudy but no product was recovered. It seems that DMSO acted as a co-solvent

32 and enhanced the solubility of IMC in Sc-CO2 . DMSO’s polar, aprotic and highly basic

33 nature can explain the enhanced dissolution of IMC in Sc-CO2 . DMSO is more basic compared to acetone and so is favorable to dissolve the weakly acidic IMC. As the solvent was replaced from DMSO to acetone and DCM precipitation of particles was observed. The precipitation of particles with acetone can be explained by its polar,

33 aprotic nature and a lower basicity (B = 6.1) compared to DMSO (B = 10.6) . Based on the particle size distribution data, DCM and acetone have median diameters (d50) of

1.01 +/- 1.14 m and 4.14 +/- 5.37 m, respectively (table 2). The smaller particles with

DCM maybe attributed to the fact that SC-CO2 is known to mix very well with DCM compared to acetone. To further optimize the process conditions by varying the concentration, pressure and temperature, DCM was selected as the solvent.

Sample d10(m) d50(m) d90(m)

-IMC 11.47+/- 0.02 40.04+/-0.67 112.69+/-4.87

-IMC (micronized) 6.28+/-0.22 15.18+/-0.58 35.35+/-1.51

-IMC (recrystallized) 0.24+/-0.08 26.85+/-2.89 156.25+/-4.37

-IMC (SAS-acetone) 0.15+/-0.08 4.14+/-5.37 66.43+/-5.39

-IMC (SAS-DCM) 0.10+/-0.01 1.01+/-1.14 83.98+/-80.77

Table 2: D10, D50, D90 of unprocessed and processed -IMC.

2.4.2 Effect of Concentration

Particle size dependence on solution concentration was studied from 0.2 to 1.5% w/v. An increase in the concentration from 0.2% w/v to 1.0% w/v resulted in a significant increase in particle size as shown in SEM images in figure 2.2. Particle size distribution was unattainable with the solution concentration of 0.2%w/v because the product yield and density were too low at this condition. Particle size doubled as the initial solution concentration was increased from 1.0% w/v to 1.5% w/v as indicated by the d50 and d90 values in table 2.1. At 1.5% w/v, the highest equilibrium solution concentration, IMC reaches its solubility limit in DCM. The direct increase in particle size with concentration is a trend that is similar to what has been observed in literature. When the solution concentration is increased the fast reduction of solvent power by the antisolvent CO2 allows homogeneous particles to nucleate. As the solution concentration is increased beyond a certain point, particle growth dominates the nucleation process, which results in larger particles34, 35.

B C

Figure 2.2 SEM images of A) -IMC, IMC dissolved in DCM at solution concentrations of B) 1.0%w/v and C) IMC at 0.2% w/v.

2.4.3 Effects of Pressure And Temperature

Particle size and size distribution can be affected by changes in pressure and temperature by a resulting shift the mixture critical point of the binary system 31. At a constant concentration of 1.0% w/v and temperature of 35.0C, precipitation at a pressure around 83 bar led to droplet formation. A single fluid phase was observed with an increase in pressure from 83 bar to 103 bar. A further increase in pressure from 103 to 117 bar (+/- 3.4 bar) shifts the ternary system away from its mixture critical point. This resulted in an increase in particle size (see figure 2.3). An increase in pressure to 117 bar resulted in an increase in the median diameter (d50) of 14.83 +/- 6.51m (table 2).

This maybe explained by the fact that, mass transfer between DCM and CO2 is fast at

103 bar but slow at the higher pressure of 117 bar. Smaller particles result when the rapid mass transfer leads to high nucleation rates of the drug 36, 37.

At all other parameters held constant, temperature was varied from 35.0C to

55.0C. Particle size increased (d50 of 20.11+/-0.31m) and the particle size distribution broadened with an increase in temperature (see figure 2.3). An increase in temperature is known to affect the mixture critical point. So to operate in the supercritical state, an increase in temperature is to be followed by simultaneous increase in pressure 31.

Sample d10(m) d50(m) d90(m)

-IMC (SAS-DCM) 0.10+/-0.01 1.01+/-1.14 83.98+/-80.77 35°C, 103 bar, 1.0% w/v

Concentration 0.11+/-0.01 2.19+/-1.68 40.15+/-12.71 1.5%w/v

Pressure 0.85+/-1.22 14.83+/-6.51 94.31+/-53.57 117 bar

Temperature 6.53+/-0.10 20.11+/-0.314 57.53+/-1.31 55°C

Table 2.1: D10, D50, D90 of SAS processed IMC in DCM at 35C, 103 bar, 1.0%w/v with varying concentration, pressure and temperature.

12 A 1.0 %w/v ( )

10 1.5 %w/v ( )

6 Volume%

0 0.010 0.100 1.000 10.000 100.000 1000.000 10000.000

Particle Size (m) 10 14 35°C ( ) 9 B 103 bar ( ) C 12 8 117 bar ( ) 55°C ( ) 10 7

6 8 5

Volume% 6 Volume% 4

3 4

2 2 1

0 0 0.010 0.100 1.000 10.000 100.000 1000.000 10000.000 0.010 0.100 1.000 10.000 100.000 1000.000 10000.000

Particle Size (m) Particle Size (m)

Figure 2.3: Particle size distribution as A) concentration, B) pressure and C) temperature are varied.

2.4.4 Powder X-ray Diffraction

X-ray diffraction patterns of indomethacin before and after SAS processing are shown in Figure 2.4. The diffraction pattern of unprocessed drug showed high-intensity diffraction peaks characteristic of the -form. Reported peak positions are at 8.4, 11.9,

14.4, and 22.1 for the -form whereas 11.6, 19.6, 21.9, 26.6 and 29.4 are attributed to the -form23. The polymorphs are further confirmed with differential scanning calorimetry, where the melting point was measured to be approximately 154-155C for the

51 metastable -form and 164C for the highly stable -form (figure 2.5). In addition, the melting temperatures (Tm) indicated that the metastable -form with high purity was obtained with the SAS process. The -form is obtained consistently when IMC alone was processed with SAS, and this is attributed to the fast supersaturation process of

SAS.

D Intensity(Counts)

5 10 15 20 25 30 35 40 2degree

Figure 2.4: Powder X-ray diffraction of IMC in the A) form, B) form, C)

IMC as is, D) IMC processed with DCM (SAS).

3.00E+00

-2.00E+00120 130 140 150 160 170 180 190

-7.00E+00 A

-1.20E+01 B

-1.70E+01 C

-2.20E+01 Heat flow Heat (W/g) -2.70E+01

-3.20E+01

-3.70E+01

-4.20E+01

-4.70E+01 Temperature (°C)

Figure 2.5: Differential Scanning Calorimetry: Thermal properties of A) -IMC and SAS processed IMC in B) Acetone and C) DCM.

2.4.5 Solubility

At the buffer pH of 5.0 the reported solubility of IMC is about 0.01mg/ml. Since

IMC solubility is pH dependent its solubility increases to about 0.14 mg/ml at pH 7.4 18.

The solubility of IMC processed with SAS (DCM at 35C 103 bar) was run under HPLC for 24 and 48 hours. At a buffer pH of 7.2, IMC as is has a solubility of around

0.137mg/ml. IMC processed with SAS resulted in a solubility of 0.363 mg/ml (see Table

2.2). A solubility increase of approximately 3 times was observed when IMC was processed with SAS. Solubility measurements were repeated in duplicate.

Sample Process conditions Saturation Solubility In deionized water -IMC As is 0.137mg/ml

-IMC (SAS 1.0% w/v, 35°C, 103 bar, 0.363 mg/ml processed) dissolved in DCM

Table 2.2: A summary of the process conditions and saturation solubility of -IMC.

2.4.6 Dissolution Studies

IMC dissolution profiles of unprocessed and processed drug are shown in Figure

2.6. Unprocessed IMC dissolution profile is comparable to reported experiments15. The

Noyes-Whitney equation relates high surface area, a result of reduced particle size, in a direct relationship with dissolution rates. Micronized - IMC compared to - IMC showed a slight increase in its dissolution profile. An increase in dissolution is attributed to the reduction in particle size, see figure 2.7 and table 2. The modified crystalline form of IMC is another parameter that may contribute to an enhancement in dissolution rate.

Recrystallization of -IMC resulted in the -polymorph of IMC with reduced particle size and a bimodal size distribution. The dissolution rate of recrystallized -IMC was higher

54 than both micronized and unmicronized - IMC. The fastest dissolution rate was observed with SAS processed IMC. Within approximately 20 minutes, with either acetone or DCM as the solvent more than 85% of the IMC is dissolved when processed with SAS. For all other conditions the dissolution rate is less than 40% within the first 20 minutes. This enhancement in dissolution compared to -IMC can be attributed both to the reduction in size and the change in polymorphic form of IMC. A slight difference between acetone and DCM processed particles is observed in the dissolution with a reduction in the intensity of the bimodal particle size distribution as well. The dissolution tests were conducted on at least 3 different batches for the processed Indomethacin samples. SAS processed -IMC yielded particles in the -polymorph with an increased reduction in particle size and a narrow particle size distribution. The overall enhanced dissolution of SAS processed IMC can be attributed to the combined effect of particle size reduction in concert with the -polymorphic form, confirmed by PXRD.

120 E D C 100 B A

40 % Indomethacin dissolved Indomethacin %

0 0 10 20 30 40 50 60

Time (mins)

Figure 2.6: Dissolution Profiles: A) -IMC, B) micronized -IMC, C) -IMC (recrystallized), D) -IMC (SAS-acetone) and E) -IMC (SAS-DCM).

18.00

16.00 B 14.00

12.00 A 10.00 E

8.00 Volume % Volume

6.00 D 4.00

2.00

0.00 C 0.010 0.100 1.000 10.000 100.000 1000.000 Particle size (m)

Figure 2.7: Particle size distribution of A) -IMC, B) micronized -IMC, C) recrystallized IMC, and IMC processed in SAS with D) acetone and E) DCM as the solvent.

2.5 Conclusion

Precipitation with supercritical antisolvent process is a promising technique to tune particle size as well as to enhance solubility and dissolution rate. IMC particle size was tuned in the SAS process by variation of process parameters. Only the -form is consistently observed for IMC alone processed with SAS, and this is attributed to the fast supersaturation process of SAS. In addition, an increase in solubility was observed for IMC processed in SAS. Dissolution profiles in accord with particle size ranges confirm that SAS process enhanced IMC dissolution rate by control of particle size and crystallinity (its polymorphic form). Both α and γ phase indomethacin maintain biological activity38. Future studies on this system would include stability testing.

