Enhancement of the Rate of Solution of Relatively Insoluble Drugs from Solid-Solid Systems Prepared by Supercritical Fluid Technology

Home , Poloxamer 407

ENHANCEMENT OF THE RATE OF SOLUTION OF RELATIVELY INSOLUBLE DRUGS FROM SOLID-SOLID SYSTEMS PREPARED BY SUPERCRITICAL FLUID TECHNOLOGY

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Carmen H. Ramirez, M.S.

* * * * *

The Ohio State University 2007

Dissertation Committee: Approved by Professor Sylvan G. Frank, Advisor

Professor William L. Hayton

Professor James T. Dalton ______Advisor Professor Robert W. Curley Graduate Program in Pharmacy

ABSTRACT

Supercritical fluid technology, specifically the method of rapid expansion of supercritical solutions (RESS), has been used to prepare small particles consisting of solid solutions of a relatively insoluble drug and a water-soluble excipient. With an increasing number of relatively insoluble compounds being discovered, a general process for enhancing drug dissolution rates would assist formulation of these compounds for therapeutic use. Solid solutions could serve as a means for enhancing drug dissolution rates, since the drug is dispersed in a solid solvent in its smallest form, i.e., a molecule, prior to entering into solution.

Therefore, solid solutions consisting of the relatively insoluble model drugs lidocaine or probucol and a water-soluble surfactant, poloxamers 407, 188, or

403 were prepared by RESS processing. Dissolution studies of these systems were performed and evaluated for their ability to enhance drug release rates.

Furthermore, the mechanism by which solid solutions form in these systems was determined using differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy. Scanning electron microscopy (SEM) was also used to study the surface characteristics of these particulate systems.

Dissolution studies of these particles showed an enhanced rate of release of drug in the presence of poloxamer. This enhancement was due to the apparent formation of solid solutions. In addition, poloxamers served to improve the wettability of the drug particles by reducing the interfacial tension and contact angle at the solid/liquid interface, thereby helping enhance the dissolution rate of the drug. DSC of these particulate systems indicated the formation of solid solutions of drug and poloxamer with increasing proportion of poloxamer. At the point where all of the drug is molecularly dispersed forming a solid solution, no endotherm for the drug appears on the DSC thermogram. Therefore, in a phase diagram, linear extrapolation of the enthalpies of drug as functions of mole fraction to zero enthalpy could serve as a novel means for predicting the mole fraction of drug at which a solid solution should form. With the formation of solid solutions, hydrogen bonding occurred between the drug and poloxamer. This bonding was dependent on the polyoxyethylene chain length of the three poloxamers, i.e., where hydrogen bonding primarily occurs. Solid solutions formed for systems consisting of drug and poloxamers 407 or 188, which have similar polyoxyethylene lengths and hence similar amounts of available sites for bonding. Solid solutions however did not form for systems consisting of drug and poloxamer 403 since poloxamer 403 has approximately half the polyoxyethylene length of poloxamers 407 and 188. Lastly, SEM shows the formation of small particles that vary in appearance as functions of poloxamer concentration, i.e., from smooth spherical surfaces to structures similar to those of the drug alone.

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Dedicated to my mother, husband, and daughter

ACKNOWLEDGMENTS

I would like to give my special thanks to:

My adviser, Dr. Sylvan G. Frank for his guidance, mentorship, passion and support of this project, for his dedication to my personal growth, and most importantly, his patience of which I am ever so grateful.

Members of my committee, Dr. William Hayton, Dr. James Dalton, and Dr. Robert Curley for their suggestions and time.

Dr. Arne Brodin for his intellectual contribution to this project and invaluable discussions.

My labmates: Chao, Jessica, and Yong for their help and friendship.

Kathy Kelley for her friendship, laughter, and putting up with my noise in the lab.

My brother and sister and their families for their unending support and willingness to share our mother so that I could pursue my goals.

My mother for leaving it all behind to come to Ohio. Thank you for your love, support, and undying faith in me.

My husband David for your love, support, and especially your patience.

My daughter Elena for your love, laughter, and smiles, which have kept me going. You are my inspiration. Always remember that no dream is ever too big.

For all my friends and family who are not mentioned here, you do not go unrecognized by me.