2.6. References

1. Keck, C. M.; Muller, R. H., Drug nanocrystals of poorly soluble drugs produced by high pressure homogenisation. European Journal of Pharmaceutics and Biopharmaceutics 2006, 62 (1), 3. 2. Cavallari, C.; Luppi, B.; Di Pietra, A. M.; Rodriguez, L.; Fini, A., Enhanced Release of Indomethacin from PVP/Stearic Acid Microcapsules Prepared Coupling Co-freeze-drying and Ultrasound Assisted Spray-congealing Process. Pharmaceutical Research 2007, 24 (3), 521-529. 3. Liu, R., Water-Insoluble Drug Formulation. CRC Press: 2000. 4. Ghaderi, R., A supercritical fluids extraction process for the production of drug loaded biodegradable microparticles. Acta Universitatis Upsaliensis:: 2000. 5. Debenedetti, P. G.; Tom, J. W., Particle Formation with supercritical Fluids—A review. Journal of Aerosol Science 1991, 22, 555–584. 6. Rantakyla, M. J., Particle Production by Supercritical Antisolvent Processing Techniques [thesis]. Helsinki, Finland: University of Technology, Helsinki (Espoo), Finland 2004. 7. York, P.; Kompella, U. B.; Shekunov, B. Y., Supercritical Fluid Technology for Drug Product Development. Informa Healthcare: 2004. 8. Elvassore, N.; Parton, T.; Bertucco, A.; Di Noto, V., Kinetics of particle formation in the gas antisolvent precipitation process. 2003; Vol. 49, pp 859-868. 9. Jung, J.; Perrut, M., Particle design using supercritical fluids: Literature and patent survey. Journal of Supercritical Fluids 2001, 20 (3), 179-219. 10. Yeo, S. D.; Kiran, E., Formation of polymer particles with supercritical fluids: A review. Journal of Supercritical Fluids 2005, 34 (3), 287-308. 11. Bodmeier, R.; Wang, H.; Dixon, D. J.; Mawson, S.; Johnston, K. P., Polymeric Microspheres Prepared by Spraying into Compressed Carbon Dioxide. Pharmaceutical Research 1995, 12 (8), 1211-1217. 12. Bleich, J.; Müller, B. W., Production of drug loaded microparticles by the use of supercritical gases with the aerosol solvent extraction system (ASES) process. Journal of microencapsulation 1996, 13 (2), 131-139. 13. Gong, K.; Rehman, I. U.; Darr, J. A., Synthesis of poly (sebacic anhydride)-indomethacin controlled release composites via supercritical carbon dioxide assisted impregnation. International journal of pharmaceutics 2007, 338 (1-2), 191-197.

14. Warwick, B.; Dehghani, F.; Foster, N. R.; Biffin, J. R.; Regtop, H. L., Micronization of copper indomethacin using gas antisolvent processes. Ind. Eng. Chem. Res 2002, 41 (8), 1993-2004. 15. Bandi N., W. W., Roberts C. B., Kotra L. P., Kompella U. B., Preparation of budesonide–and indomethacin–hydroxypropyl-ß-cyclodextrin (HPBCD) complexes using a single-step, organic-solvent-free supercritical fluid process. European Journal of Pharmaceutical Sciences 2004, 23 (2), 159-168. 16. Backensfeld T., M. B. W., Wiese, M., Seydel J. K., Effect of Cyclodextrin Derivatives on Indomethacin Stability in Aqueous Solution. Pharmaceutical Research 1990, 7 (5), 484-490. 17. Rusu, D.; Cimpoiu, C.; Hodisan, T., The control over the new obtaining procedeum of indomethacin. Journal of Pharmaceutical and Biomedical Analysis 1998, 17 (3), 409-413. 18. Yazdanian, M.; Briggs, K.; Jankovsky, C.; Hawi, A., The ―High Solubility‖ Definition of the Current FDA Guidance on Biopharmaceutical Classification System May Be Too Strict for Acidic Drugs. Pharmaceutical Research 2004, 21 (2), 293-299. 19. Jain, A. K., Solubilization of indomethacin using hydrotropes for aqueous injection. European Journal of Pharmaceutics and Biopharmaceutics 2008, 68 (3), 701-714. 20. Van De Waterbeemd, H.; Testa, B., Drug bioavailability: estimation of solubility, permeability, absorption and bioavailability. Vch Pub: 2008. 21. Tozuka, Y.; Miyazaki, Y.; Takeuchi, H., A combinational supercritical CO2 system for nanoparticle preparation of indomethacin. International Journal of Pharmaceutics 2009, 386 (1-2), 243-248. 22. Imaizumi, H.; Nambu, N.; Nagai, T., Stability and several physical properties of amorphous and crystalline form of indomethacin. Chem Pharm Bull (Tokyo) 1980, 28 (9), 2565-9. 23. Okumura, T.; Ishida, M.; Takayama, K.; Otsuka, M., Polymorphic transformation of indomethacin under high pressures. Journal of Pharmaceutical Sciences 2006, 95 (3), 689-700. 24. Kaneniwa, N., Otuska, M.,Hayashi, T., Physicochemical characterization of indomethacin polymorphs and the transformation kinetics in ethanol. Chem. Pharm. Bull 1985, 33, 3447-3455. 25. Wu, K.; Li, J., Precipitation of a biodegradable polymer using compressed carbon dioxide as antisolvent. Journal of Supercritical Fluids 2008, (In press). 26. Banakar, U. V., Pharmaceutical dissolution testing. Informa Healthcare: 1991.

27. Schmitt, W. J.; Salada, M. C.; Shook, G. G.; Speaker, S. M., Finely- divided powders by carrier solution injection into a near or supercritical fluid. AIChE Journal 1995, 41 (11), 2476-2486. 28. Reverchon, E.; Della Porta, G., Production of antibiotic micro-and nanoparticles by supercritical antisolvent precipitation. Powder Technol 1999, 106 (1- 2), 23-29. 29. Gokhale, A.; Khusid, B.; Dave, R. N.; Pfeffer, R., Effect of solvent strength and operating pressure on the formation of submicrometer polymer particles in supercritical microjets. Journal of Supercritical Fluids 2007, 43 (2), 341-356. 30. Andreatta, A. E.; Florusse, L. J.; Bottini, S. B.; Peters, C. J., Phase equilibria of dimethyl sulfoxide (DMSO)+ carbon dioxide, and DMSO+ carbon dioxide+ water mixtures. The Journal of supercritical fluids 2007, 42 (1), 60-68. 31. Reverchon, E.; Caputo, G.; De Marco, I., Role of Phase Behavior and Atomization in the Supercritical Antisolvent Precipitation. ACS AMERICAN CHEMICAL SOCIETY: 2003; Vol. 42, pp 6406-6414. 32. Reverchon, E.; Adami, R.; Marco, I. D.; Laudani, C. G.; Spada, A., Pigment Red 60 micronization using supercritical fluids based techniques. Journal of Supercritical Fluids 2005, 35 (1), 76-82. 33. Sauceau, M.; Letourneau, J. J.; Freiss, B.; Richon, D.; Fages, J., Solubility of eflucimibe in supercritical carbon dioxide with or without a co-solvent. Journal of Supercritical Fluids 2004, 31 (2), 133-140. 34. Reverchon, E.; Della Porta, G.; Di Trolio, A.; Pace, S., Supercritical antisolvent precipitation of nanoparticles of superconductor precursors. Ind. Eng. Chem. Res 1998, 37 (3), 952-958. 35. Dixon, D. J.; Johnston, K. P.; Bodmeier, R. A., Polymeric materials formed by precipitation with a compressed fluid antisolvent. AIChE Journal 1993, 39 (1), 127-139. 36. Henczka, M.; Baldyga, J.; Shekunov, B. Y., Particle formation by turbulent mixing with supercritical antisolvent. Chemical Engineering Science 2005, 60 (8- 9), 2193-2201. 37. Miguel, F.; Martin, A.; Gamseb, T., Supercritical anti solvent precipitation of lycopene Effect of the operating parameters. Journal of Supercritical Fluids 2006, 36, 225-235. 38. Arisawa, M.; Kasaya, Y.; Obata, T.; Sasaki, T.; Ito, M.; Abe, H.; Ito, Y.; Yamano, A.; Shuto, S., Indomethacin Analogues that Enhance Doxorubicin Cytotoxicity in Multidrug Resistant Cells without Cox Inhibitory Activity. ACS Medicinal Chemistry Letters.

CHAPTER THREE

Dissolution rate enhancement of indomethacin with one-step precipitation of indomethacin and cyclodextrin complexation

3.1 Introduction

Cyclodextrins are cyclic oligosaccharides that have been around since their discovery in 1891. They are beneficial in many ways such as enhancing the solubility of compounds, providing stability against oxidation, heat and light, taste masking and other unwanted physiological effects1. Cyclodextrins have wide application from food and flavors, cosmetics, packing, textiles, separation processes, environment protection, fermentation and catalysis, to pharmaceuticals 2. These cyclic oligosaccharides are linked by (1,4) glucopyranose subunits which create a cavity with a hydrophobic interior and hydrophilic exterior, a ―micro heterogenous environment‖. Interest in cyclodextrins is mainly in their ability to form inclusion complexes with compounds3, 4.

Complexation with cyclodextrin is a well-known approach to enhance the dissolution properties of BCS class II, poorly water soluble APIs by encasing the entire or part of the desired drug molecule 5-7. , , and  cyclodextrins are the most common cyclodextrins that differ in the number of glucose molecules. Of these - CDs are the most suitable for APIs of medium size. However, - CD presents several challenges as it is has the lowest water solubility in water and only a limited amount of it can be taken within the body since its toxicity level is high. To alleviate this challenge hydroxylated derivates of cyclodextrin were synthesized. Hydroxypropyl β- cyclodextrin is an example

61 of this derivative which unlike its parent molecule is highly water soluble and has much lower toxicity levels2, 8.

Numerous approaches to form cyclodextrin – drug complexes include kneading, coprecipitation, slurry method 1, ammonium method 9, lyophilization10, and supercritical

11 CO2 as a solvent . Most of the conventional methods applied require multiple steps and are time consuming. The use of supercritical CO2 as a solvent is safe and is carried out in a single step. However, it requires high pressure and temperature for processing.

Instead, the use of supercritical CO2 as an antisolvent allows for mild conditions for the processing of a wide range of materials.

Indomethacin (IMC), is an NSAID that inhibits the enzyme cyclooxygenase which in turn inhibits prostaglandin biosynthesis- This inhibition allows IMC to serve as an antipyretic, and analgesic agent in alleviating ailments such as rheumatoid arthritis, and gout12, 13. This BCS Class II API is associated with side effects in the gastrointestinal tract, central nervous system and skin14, 15.

At high pressures Sc-CO2 is reported to dissolve IMC. IMC-HPβCD has been reported to be sucessfully processed with a 3-fold enhancement in dissolution with the

RESS process at 20C and 211 bars for 20 hours11. SAS process offers a milder condition with a shorter time to process IMC in. Previously IMC, the drug alone was successfully processed with the SAS method16. In this study IMC is processed with

HPCD and compared with single step processes spray drying and supercritical CO2 as an antisolvent in enhancing the dissolution of IMC by complexation. We show that dissolution rate can be enhanced by micronization and modification of the API with SAS.

3.2 Background

Inclusion complexation is the insertion of a guest molecule(s) into a cavity formed by the complexing agent. A requirement of the complexing agent is a nonpolar core and a polar exterior. The guest molecule’s nonpolar area is excluded from water because of its insertion into the complexing agent. Complex formation is largely independent of the chemical properties of the guest molecule. Cyclodextrins (CDs) allow the formation of both inclusion and non-inclusion complexes. Non-inclusion complexes involve interaction with CD without the insertion of the guest molecule inside the CD cavity.

CDs are naturally formed as a product during bacterial digestion of cellulose.

These cyclic oligomers are composed of - D glucose units joined by glycosidic/ether linkages) that result in a hollow truncated cone/ doughnut shape. This truncated cone shape is a result of the chair conformation taken up by the glucopyranose units. The glucose units, molecular weight, cavity volumes and water solubility of cyclohexamylose(), cycloheptamylose() and cyclooctamylose() are compared in

Table 3.

α β γ No of glucopyranoseunits 6 7 8 Molecular weight (Daltons) 972 1135 1297 Cavity size (Å) 4.7-5.3 6.0-6.5 7.5-8.3 Water solubility 25⁰C 14.5 1.85 23.2 (g/100ml)

Table 3: Properties of α, β, γ cyclodextrins.

The parent CDs , , and  are limited in their practical use because of low drug

solubility. CD derivatives exhibit higher solubility than their parent CDs and are formed

by substitution of the hydroxyl groups. They include those of pharmaceutical interest

such as the hydroxypropyl derivatives of β- and γ-cyclodextrin, the randomly methylated

β-cyclodextrin, sulfobutylether β-cyclodextrin, and branched cyclodextrins such as

glucosyl-β cyclodextrin.

Figure 3 depicts the structure of β-cyclodextrin. The glucopyranose units take up

the chair conformation and render the CD molecule its cone shape. Primary and

secondary hydroxyl groups on the outer narrow and wide edges of the cone,

respectively. This gives CDs their hydrophilic character. The lipophilic CD cavity is lined

by skeletal carbons and ethereal oxygens.

CH 2OH

O O O HOH 2C HO O OH OH

HO Edge of primary hydroxyls CH 2OH Edge of secondary hydroxyls O HO

O O OH OH

Apolar cavity HOH 2C O OH OH OH

O H CH 2OH OH O O OH OH O O

CH 2OH O

O CH 2OH

Figure 3: The molecular shape and chemical structure of β-cyclodextrin.

In aqueous solutions, the majority of water molecules are outside the CD

molecule and very few molecules are inside the CD cavity. The water molecules inside

64 the CD are of higher enthalpy. Energetically they are unfavorable because of polar- apolar interactions between the water molecules and the apolar interior. When these enthalpy rich water molecules are replaced by suitable guest molecules which are less polar than water, the energy of the system is lowered. The driving force to replace the enthalpy rich water molecules in larger CDs is weaker3. So, complex formation is enthalpically driven and is always associated with a relatively large negative H and a

S that can be either positive or negative. Figure 3.1 illustrates the inclusion complex formation of salicylic acid. Salicylic acid and β-CD, with interior water molecules, yield a salicylic acid -β-CD complex without interior drug molecules. Kc represents the equilibrium constant that this equilibrium process is governed by.

COOH COOH Kc +

OH OH

Figure 3.1: Formation of an inclusion complex of salicylic acid with β-cyclodextrin.

Higuchi and Connors phase-solubility diagram (Figure 3.2) is a well-known quantitative method to evaluate the solubilization ability of CDs17. Type A plot indicates that a soluble complex was formed. Type B indicates that a complex with finite solubility is formed which is typical of complexation with parent CDs. Further classification of type

A into subtype AL,indicates a linear relationship between guest solubility and CD

concentration solubility, a stoichiometry of 1:1. Subtype AP, indicates higher order complex formation which results when more than one CD is involved in complex formation, AN interaction mechanism is complicated because of significant contribution of solute-solvent interaction to the complexation. Subtype BS indicates that solubility increases initially followed by a plateau region which leads to the microcrystalline precipitation of the complex, which is indicated by the decrease in solubility. Finally, the

BI - subtype indicates the formation of insoluble complexes in water.

Bs Concentration of guest molecule guest of Concentration Bl Concentration of Cyclodextrins(M)

Figure 3.2: Higuchi and Connors phase solubility diagram

3 .3 Materials and Methods

3.3.1 Materials

99% pure Indomethacin from Sigma Aldrich (Saint Louis, MO) was generously supplied by Johnson and Johnson pharmaceuticals, Pharmaceutical Research and

Development Raritan, NJ. HP-β-CD with molecular weight 1380 (both with 0.6 and 1.0 substitution), molecular weight 1540, and Polyvinylpyrrolidone (29-30K) were purchased from Sigma Aldrich (Saint Louis, MO). 200 proof, ethyl alcohol was purchased from

Pharmaco (Brookfield, CT). 99.9% pure CO2 was purchased from GTS (Allentown, PA).

(0.5 M) and sodium hydroxide pellets into distilled water to a final pH of 7.2. 1:1 ratio of binary mixture was used.

3.3.2 Methods

3.3.2.1 Preparation of Physical Mixtures

Dry physical mixtures were prepared at a 1:1 ratio of IMC: HP-β-CD. The

Turbula® TF2 shaker mixer (Glen Mills) was set for 10mins at 46 revolutions

per minute.

3.3.2.2 Spray Dryer (Buchi 290)

The mini spray dryer Buchi 290 (Flawil, Switzerland) was injected with IMC and

HP-β-CD solutions. Considering the solvent that was used, ethanol, the inlet temperature was set at 100 ±2C at the drying chamber with an outlet temperature that was maintained around 58 ± 5C. Both the aspirator setting and spray feed were set at

100 and 10%, respectively. Each experiment was repeated at least twice.

3.3.2.3 High Pressure System

Processing conditions were set at a temperature of 35C with pressure of 103 bars. Ethanol was the selected solvent since it is pharmaceutically acceptable and IMC,

HP-β-CD and PVP are soluble in it. Solution injection rate was set at 1.0ml/min. CO2 flow rate was set at 30L/min.

3.3.2.4 Powder X-ray Diffraction (RigakuD-Max/2200)

Structure characterization was carried out on a powder X-ray diffractometer. Data were collected between a 2θ of 5-50 with a step size of 0.01 at a scanning speed of

11/min.

3.3.2.5 Differential Scanning Calorimetry (Perkin Elmer)

The measurements were carried out in aluminum pans with a pinhole on the lid at a scanning rate of 10°C/min using approximately 2-3 mg samples.

3.3.2.6 Scanning Electron Microscopy (SEM AMRAY 1830I)

Samples were placed on an adhesive tape attached to a metal stub. This was then sputter coated with Au-Pd mixture under an Argon atmosphere. The coated samples were then placed in the SEM microscope and viewed under a voltage of 15KV.

3.3.2.7 HPLC Analysis

The powders were rotated for 24 hours in dissolution medium and then filtered.

HPLC was used to determine the concentration of Indomethacin. Mobile phase was composed of acetonitrile: triethylamine: glacial acetic acid in a ratio of 505:0.65:495. The column used was 150 X 4.6mm, 3.5µm, Eclipse XDB-C. It was maintained at a temperature at 30C at a flow rate of 1ml/min with the UV detector set at 318nm.

3.3.2.8 Vibrational Spectroscopy

The attenuated total reflection (ATR)- Infrared, and Raman Spectrometers

(Nicolet FTIR 320 and Bruker RamanScopeIII) were available for use. Both ATR- IR and

Raman spectras were measured at a set wavelength range of 400-4000 cm-1. Each measurement was obtained after 100 scans. Laser strength used for Raman spectra was 400mW.

3.3.2.9 Dissolution (Vankel VK7010/ VK 8000)

The dissolution medium consisted of 900ml of pH 7.2 phosphate buffer maintained at 37°C. 2.5 mg equivalent IMC was placed into 1‖ gray capsules and then placed into the dissolution medium. The USP paddle dissolution method was employed with continuous stirring at 50rpm.

3.4 Results and Discussion

3.4.1 Phase Solubility of IMC with HP-β-CD

The phase solubility diagram of IMC-HPβCD (figure 3.3) showed an AL- type linear relation. According to the Higuchi and Connors classification the AL- type implies that an increase in HP-β-CD concentration increases linearly with the amount of IMC that can be dissolved in it. The slope value 0.0039 which is less than one indicates that an inclusion complex was formed18, 19.

Figure 3.3: Phase-solubility plot of Indomethacin in increasing amount of HP--CD.

3.4.2 PXRD Analysis

The angle of diffraction, that is the peak position, indicates the crystal structure whereas the peak height is a measure of the sample crystallinity. IMC has characteristic peaks at 11.6, 19.6, 21.9, 26.6 and 29.4° for the γ-form (Figure 3.4). HP-β-CD is amorphous and is marked by a halo-like profile. Both spray dried and SAS processed particles have a halo like profile as well which indicates that there is a loss of crystallinity. This is an indication that a complex may have formed with IMC and HP-β-

CD 7. Further supporting data for complex formation between IMC and HP-β-CD is provided by DSC, IR and Raman spectroscopy.

C Intensity (Counts) Intensity B

2θ

Figure 3.4: PXRD pattern of A) γ-IMC as is B) HP-β-CD, IMC- HP-β-CD C) physical mixture D) spray dried, E) SAS processed and F) SAS processed ternary mixture of IMC- HP-β-CD-PVP

3.4.3 Thermal Analysis

The formation of an inclusion complex is reflected in a shift of the melting, boiling and sublimation temperature points 20. The highly stable γ – Indomethacin has a sharp melting peak at 164C (figure 3.5). HP-β-CD displays a broad band at 86-88C due to the evaporation of absorbed water. The broad band reveals that the water molecules are bond in varying bond strengths to the CD21. The physical mixture indicates a shift in the sharp IMC melting peak from 164C to a broad peak at159C. For the physical mixture, the dehydration and melting peak indicate a weak interaction between IMC and

HP-β-CD. No endothermic peaks were observed for both the SD and SAS processed particles. This implies a loss in crystallinity for the mixture that is processed by both these one-step processing methods22.

Temperature (C)

80 100 120 140 160 180 200 D C

B A

Figure 3.5 Observing the ibuprofen crystallization peak in the differential scanning calorimeter of A) - Indomethacin as is, B) IMC+HP-β-CD physical mixture, C) Spray dried and D) SAS processed.

3.4.4 Raman/ATR-IR

Both raman and infrared (IR) spectroscopy provide supporting data for complex formation. Complex formation is reported to be indicated by a significant blue or red shift in peak frequency or a disappearance or broadening of relevant peaks23,24. Indomethacin has two carbonyls that can be involved in hydrogen bonding, the benzoyl and acidic carbonyls. In raman spectra (see figure 3.6), the benzoyl carbonyl stretch for -IMC is observed at 1697 cm-1 and is assigned as a cyclic symmetric stretch. A shift in this peak to lower frequency is observed in both the spray-dried and the SAS processed particles from 1697 to 1675 cm-1. This frequency is typical of what is observed for both  and amorphous indomethacin24, 25. The peak shift suggests the interaction of IMC with HP-β-

CD. The peak also broadens, which indicates a decrease in crystallinity.

Wavelength (cm-1)

Figure 3.6 Observing a shift of the benzoyl peak in the raman spectra of

A) - Indomethacin as is, B) IMC+ HPβCD physical mixture, C) Spray dried, and D) SAS processed.

The benzoyl stretch is also observed to shift in peak frequency and is broadened in IR

(see figure 3.7). This reconfirms the Raman results for the benzoyl carbonyl. Asymmetric stretches are inactive in Raman spectroscopy and are observed instead in infrared spectroscopy. A blue shift and broadening of the acidic carbonyl peaks was observed for both the spray dried and supercritical antisolvent processed particles in IR. In the infrared range, the acid carbonyl stretch is typically observed at 1717cm-1 for -IMC.

When IMC was processed the acidic carbonyl’s peak broadened which indicates a decrease in crystallinity.

-1 Wavelength (cm )

Figure 3.7 Observing a shift of the benzoyl peak in the infrared spectra of A) -Indomethacin as is, IMC- HPβCD B) physical mixture,

C) Spray dried and D) SAS processed.

3.4.5 Dissolution

All complexed indomethacin particles show higher dissolution than IMC as is

(see figure 3.8). For the physical mixture, the slightly higher dissolution compared to IMC as is, can be attributed to the some weak interaction of IMC with HP--CD. This is confirmed by the IMC peak shift observed for PM with thermal analysis. The dissolution of complex particles prepared with spray drying was enhanced more than the PM and

IMC as is particles. This can be attributed to better complexation where the drug is complexed in an amorphous form with a reduction of overall particle size10. The fastest release, observed by the initial slope, was obtained with the SAS processed IMC-

HPCD. This initial increase is dissolution rate is higher than that previously reported with RESS processed IMC-HPCD. For the RESS process 3mg IMC was processed in a

1:10 ratio of IMC: HPCD11. In the SAS process, the initial slope is attributed to the fully dispersed particles because of their larger surface area. However, dissolution is incomplete both in the SAS and SD processes. This steady incomplete IMC release can be attributed to aggregation26. It is well known that CD and CD complexes self- aggregate. The addition of water-soluble polymers serves to enhance the dissolution by forming non-inclusion complexes with the cyclodextrin complex27-31. The enhanced dissolution rate of IMC-HPBCD binary mixture is observed with the addition of 0.5% w/v

Polyvinylpyrrolidone (PVP).

Dissolution of binary and ternary IMC mixtures

100

90 F E 80

D 60

40 C

30 % Indomethacin Dissolved Indomethacin %

B 10 A

0 0 5 10 15 20 25 30 35 40 45 50 Time (mins)

Figure 3.8 Dissolution profile of A) - Indomethacin as is, B) IMC- HPβCD physical mixture, C) Spray dried IMC- HPβCD, D) SAS processed IMC- HPβCD E) Spray dried IMC- HPβCD-PVP, and F) SAS processed IMC- HPβCD -PVP .

3.4.6 Scanning Electron Microscopy

Scanning electron microscopy (SEM) provides visual images of desired areas. A decrease in particle size is observed for both spray dried and SAS processed - IMC particles (see Figure 3.5). The smallest particles were obtained with SAS processing but the particles are agglomerated.

Figure 3.9: SEM images of - Indomethacin as is A), IMC processed with Spray drying zoomed in view B) and zoomed out view C), IMC processed with SAS zoomed in view D) and zoomed out view E)

3.4.7 Drug Loading

Drug Loading was determined using HPLC. Spray dried particles were detected to contain about 89% Indomethacin whereas the SAS processed particles had 42% indomethacin. The use of ethanol probably acts as a cosolvent and dissolves

Indomethacin in supercritical CO2.

3.5 Summary

IMC was complexed with HP-β-CD to enhance it solubility. The SAS process had the highest initial dissolution rate, higher than the RESS process reported earlier for this system and spray drying. However, the high initial dissolution rate was followed by a steady incomplete dissolution for the binary mixtures. The smaller particles from the SAS

77 and spray drying processes probably resulted in increased aggregation of these particles. The addition of PVP to the binary IMC- HPβCD system enhanced the dissolution of the binary complex further. Only a small difference is obtained with the addition of PVP to the binary mixture between SAS and spray-drying technique. Spray- drying is the better option for drug loading since most of the drug was dissolved in SAS because ethanol probably acted as a cosolvent.

3.6 References:

1. Hedges, A. R., Industrial applications of cyclodextrins. Chem Rev 1998, 98, (5), 2035-2044.

2. Del Valle, E. M., Cyclodextrins and their uses: a review. Process Biochemistry 2004, 39, (9), 1033-1046.

3. Szejtli, J., Introduction and general overview of cyclodextrin chemistry. Chemical Reviews 1998, 98, (5), 1743-1754.

4. Szejtli, J., Past, present, and future of cyclodextrin research. Pure and applied chemistry 2004, 76, (10), 1825-1846.

5. Miro, A.; Quaglia, F.; Sorrentino, U.; La Rotonda, M. I.; D'Emmanuele Di Villa Bianca, R.; Sorrentino, R., Improvement of gliquidone hypoglycaemic effect in rats by cyclodextrin formulations. European journal of pharmaceutical sciences 2004, 23, (1), 57-64.

6. Dias, M. M. R.; Raghavan, S. L.; Pellett, M. A.; Hadgraft, J., The effect of [beta]- cyclodextrins on the permeation of diclofenac from supersaturated solutions. International journal of pharmaceutics 2003, 263, (1-2), 173-181.

7. Fernandes, C. M.; Teresa Vieira, M., Physicochemical characterization and in vitro dissolution behavior of nicardipine-cyclodextrins inclusion compounds. European journal of pharmaceutical sciences 2002, 15, (1), 79-88.

8. Loftsson, T.; Brewster, M. E., Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. Journal of pharmaceutical sciences 1996, 85, (10), 1017-1025.

9. Jambhekar, S.; Casella, R.; Maher, T., The physicochemical characteristics and bioavailability of indomethacin from [beta]-cyclodextrin, hydroxyethyl-[beta]-cyclodextrin, and hydroxypropyl-[beta]-cyclodextrin complexes. International journal of pharmaceutics 2004, 270, (1-2), 149-166.

10. Wang, Z.; Deng, Y.; Sun, S.; Zhang, X., Preparation of hydrophobic drugs cyclodextrin complex by lyophilization monophase solution. Drug development and industrial pharmacy 2006, 32, (1), 73-83.

11. Bandi, N.; Wei, W.; Roberts, C. B.; Kotra, L. P.; Kompella, U. B., Preparation of budesonide-and indomethacin-hydroxypropyl-[beta]-cyclodextrin (HPBCD) complexes using a single-step, organic-solvent-free supercritical fluid process. European journal of pharmaceutical sciences 2004, 23, (2), 159-168.

12. Backensfeld, T.; Müller, B. W.; Kolter, K., Interaction of NSA with cyclodextrins and hydroxypropyl cyclodextrin derivatives. International journal of pharmaceutics 1991, 74, (2-3), 85-93.

13. Rusu, D.; Cimpoiu, C.; Hodian, T., The control over the new obtaining procedeum of indomethacin1. Journal of pharmaceutical and biomedical analysis 1998, 17, (3), 409-413.

14. Boardman, P. L.; Hart, F. D., Side-effects of indomethacin. Annals of the Rheumatic Diseases 1967, 26, (2), 127.

15. Yazdanian, M.; Briggs, K.; Jankovsky, C.; Hawi, A., The ―high solubility‖ definition of the current FDA guidance on biopharmaceutical classification system may be too strict for acidic drugs. Pharmaceutical research 2004, 21, (2), 293-299.

16. Varughese, P.; Li, J.; Wang, W.; Winstead, D., Supercritical antisolvent processing of [gamma]-Indomethacin: Effects of solvent, concentration, pressure and temperature on SAS processed Indomethacin. Powder Technology.

17. Uekama, K.; Hirayama, F.; Irie, T., Cyclodextrin drug carrier systems. Chemical Reviews 1998, 98, (5), 2045-2076.

18. Hirlekar, R.; Kadam, V., Preparation and characterization of inclusion complexes of carvedilol with methyl- -cyclodextrin. Journal of Inclusion Phenomena and Macrocyclic Chemistry 2009, 63, (3), 219-224.

19. Ribeiro, L. S. S.; Ferreira, D. C.; Veiga, F. J. B., Physicochemical investigation of the effects of water-soluble polymers on vinpocetine complexation with [beta]- cyclodextrin and its sulfobutyl ether derivative in solution and solid state. European journal of pharmaceutical sciences 2003, 20, (3), 253-266.

20. Marques, H. M.; Hadgraft, J.; Kellaway, I. W., Studies of cyclodextrin inclusion complexes. I. The salbutamol-cyclodextrin complex as studied by phase solubility and DSC. International journal of pharmaceutics 1990, 63, (3), 259-266.

21. Fini, A.; Ospitali, F.; Zoppetti, G.; Puppini, N., ATR/Raman and Fractal Characterization of HPBCD/Progesterone Complex Solid Particles. Pharmaceutical research 2008, 25, (9), 2030-2040.

22. Gajare, P.; Patil, C.; Kalyane, N.; Pore, Y., EFFECT OF HYDROPHILIC POLYMERS ON PIOGLITAZONE COMPLEXATION WITH HYDROXYPROPYL- - CYCLODEXTRIN. Digest Journal of Nanomaterials and Biostructures.

23. Sinha, V. R.; Anitha, R.; Ghosh, S.; Kumria, R.; Bhinge, J. R.; Kumar, M., Physicochemical characterization and in vitro dissolution behaviour of celecoxib- - cyclodextrin inclusion complexes. Acta Pharmaceutica 2007, 57, (1), 47-60.

24. Taylor, L. S.; Zografi, G., Spectroscopic characterization of interactions between PVP and indomethacin in amorphous molecular dispersions. Pharmaceutical research 1997, 14, (12), 1691-1698.

25. Fini, A.; Cavallari, C.; Ospitali, F., Raman and thermal analysis of indomethacin/PVP solid dispersion enteric microparticles. European Journal of Pharmaceutics and Biopharmaceutics 2008, 70, (1), 409-420.

26. Kale, K.; Hapgood, K.; Stewart, P., Drug agglomeration and dissolution-What is the influence of powder mixing? European Journal of Pharmaceutics and Biopharmaceutics 2009, 72, (1), 156-164.

27. Loftsson, T.; Konradsdottir, F.; Másson, M., Influence of aqueous diffusion layer on passive drug diffusion from aqueous cyclodextrin solutions through biological membranes. Pharmazie 2006, 61, (2), 83-89.

28. Loftsson, T.; Magnúsdóttir, A.; Másson, M.; Sigurjónsdóttir, J. F., Self association and cyclodextrin solubilization of drugs. Journal of pharmaceutical sciences 2002, 91, (11), 2307-2316.

29. Loftsson, T.; Másson, M.; Brewster, M. E., Self association of cyclodextrins and cyclodextrin complexes. Journal of pharmaceutical sciences 2004, 93, (5), 1091-1099.

30. Loftsson, T.; Össurardóttir, Í. B.; Thorsteinsson, T.; Duan, M.; Masson, M., Cyclodextrin solubilization of the antibacterial agents triclosan and triclocarban: effect of ionization and polymers. Journal of Inclusion Phenomena and Macrocyclic Chemistry 2005, 52, (1), 109-117.

31. Quan, P.; Liu, D.; Li, R.; Zhang, Q.; Qian, Y.; Xu, Q., The effects of water-soluble polymers on hydroxypropyl- -cyclodextrin solubilization of oleanolic acid and ursolic acid. Journal of Inclusion Phenomena and Macrocyclic Chemistry 2009, 63, (1), 181-188.

CHAPTER FOUR

Coordination drug polymers, a new approach to enhance drug dissolution

4.1 Introduction

Active pharmaceutical ingredients (APIs) are commonly found to occur as crystalline solids. This is also the preferred form to administer drugs in because of the relative ease of isolation of crystalline forms, rejection of impurities and their stability1, 2.

However, crystalline APIs continue to be a challenge because of their poor water solubility which in turn leads to poor bioavailability. Salt formation is a conventional technique that relies on ionizable components and has resulted in marketable drugs3.

Cocrystal formation1 have been known since the 19th century and have been applied in conductive organic and non-linear optical crystals, pigments, dyes, etc., but has only recently gained much attention in the pharmaceutical industry4. Cocrystals exist in the solid state and are applicable for a wider group of APIs, with varying physicochemical properties, including ionizable APIs.This makes this approach more favorable for pharmaceutics compared to the well-know salt formation technique5.

Cocrystals, despite the controversies over its definition are broadly defined as crystals with more than one component combined together, where the component is defined as atoms, molecules, hydrates, etc6-8. The difference in the components lead to the difference in the subsets of multi-component crystals which include cocrystals, clathrates, hydrates and solvates. In cocrystals, the API and cocrystal formers which can be either another API or a non-active pharmaceutical ingredient, are both in the solid form and are bonded through non-covalent bonds, based on the

82 supramolecular synthon, which consists of arrangements that involve specific intermolecular interactions mainly through hydrogen bonds of functional groups such as carboxylic acids, amides, alcohols 1, 9. Cocrystals are advantageous because they are easily synthesized, able to form into different solid crystal forms with different physicochemical properties such as solubility, despite the need for ionizable groups.

Solubility enhancement of an anticancer drug has been reported to have increased 2.5 fold compared to the drug alone10. Cocrystal design by the formation of reversible hydrogen bonds can enhance the possibility of rationally designing new crystals to enhance solubility without breaking or forming covalent bonds 1, 11. Cocrystallization opens up avenues to other non-conventional routes to enhance solubility and dissolution rate of poorly water soluble APIs. The design of coordination drug polymers (CDPs) is a new approach to enhance solubility.

Metal coordination polymers, in general are metal ions linked by coordinated ligands into an infinite array and are well known for their use in catalysis, as sensors, gas storage, conductivity, magnetism, etc.12, 13. The synthesis of coordination drug polymers (CDP), where the active pharmaceutical ingredient is incorporated as a ligand, to enhance the solubility of APIs, was discovered for the first time by Joyce Pan in

Professor Jing Li’s Laboratory. The dissolution mechanism of CDPs is proposed to be as follows: when CDPs are placed in an aqueous solution the N….Zn…N bonds are broken. This results because the affinity of 4,4’ bipyridine (BPY) and water molecules to zinc metal is comparable. When CDPs are placed in water, the BPY molecules are displaced by the water molecules. The BPY molecules are displaced by the water molecules which outnumber them. This proposed mechanism is supported by proton nuclear magnetic resonance (1H NMR). References were obtained for both BPY and

Ibuprofen (HIBP) dissolved in deuterated water at 300MHz. For BPY there are two

83 peaks obtained at 7.66 and 8.55 ppm. HIBP has seven active hydrogen peaks according to figure 4 with some additional peaks which are attributed to impurities. For all spectra obtained the presence of deuterated water is indicated at 4.68ppm. The dissolution

1 mechanism is supported by the similar H NMR spectra of Zn(IBP)2 (BPY)2H2O (CDP1) and Zn(IBP)2 (BPY)2 (CDP2) (see figure 4). CDP1 is differentiated from CDP2 by the presence of water molecules in the former. Both CDP1 and CDP2 are indistinguishable when placed in deuterated water and display their characteristic peaks for HIBP and

BPY.

Figure 4: 1H NMR of BPY (top left), HIBP (bottom left) and CDP1 (top right) and CDP2 (bottom right) in deuterated water.

The proposed dissolution mechanism was confirmed further by additional 1H

NMR data for 1,2- bis (4-pyridyl) ethane (BPE) and CDP3 which is Zn(IBP) 2 (BPE)2 in deuterated water (see figure 4.1). BPE has 3 characteristic peaks located at 8.26, 7.12 and 2.94 ppm. CDP3 has poor dissolution compared to CDP2 and CDP1 which is why a

84 low concentration of CDP3 was obtained. 1H NMR of CDP3 indicates characteristic peaks of BPE in addition to other peaks from CDP3 at low intensity. These results further confirm the proposed mechanism that the CDP bridging ligands when placed in water are replaced by the water molecules. This is confirmed by the presence of the characteristic peaks of BPE along with CDP3 without any shifts or absence of these peaks.

Figure 4.1: 1H NMR of BPE (left), and CDP3 (right) in deuterated water.

CDPs are bonded by covalent coordination bonds that are stronger than the moderate hydrogen bonds13, 14. CDPs which involve the formation of covalent coordination bonds are synthesized by a one-pot, solvothermal reaction. This is advantageous over the synthesis of linear chain prodrugs where the formation of covalent ester bonds involves several steps and days14. In addition, CDPs also incorporate metals essential for human development. These essential elements can be supplemented with an appropriate diet or may result in the immunity and growth being compromised. For example zinc metal was used for all the CDPs synthesized so far.

Zinc metal is an essential micronutrient that is used for tissue repair, cell growth reproduction, skin disorders15. The choice of metal can be extended to other useful metals such as iron (Fe2+) and calcium (Ca2+). Properties of these polymers can be

85 tuned further by varying the ligands and selecting different pharmaceutically relevant metals as well.

One-dimensional CDPs where the API of interest are Ibuprofen and

Indomethacin, two anti-inflammatory nonsteroidal drugs have been synthesized.

Ibuprofen is considered a class II API. It has poor water solubility of 0.14mg/ml at a pH of 5.0 with decreasing solubility as the pH lowers16. It possess a monocarboxylic group

(see figure 4.2) which makes it suitable to form CDP. Please see previous chapters for details on indomethacin. Overall, the CDP structure consists of these APIs that are coordinatively bonded to the zinc metal ion as ligands by the oxygen of the API carboxylic group. Based on the principal of coordination polymer, which is directed by the coordination bond12, the metal bridging ligand 4,4’bpy was used to connect from metal to metal. The Lewis base, the carboxylic OH of Ibuprofen forms a coordination bond with the Lewis acid, the metal atom. The structure and solubility properties of these new coordination polymers are compared and contrasted. Some CDPs exhibit higher solubility over the drug alone. The aim of this study was to demonstrate 1) successful formation of new CDP crystals 2) show CDP structure- property relationship and 3) show enhanced solubility of CDPs compared to the drug as is.

Figure 4.2: Structure of Ibuprofen.

4.2 Materials And Methods

4.2.1 Materials

Zn(NO3)2H2O (Acros, 98%), 4,4’bipyridine (Acros, 98%), 1,2-bis(4-pyridyl)-ethane

(BPE) (Sigma-Aldrich 99%) and 1,2-bis(4-pyridyl)-ethylene (BPEE) ((Sigma-Aldrich

99%)) were purchased. Ibuprofen (2-(4-Isobutylphenyl)propionic Acid) was purchased from TCI (Portland, OR). Indomethacin with 99 % purity was purchased from Sigma

Aldrich (Saint Louis, MO). Reagent grade N,N-dimethylformamide (DMF) 99%+ purity was purchased from Fisher as well.

4.2.2 Methods

4.2.2.1 CDP synthesis

CDPs were synthesized by varying the bridging ligands to obtain a systematic understanding of the dissolution mechanism. Ligand solubility in water, its basicity and water solubility probably play a key factor in determining the dissolution of CDPs. Table

4 lists the basicity and solubility of the bridging ligands that have been initially investigated. In regards to basicity, aromatic amines have lower basicity than aliphatic amines. This is because the aromatic ring delocalizes the electron density throughout the ring rather than it being concentrated on nitrogen. This makes nitrogen less electron donating. So, the less electron donating the ligand is, the weaker its bond with the metal ion. A bridging ligand with lower basicity should result in faster dissolution of the CDP

87 because of its weaker bond with the metal ion. BPY is similar in structure to BPE and

BPEE. According to table 4, BPY has the lowest basicity compared to BPE and BPEE.

Ligand Structure pKa(pKb)/ Solubility

4,4’ bipyridine (BPY) 4.82(9.18)17/4.5g/L18

1,2- bis (4-pyridyl) ethane 5.9(8.1)19/5.3g/L20 (BPE)

1,2- bis (4-pyridyl) ethylene 5.65(8.35)19/1.8g/L20 (BPEE)

Table 4: BPY, BPE and BPEE ligands, their structure, basicity and solubility.

Solubility and hydrophilicity of the bridging ligands are other factors that can influence the dissolution or solubility of CDPs in water. The more hydrophilic the bridging ligands are the more readily these ligands will go into aqueous solution. These hydrophilic ligands will be more readily replaced by water molecules which would result in a faster dissolution of the CDPs.

CDPs were synthesized by the solvothermal method with varying temperature and times. Single crystals of these materials were obtained as follows. The starting reagents were added in a stoichiometric ratio of 1:2:1 of metal hydrate: API: bridging ligand into a glass vial. Dimethlyformamide was then added to this mixture, capped and then heated at varying temperatures and times. After heating, all the solutions were

88 cooled to room temperature, uncapped and set to evaporate for about two to three weeks. Zn(IBP)2(BPY) 2 (H2O) (CDP1) and Zn(IBP)2BPY (CDP2) were heated at 50°C for 4 hours and 100°C for 2 hours, respectively. Clearless rod-like crystals were obtained for CDP1. Single crystals of Zn(IBP)2BPE (CDP3) were obtained after heating at 50°C for 4 hours. Zn(IBP)2BPY (CDP2) and CDP5 were heated at 100°C for 2 hours.

Clearless plate like crystals were obtained for CDP5. CDP6 crystals were obtained with a 1:2:2 stoichiometric ratio of Zinc nitrate hexahydrate: indomethacin: bpe.

For Ibuprofen, CDP1 and CDP2 are obtained with the ligand BPY. They differ from each other mainly because of the presence of water molecules in CDP1. Both

CDP1 and CDP2 are one-dimensional chains. CDP1 is coordinated to zinc (Zn2+), 2

Ibuprofen, 2 BPY ligands and 2 water molecule. The resulting chain is a linear backbone for CDP1. CDP2 on the other hand, has a four coordinated Zn2+ without the presence of water. The CDP2 results in a zigzag chain.

When the bridging ligand was changed to BPE, Zn(IBP)2BPE (CDP3) was obtained. CDP3 was confirmed with single crystal and powder xray diffraction analyses to be isostructural to CDP2. Yin Wang obtained the single crystal with BPEE.

By changing the API to Indomethacin, another carboxylic acid ligand, two new coordination polymers were obtained. Zn(IMC)2BPY(H2O)2 (CDP5) and

Zn(NO3)(IMC)BPE (CDP6) resulted by varying the bridging ligand from BPY to BPE,

CDP5 has a similar structure to CDP1. CDP6 however is different in that each indomethacin is coordinated to two different Zn2+ ions. Each Zn2+ is also coordinated to 2 oxygen atoms of the nitrate ion making zinc octahedrally coordinated.

BPY is similar in structure to BPE and BPEE. CDPs were synthesized with these varying ligands to obtain a systematic understanding of the dissolution mechanism.

4.2.2.2 Ultrasonic Processor

The ultrasonic processor was used to reduce particle size. Ultrasonic processors use acoustic sound to propagate through the liquid or solvent of interest to create alternating high and low pressures. Acoustic cavitation is the process in which vacuum bubbles form, grow and collapse as a result of alternating high and low pressure cycles.

When a low pressure cycle creates a bubble it grows until a high pressure cycle collapses the bubble. These cycles of alternating high and low pressures, exert stress on the liquid and the attractive forces between the particles. The mechanical stress causes the particles to separate and results in intense heating21, 22.

There are many parameters to control particle size. Some of the most important parameters to control particle size are amplitude (intensity at which the pressure lowers and increases), temperature, viscosity, concentration and time23. Kaolanite ( an industrial raw material ) showed reduced particle size as a function of pressure, concentration and increasing time of ultrasonication. With the ultrasonic processor particle size reduction occurs in solution since nanoparticles with very high surface area have a tendency to agglomerate when placed in solution to be delivered into the body.

Ultrasonic processor and microtip (VCX 750 Watts and 13mm tip diameter)

(Sonics and Materials, Inc., Newton, CT) were used. The ultrasonic processor was used at a high frequency of 20KHz with a set amplitude and temperature of 30% and 25C, respectively. The pulsation was set at 2.0 seconds on and 1.0 seconds off. Time or duration was varied from 10, 20 and 45 minutes.

4.2.2.3 Powder X-ray Diffraction

PXRD and CCD database were used to confirm crystallinity, purity, new and modified CDP structures. Suitable single crystals were obtained for single crystal X-ray diffraction analysis. A summary of unit cell parameters for the CDPs 1-3,5,6 are also provided in table 4.1. CDPs 1-4 are synthesized with the incorporation of Ibuprofen within it, whereas CDP 5 and 6 incorporate indomethacin

CDP-1 CDP-2 CDP-3 CDP-5 CDP-6 Zn(IBP)2(BPY) 2H2O Zn(IBP)2BPY Zn(IBP)2BPE Zn(IMC)2(bpy)2(H2O)H2O Zn(NO3)(IMC)(bpe)

a(Å) 5.6816(5) 33(2) 29.545(2) 7.5621(5) 25.1979(13) b(Å) 11.5190(11) 5.729(4) 5.7623(4) 13.1623(9) 7.5854(4) c(Å) 13.690(2) 16.6801(8) 23.241(2) 15.5868(11) 22.8277(11)

° 89.014(19) 90 90 71.686(1) 90 ° 78.501(18) 92.55 121.993(1) 77.062(1) 97.034(2) ° 79.057(15) 90 90 77.135(1) 90 Volume(Å3) 861.83(17) 3137(191) 3355.7(4) 1415.85(17) 4330.2(4) Space group P-1 C2/c C2/c P-1 C2/c

Crystal System Triclinic Monoclinic Monoclinic Triclinic Monoclinic

Z 1 4 4 2 4

Table 4.1: Unit cell parameters of CDPs

4.2.2.4 Ultraviolet-Visible Spectroscopy

Ibuprofen in distilled water has an absorbance peak at 239nm that is attributed to its benzoic ring. The ligands BPY and BPE have peaks that overlap with Ibuprofen’s peak (see figure 4.3). Based on the CDP1 and CDP2 structures it was determined that

IBP and BPY occurred in a fixed ratio of 2:1 similar to IBP and BPE. Ibuprofen was detected by measuring the absorption intensity of BPY and BPE depending on the CDP.

On the other hand, Indomethacin has its absorbance maximum at 320nm. The ligands

BPY and BPE lack overlap with indomethacin at this wavelength (see figure 4.4).

HIBP Absorbance Absorbance 2 BPE

1 BPY

0 210 260 310 360 Wavelength (nm)

Figure 4.3: Absorbance peaks of HIBP (solid line), BPY (solid gray line) and BPE

(dotted line).

3 IMC 2

BPE

Absorbance Absorbance 1 BPY 0 210 260 310 360 410 460 -1 Wavelength (nm)

Figure 4.4: Absorbance peaks of HIMC (solid line), BPY (solid gray line) and BPE (dotted line).

4.2.2.5 Solubility

Time dependent solubility was conducted at room temperature. 20ml of distilled water was saturated with the desired compound and stirred with a stirring bar continuously. Time points between 0-72 hours were taken from a clear solution after the stirring in the saturated solution was stopped. Three trials were taken per compound.

After creating a calibration curve at 239nm, the concentrations for a 72 hour period were calculated based on the peak for the ligand 4,4’bipyridine, because there was an overlap with the Ibuprofen and 4,4’bipyridine peak. The ratio of 4,4’bipyridine to Ibuprofen is 1:2.

Solubility testing for nanoparticles were conducted using 0.0020 and 0.0021mM concentrations of CDP2 and the new CDP, respectively. HIBP was used in an equivalent amount of 0.0041mM.

4.3 Results And Discussion

Understanding the similarities and differences between the CDP structures aids to tune these CDPs as desired. In the CDP1 structure, zinc maintains an octahedral coordination within a triclinic system with a linear polymer backbone (see figure 4.5a-c).

CDP2 on the other hand is tetrahedrally coordinated into a monoclinic system with a zigzag backbone (see figure 4.6a-c). CDP2, 3 and 4 are all isostructurally related. Larger volumes are observed as larger ligands are incorporated as we go from bpy, bpe, and bpee.

CDP 1

BPY

HIBP

Zn(NO3)2 6H2O

2θ

Figure 4.5: a) Structure of CDP1 b) Linear polymer backbone of CDP1 and c) PXRD of CDP1.

CDP 2

BPY

HIBP

Zn(NO3)2 6H2O

2θ c

Figure 4.6: a) Structure of CDP2 b) Zigzag chain of CDP2 and c) PXRD of CDP2.

CDP 3

BPE

HIBP

Zn(NO3)2 6H2O

2θ

Figure 4.7c: a) Structure of CDP3 b) Linear polymer backbone of CDP3 and c) PXRD of CDP3.

CDP 5

BPY HIBP

Zn(NO3)2 6H2O

2θ

Figure 4.8: Structure of CDP5 b) Linear polymer backbone of CDP5 c) PXRD of CDP5.

CDP 6

BPE

HIBP

Zn(NO3)2 6H2O

2θ c

Figure 4.9: a) Structure of CDP6 b) Linear polymer backbone of CDP6 c) PXRD of CDP6.

Obtaining the structural information is useful in relating it to other properties or behaviors that are exhibited by these CDPs. Figure 4.10a displays the time dependent concentration of CDP2 compared to CDP1 and CDP3. CDP2 displayed the highest

98 solubility so far. The UV3101pc and UV3600 from Shimadzu have absorbance that are valid from 4~5 and 5-6, respectively (see the respective manuals). A 20% increase in solubility was observed with CDP2 compared to Ibuprofen as is (see figure 4.10b).

CDP1 and CDP2 are similar because both are composed of zinc, ibuprofen and BPY in a 1:2:1 ratio. They are different because CDP1 incorporates two water molecules, whereas these water molecules are missing in CDP2. One possible reason for the difference in solubility between CDP1 and 2 may be the steric hindrance in CDP1 that probably prevents the water molecules from easy access to coordinate with zinc. The tetrahedral coordination in CDP2, on the other hand allows water molecules to coordinate to zinc more readily.

The lower solubility of CDP3 compared to CDP1 and CDP2 can be attributed to the BPE ligand which has a higher basicity compared to BPY. This allows BPE to maintain a stronger coordination bond with zinc metal than BPY. This poor solubility is also confirmed with the 1H NMR data of BPE and CDP3.

Absorbance

Time (mins)

Figure 4.10: Time dependent concentration of a) CDP1 (black), 2 (red), 3 (blue) in distilled water and b) CDP2 (black), and Ibuprofen as is (red) in distilled water.

Time dependent solubility studies were conducted for indomethacin based CDP as well (see figure 4.11). CDP6 was easily obtained in bulk form unlike CDP5. CDP6 compared to indomethacin as is in distilled water indicated that CDP6 has very high

100 solubility initially followed by a decrease in solubility after only 10 minutes. A decrease in solubility like this is commonly observed when a metastable or amorphous material crystallizes out of solution to its more stable form. Further studies need to be carried out to understand the reason for the decrease in solubility when in a CDP system.

Figure 4.11: Time dependent concentration of CDP6 (blue) compared to Indomethacin alone (red) in distilled water.

Controlling the physical size of particles allows material properties to be tuned. A reduction in particle size is known to enhance solubility or dissolution rate. Nanoparticles have a higher surface area than microparticles and so they exhibit much higher solubility and or dissolution rate24. CDP2 had the fastest dissolution of all the ibuprofen based

CDPs. CDP2 was selected for further modification by a reduction in particle size.

Attempts to synthesize nanoparticles based on room temperature solution based syntheses25, 26 failed. The ultrasonic processor was successfully used to achieve a reduction in particle size.

SEM images confirm the formation of CDP2 nanoparticles in figure 4.12. After ultrasonication particles 500nm or less were obtained. PXRD patterns (figure 4.13) of

101 these ultrasonicated CDPs were compared with that of CDPs before ultrasonication.

CDP2 particles after ultrasonication indicate a removal of impurities in the nanoCDP2 with the presence of a minor phase at low angle. In addition, the ultrasonicated particles of the new CDP (see figure 4.14) resulted in a similar minor phase along with the removal of impurities. The new CDP was synthesized with the same stoichiometric ratio and starting reagents as CDP1 and CDP2. The starting concentration of the

Zn(NO3)2H2O was 0.3mM and the remaining reagents were adjusted accordingly. Similar to CDP2 the new CDP was heated at 100C for 2 hours and set to cool for 3 weeks or more. The structure of this new CDP can be determined once a single crystal is obtained. In addition a better understanding of its solubility and structure and be determined once the structure is confirmed and SEM images are obtained to assess particle size.

Figure 4.12: SEM images confirming particle size reduction: Drug as is (top left), CDP2 (top right), ultrasonicated CDP2 (bottom).

102

CDP 2 (after ultrasonication) CDP 2 (before ultrasonication)

CDP 2

BPY

HIBP

Zn(NO3)2 6H2O

2θ

Figure 4.13: Powder x-ray pattern of CDP2 before and after ultrasonication.

New CDP (after ultrasonication) New CDP (before ultrasonication)

CDP 2

CDP 1

BPY

HIBP

Zn(NO3)2 6H2O

2θ

Figure 4.14: Powder x-ray pattern of the new CDP before and after ultrasonication.

Time dependent solubility was measured for Ibuprofen, and the nanoparticles of

CDP2 and the new CDP. Basically the nano CDPs display better dissolution compared to the drug alone (see figure 4.15). A proper assessment of the percentage of drug

103 released can be made after the time-dependent solubility is repeated. Care must be taken when retesting since the drug amount is weighed at low milligram levels. Much more significant difference in solubility might occur when a larger amount of equivalent drug is used. Overall, these CDPs and modified CDPs release rate is faster than that observed for the approximately one week release of polyanhydride esters. These CDPs provide potential for being tuned for faster release for instance with the synthesis of a more hydrophilic system as opposed to a more hydrophobic system that would be

required for applications requiring a slower release rate. Absorbance

Time (mins)

Figure 4.15: Absorption vs. time of the ultrasonicated particles of the new CDP (red), CDP2 (blue) and HIBP as is (black).

Toxicity assessment

For a 400mg dose of Ibuprofen for a typical average adult body weight of 70 kilograms the mg/kg dosage is about 5.7mg Ibuprofen. The equivalent amount of CDP2 would be about 8.7mg. This contains 5.7mg Ibuprofen, 0.903mg zinc and 4.31mg BPY.

The daily recommended dosage for zinc supplements is a maximum of two 10mg doses27. Toxicity studies revealed a 100mg/kg of BPY to be safe for humans28. This

104 implies that ten times the CDP2 dosage of 8.7mg can be taken safely without overdosage of zinc but then overdosage of Ibuprofen needs to be considered.

4.4 Summary

CDPs were successfully synthesized and provide pathway for the synthesis of additional relevant CDPs. Synthesis conditions, including temperature, duration and ligands result in CDPs with varying properties. A solubility property test also revealed higher solubility for CDPs compared to Ibuprofen as is. Successful particle size reduction of CDP2 was obtained by ultrasonication to further enhance CDP solubility. Further experiments need to be conducted to confirm the enhanced solubility of CDP2 nanoparticles. Toxicity evaluation suggests CDP2 to be safe for a 400mg ibuprofen dosage.

105

4.5 References

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2. Vippagunta, S. R.; Brittain, H. G.; Grant, D. J. W., Crystalline solids. Advanced Drug Delivery Reviews 2001, 48 (1), 3-26.

3. Serajuddin, A. T. M., Salt formation to improve drug solubility. Advanced drug delivery reviews 2007, 59 (7), 603-616.

4. Trask, A. V., An Overview of Pharmaceutical Cocrystals as Intellectual Property†. 2007.

5. Schultheiss, N.; Newman, A., Pharmaceutical Cocrystals and Their Physicochemical Properties. Crystal Growth & Design 2009, 9 (6), 2950.

6. Dunitz, J. D., Crystal and co-crystal: a second opinion. In CrystEngComm, Royal Society of Chemistry: 2003; Vol. 5, pp 506-506.

7. Stahly, G. P., Diversity in single-and multiple-component crystals. The search for and prevalence of polymorphs and cocrystals. Crystal growth & design 2007, 7 (6), 1007-1026.

8. Shan, N.; Zaworotko, M. J., The role of cocrystals in pharmaceutical science. Drug discovery today 2008, 13 (9-10), 440-446.

9. Desiraju, G. R., Chemistry beyond the molecule. Nature 2001, 412 (6845), 397- 400.

10. Aakero y, C. B.; Forbes, S.; Desper, J., Using cocrystals to systematically modulate aqueous solubility and melting behavior of an anticancer drug. ACS Publications: 2009; Vol. 131, pp 17048-17049.

11. Vishweshwar, P.; McMahon, J. A.; Bis, J. A.; Zaworotko, M. J., Pharmaceutical co-crystals. Journal of pharmaceutical sciences 2006, 95 (3), 499.

12. Robin, A. Y.; Fromm, K. M., Coordination polymer networks with O-and N- donors: What they are, why and how they are made. Coordination chemistry reviews 2006, 250 (15-16), 2127-2157.

13. Batten, S. R.; Turner, D. R.; Neville, S. M., Coordination Polymers: Design, Analysis and Application. Royal Society of Chemistry: 2009.

14. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Jarvinen, T.; Savolainen, J., Prodrugs: design and clinical applications. Nature Reviews Drug Discovery 2008, 7 (3), 255-270.

15. Hambidge, M., Human zinc deficiency. Journal of Nutrition 2000, 130 (5), 1344S.

16. Yazdanian, M.; Briggs, K.; Jankovsky, C.; Hawi, A., The ―high solubility‖ definition of the current FDA Guidance on Biopharmaceutical Classification System may be too strict for acidic drugs. Pharmaceutical research 2004, 21 (2), 293-299.

106

17. Dean, J. A., Lange's Handbook of Chemistry. 14th. New York: McGraw-Hill, Inc: 1992.

18. Acros Organics MSDS and Alfa Aesar MSDS.

19. Szalda, D. J.; Fagalde, F.; Katz, N., Pentaammine-25N-(-4, 4'-bipyridine-1N: 2N')(2, 2'-bipyridine-12N, N')(2, 2': 6', 2''-terpyridine-13N, N', N'') diruthenium Tetrakis (hexafluorophosphate) Acetonitrile Solvate. Acta Crystallographica Section C: Crystal Structure Communications 1996, 52 (12), 3013-3016.

20. Predicted properties by Scifinder (Calculated using Advanced Chemistry Development (ACD/Labs) Software V8.14 for Solaris ( 1994-2009 ACD/Labs)).

21. Suslick, K. S.; Doctycz, S. J., Interparticle collisions driven by ultrasound. Science 1990, 247 (4), 946.

22. Suslick, K. S.; Didenko, Y.; Fang, M. M.; Hyeon, T.; Kolbeck, K. J.; McNamara, W. B.; Mdleleni, M. M.; Wong, M., Acoustic cavitation and its chemical consequences. Philosophical Transactions of the Royal Society of London. Series A: Mathematical, Physical and Engineering Sciences 1999, 357 (1751), 335.

23. Hielscher, T., Ultrasonic production of nano-size dispersions and emulsions. Arxiv preprint arXiv:0708.1831 2007.

24. Mohanraj, V.; Chen, Y., Nanoparticles-a review. Tropical Journal of Pharmaceutical Research 2007, 5 (1), 561-573.

25. Pan, Y.; Liu, Y.; Zeng, G.; Zhao, L.; Lai, Z., Rapid synthesis of zeolitic imidazolate framework-8 (ZIF-8) nanocrystals in an aqueous system. Chem. Commun. 2011.

26. Cravillon, J.; Mu nzer, S.; Lohmeier, S. J.; Feldhoff, A.; Huber, K.; Wiebcke, M., Rapid Room-Temperature Synthesis and Characterization of Nanocrystals of a Prototypical Zeolitic Imidazolate Framework. Chemistry of Materials 2009, 21 (8), 1410- 1412.

27. Berger, A., Science commentary: What does zinc do? BMJ: British Medical Journal 2002, 325 (7372), 1062.

28. Li, S.; Crooks, P. A.; Wei, X.; Leon, J., Toxicity of dipyridyl compounds and related compounds. CRC Critical Reviews in Toxicology 2004, 34 (5), 447-460.

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CHAPTER FIVE

5. Closing remarks

This thesis examines the enhancement of the solubility and dissolution rate of poorly water soluble active pharmaceutical ingredients mainly by particle size reduction. Complexation of drug with the addition of hydrophobic excipients offers another avenue to enhance dissolution rate. By proper tuning of the experimental parameters, supercritical CO2 antisolvent modified products with enhanced dissolution rates were obtained. The alternate method of investigating crystalline compounds led to the synthesis of CDPs. Ultrasonic processor is another unconventional method that was successfully used to control particle size and retain structural integrity.

Further examination of process parameters are needed to tune particle size, crystallinity and complexation abilities. The synthesis of novel crystalline

CDPs to enhance bioavailability by solubility or dissolution rate can be modified further with the replacement the current metal ions, bridging ligands with pharmaceutically relevant more soluble amine based ligands, and the incorporation of different potential APIs. Ultrasonic processing on this system needs to studied further with a variation in amplitude, temperature effect, solvent selection, concentration, etc. Comparison of SAS processing with the ultrasonicated particles remains to be studied, along with the incorporation of ultrasonication during SAS processing.

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Overall, these non-traditional techniques or approaches offer promise for tuning poorly water soluble drug with controllable particle size and the resulting modified physicochemical properties. Much more work remains in terms of understanding these physicochemical modifications for scale up purposes. The synthesis of novel crystalline CDPs serves as a potential drug form (specifically an NSAID) that incorporates a desired essential metal to be delivered into the body at the same time with enhanced dissolution.

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APPENDIX

Powder x-ray diffraction (PXRD)

Powder X-ray diffraction is one of the most pertinent analytic techniques to characterize solid materials. It used to: 1) identify phases and 2) provide the unit cell dimensions of crystal structures. Each crystalline solid has its own unique powder pattern just like a fingerprint.

When light hits a grating with a similar distance as the incident lights’ wavelength, it diffracts. Similarly, X-rays have wavelengths on the order of a few angstroms similar to the distance between atoms. When x-rays hit the angstrom level interatomic distances

(d-spacing) of a crystalline solid, a diffraction pattern occurs. This diffraction pattern provides information on the structure and phase of the compound of interest. The most common source of X-rays for inorganic materials is Copper (Cu). The strongest Cu radition is Kα with a wavelength of =1.54 angstroms.

This robust, nondestructive technique basically requires an X-ray source, a sample holder/sample and an x-ray detector (see figure below A1).

Diffracted beam

Incident beam

X-ray Source (Cu) Sample Filter

Detector Figure A1: Schematic of PXRD

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Braggs equation mathematically shows constructive interference from two different planes (see Figure A2)

2=ndsin n= integer, known as the order of the diffracted beam d= interatomic spacing in angstroms

= angle of diffraction

= wavelength of incident X-ray

  Atomic plane d

Atomic plane Figure A2: Diffraction within atomic planes

Ultraviolet –Visible Absorption Spectrometry (UV-Vis)

Ultraviolet-Visible absorption spectroscopy covers the electromagnetic spectrum from a wavelength of 200-400nm (UV) and 400-800nm (Vis). UV-Vis absorption spectroscopy measures electronic transitions from the ground to the excited state. There are two ways to measure absorption spectra; for qualitative and quantitative purposes.

Light source either a tungsten lamp (Vis) or deuterium or halogen lamp (UV) shines light of all wavelengths that pass through a grating monochromator. The monochromator separates the wavelength to the desired one. The selected wavelength

111 passes through the sample. Any light that is unabsorbed by the sample passes through to the detector. For a double beam spectrometer (see figure A3) after the light source and monochromator there is a splitter with mirrors to get the selected light to the reference and sample. Quartz cuvettes are used because it is clear in the UV range. The sample must be dissolved in the solvent that it is in otherwise scattering will result.

Beer-Lambert Law directly relates the concentration of a substance to its absorbance.

A= εcl

A=Absorbance (unitless, usually in arbitrary units)

ε= molar absorptivity or extinction coefficient (M-1 cm-1) c= concentration (M) l= path length of the sample (usually in cm)

Spectrum of light Light source Reference Detector

Halogen/Tunsgten Monochromator lamps

Sample Figure A3: Schematic of ultraviolet visible spectrophotometer

Vibrational Spectroscopy

Infrared spectroscopy is an absorption technique that is used to analyze solids, liquids and gases. It probes the vibrational modes of molecules. These vibrational modes are fingerprints for molecules. Molecules absorb frequencies that are characteristic of their structure. In order for something to be ―IR active‖ it must correspond to a change in dipole moment.

In this thesis (ATR-IR) attenuated total reflection- infrared spectroscopy was used. ATR schematic is shown below (figure A4). As the incident beam hits the crystal,

112 instead of leaving the crystal something called the total internal reflection occurs. This means that at an appropriate setting instead of leaving the crystal the infrared beam reflects off the crystal surface. The crystal has to have the proper refractive index and the light has to have the appropriate angle of incidence for this to occur. In the schematic below the infrared radiation hits the crystal three times before leaving. A standing wave is created once the radiation gets inside the crystal. This standing wave also known as an evanescent wave is unique in that it is bigger than the crystal it is confined to. The evanescent wave slightly penetrates above and below the crystal surface. Above it, the evanescent wave hits the sample. The increased pressure on the sample allows it to have very close contact with the crystal. When the sample absorbs the evanescent wave it is attenuated. The attenuated wave is then analyzed by the detector and sample information is obtained.

Pressure

Sample

IR beam Detector

Evanescent wave ATR crystal Figure A4: Illustration of the basic concept of an ATR

Raman spectroscopy is also a type of vibrational spectroscopy that involves scattering instead of absorption. When a laser light hits a sample photons of higher or lower energy are emitted or scattered. This shift in frequency of the photons (or inelastic scattering) relays information about the molecular vibrations of the sample at hand.

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Raman is complimentary to IR spectroscopy which does not require a molecule to have a change in dipole moment, but can view molecules that have the ability to show polarizability.

Differential Scanning Calorimetry (DSC)

DSC is a thermoanalytical tool to measure the changes that occur in a compound by the difference in heat flow. That is temperature is measured as a function of heat flow to the system. You can measure, crystallization or melt temperatures, glass transition temperature. For instance at the phase transition there is a peak because, energy is input in which bonds are broken or formed. As heat flow begins to both the sample and reference (usually a blank pan). As enough flows to the sample to cause a loosening of break of the bonds a change in the DSC will be observed.

Additional Works

A B

Figure A5: HP-β-CD (Hydroxypropyl-beta-cyclodextrin) is processed with the supercritical Antisolvent at 1%w/v Ethanol 35°C, 1500psi and varies from (A) 0.6 to (B) 1.0 in the degree of substitution. A significant difference in particle morphology and size is observed for (A) where there are irregular shaped nanoparticles and (B) is composed of microparticles that are spherical.

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Sample: HPBCD File: C:\TA\Data\DSC\pv\HPBCD.003 Size: 8.2000 mg DSC Operator: pv Method: Heat/Cool/Heat Run Date: 2007-05-09 15:32 Comment: as is 1380 Instrument: DSC Q100 V9.0 Build 275 0.5

0.0

258.59°C

-0.5 112.28°C Transconformation -1.0 Broad endothermic peak: corresponds to of the molecule (i.e. glass Heat Flow (W/g) Flow Heat the release of water transition) -1.5 from HP--CD

-2.0 Decomposition

341.67°C temperature

-2.5 0 50 100 150 200 250 300 350 400 Exo Up Temperature (°C) Universal V4.1D TA Instruments

Figure A6: DSC plot of HPCD

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S ample S olubility in water (ng/µl)

IMC as is 137.13

IMC processed in ethanol 263.05

(spray dried)

IMC processed in DCM 403.35 (SAS) IMC 1:1 HP βC D (P M) 214.22 IMC 1:1 HP βC D (S AS ) 356.18 IMC 1:1 HP βC D (S D) 195.35 IMC 1:2 HP βC D (S D) 259.96 IMC 1:3 HP βC D (S D) 320.66 IMC 1:4 HP βC D (S D) 457.99 IMC:PVP 29/32K 272.74 IMC :HP βCD :PVP 29/32K 225.80 1:1:29 wt%

IMC :HP βCD :PVP 25K 488.09 1:1:29 wt%

IMC :HP βCD :PVP 12K 461.67 1:1:29 wt%

Table A1: Additional solubility data

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Dissolution profile with increasing HPBCD pH 7.2 phosphate buffer 120

100

Percent Percent dissolution 40

0 0 5 10 15 20 25 30 35 40 45 50

Time (mins)

Figure A7: Spray dried IMC-HPCD in a 1:1 (solid line) and 1:4 (dashed line) ratio

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1695

IMC as is 1675 HPBCD as is PM (IMC:HPBCD) SD (IMC:HPBCD) PM (IMC:PVP29/32K) SD (IMC:PVP29/32K)

Intensity / Arb.Units PM (IMC:HPBCD: PVP 0.1) PM (IMC:HPBCD: PVP 0.5) PM (IMC:HPBCD: PVP 0.9) SD (IMC:HPBCD: PVP 0.5)

2114.2 2050 2000 1950 1900 1850 1800 1750 1700 1650 1600 1550 1500 1450 1400 1356.0 cm-1 Figure A8: Supporting raman spectra for chapter 3

heatflow 20

0 100 110 120 130 140 150 160 170 180 190 200

-10 Sample temperature(C)

Figure A9: A year old sample of IMC:HPCD(SD) stored at room temperature (DSC).

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Residual solvent analysis for Ethanol Overlaypresence Report in IMC + HPBCD Reported by User: Michelle DaltonFDA (mdalton3) requirement ≤5000 ppmProject Name: Empow er 1 Projects\JNJ28431754_2008_DS

30.00

28.00

26.00

24.00

22.00

20.00

18.00

16.00 pA 14.00

12.00 Reference

10.00 (DMAC)

8.00 SD (11,766-7,456ppm)

6.00

4.00

2.00 SAS (4,241-3347ppm)

0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 Minutes P73_R3; Date Acquired: 15-Oct-2008 16:14:59 EDT; Result Set Id 23056-41c; Date Acquired: 12-Aug-2008 20:29:15 EDT; Result Set Id 3896 Standard 1 check; Date Acquired: 12-Aug-2008 21:09:47 EDT; Result Set Id 3896

Figure A10: Residual solvent analysis indicating that the SAS process meets the FDA requirements.

Report Method: Overlay Report Printed 10:02:24 AM24-Oct-2008 US/Eastern

119

Princy A. Abraham

EDUCATION:

 Ph.D. in Chemistry and Chemical Biology at Rutgers, The State University of New Jersey (2002-2011).  B.S. (Honors) in Chemistry and Music minor at Binghamton University, The State University of New York (1998-2002).

EXPERIENCE:

PhD Thesis advisor: Professor Jing Li, Chemistry

 Graduate Assistant (2006-2011).  Internship at Johnson and Johnson Pharmaceutical Research and Development, Raritan, NJ (August-October 2006, June-August 2007, May-Sept 2008).  Graduate Fellow (2004-2006 IGERT).  Graduate Assistant (2002-2004).  Teaching Assistant for General Chemistry Lab (2007).  Teaching Assistant for Preparation for Chemistry (2006).  Teaching Assistant for General Chemistry Lab (2002-2003).  Summer 2001 visit to Poznan, Poland for collaboration on summer project  Summer research (REU) at University of Tennessee, Knoxville (2000-2001)  Research Assistant at University of Binghamton (1999-2002)

PUBLICATIONS:

 ―Supercritical antisolvent processing of γ-Indomethacin: Effects of solvent, concentration, pressure and temperature on SAS processed Indomethacin‖ Princy Varughese, Jing Li, Wayne Wang and Denita Winstead, Powder Technology, 201, 64 (2010)  ―A Three-dimensional Coordination Polymer Featuring Effective Ferromagnetic Hydroxide Bridged Manganese (II) Chains‖, Jin- Tang Li, Jun Tao, Rong-Bin Huang, Lan-Sun Zheng, Tan Yuen, C. L. Lin, Princy Varughese, and Jing Li, Inorg. Chem., 44, 4448 (2005).

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 "Local and Spacial Disorder in "-(ET)2SF5RSO3 Alloys (R=CH2CF2, CHF, and CHFCF2)", A.D. Garlach, J.L. Musfeldt, J.M. Pigos, B.R. Jones, P.A. Varughese, I. Olejniczak, A. Graja, M. Whangbo, J. Schlueter, U. Geiser, and G. Gard, Chem. Mater., 14, 2969 (2002).  "Lattice Dynamics of the One-Dimensional S=1/2 Heisenberg Antiferromagnet Copper Pyrazine Dinitrate", B.R. Jones, P.A. Varughese, I. Olejniczak, J.M. Pigos, J.L. Musfeldt, C.P. Landee, M.M. Turnbull, and G.L. Carr, Chem. Mater., 13, 2127 (2001).