VITA

August 30, 1976 ...... Born – Fort Worth, Texas

1998 ...... B.S. Chemistry, University of North Texas

1999-2001 ...... Graduate Research Associate The Ohio State University

2001-2004 ...... Professor Sylvan G. Frank Graduate Fellow in Pharmaceutics

2004 ...... M.S. Pharmaceutics, The Ohio State University

PUBLICATIONS

Research Publication

1. Hernandez, C.E. and Frank, S.G., “Enhancement Of The Rate Of Solution Of Relatively Insoluble Drugs From Solid-Solid Systems Prepared With Supercritical Fluid Technology.” Abstracts, AAPS Annual Meeting, Salt Lake City, UT, October, 2003

2. Hernandez, C.E. and Frank, S.G., “Enhancement Of The Rate Of Solution Of Relatively Insoluble Drugs From Solid-Solid Systems Prepared With Supercritical Fluid Technology.” 35th Pharmaceutics Graduate Student Research Meeting, Chicago, IL, June, 2003

FIELDS OF STUDY

Major Field: Pharmacy Drug delivery, pharmaceutics, and pharmaceutical technologies vi

TABLE OF CONTENTS

Abstract ...... ii

Dedication...... iv

Acknowledgements ...... v

Vita ...... vi

List of Tables ...... x

List of Figures...... xii

Chapters:

1. Introduction...... 1 1.1 Solubility, dissolution, and bioavailability...... 2 1.2 Solid dispersions ...... 12 1.2.1 Definitions ...... 13 1.2.2 Methods of preparation ...... 21 1.2.3 Characterization of solid dispersions ...... 24 1.2.4 Dissolution of drugs from solid dispersions...... 27 1.3 Supercritical fluid technology...... 30 1.3.1 Background...... 30 1.3.2 Applications of supercritical fluid technology...... 37 1.3.3 Supercritical fluid processing methods...... 41 1.3.3.1 Rapid expansion of supercritical solutions (RESS)...... 41 1.3.3.2 Gas anti-solvent method (GAS) ...... 42 1.3.3.3 Other methods utilizing supercritical fluids...... 43 1.3.4 Applications of supercritical fluid technology in drug delivery ...... 44 1.4 Objectives ...... 52

2. Characterization of drug / surfactant systems consisting of relatively insoluble drug and various poloxamers formed by RESS processing...... 53 2.1 Introduction ...... 54 2.2 Purpose of the study ...... 58 2.3 Experimental ...... 59 vii

2.3.1 Materials ...... 59 2.3.1.1 Lidocaine ...... 59 2.3.1.2 Probucol...... 61 2.3.1.3 Poloxamers 407, 188, and 403...... 63 2.3.2 Equipment...... 66 2.3.3 Methods of particle formation...... 69 2.3.3.1 Formation of drug / poloxamer 407, 188, or 403 particles by supercritical fluid processing...... 69 2.3.4 Methods of analysis ...... 73 2.3.4.1 Differential scanning calorimetry (DSC)...... 73 2.3.4.2 Scanning electron microscopy (SEM)...... 74 2.3.4.3 Dissolution of drug from particles consisting of drug and poloxamer...... 75 2.3.4.4 Aqueous solubility of lidocaine...... 78 2.3.4.5 Fourier Transform Infrared (FTIR) Spectroscopy...... 79 2.3.4.6 Solubility of lidocaine, probucol, or poloxamer in supercritical carbon dioxide...... 80 2.4 Results and discussion...... 81 2.4.1 Lidocaine, poloxamer 407, and lidocaine / poloxamer 407 compositions...... 81 2.4.1.1 Differential scanning calorimetry...... 81 2.4.1.2 Scanning electron microscopy...... 91 2.4.1.3 Release of lidocaine from particles containing poloxamer 407...... 98 2.4.1.4 Solubility of lidocaine in the presence of poloxamer 407...... 105 2.4.1.5 Fourier transform infrared (FTIR) analysis ...... 105 2.4.2 Lidocaine, poloxamer 188, and lidocaine / poloxamer 188 compositions...... 114 2.4.2.1 Differential scanning calorimetry...... 114 2.4.2.2 Scanning electron microscopy...... 121 2.4.2.3 Release of lidocaine from particles containing poloxamer 188...... 124 2.4.2.4 Fourier transform infrared (FTIR) analysis ...... 128 2.4.3 Lidocaine, poloxamer 403, and lidocaine / poloxamer 403 compositions...... 131 2.4.3.1 Differential scanning calorimetry...... 131 2.4.3.2 Scanning electron microscopy...... 140 2.4.3.3 Release of lidocaine from particles containing poloxamer 403 ...... 143 2.4.3.4 Fourier transform infrared (FTIR) analysis ...... 144 2.4.4 Summary: Comparison of lidocaine / poloxamer 407 lidocaine / poloxamer 188, and lidocaine / poloxamer 403 particles prepared by supercritical fluid processing ...... 147

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2.4.5 Probucol, poloxamer 407, and probucol / poloxamer 407 compositions...... 152 2.4.5.1 Solubility of probucol in supercritical carbon dioxide ...... 152 2.4.5.2 Differential scanning calorimetry...... 153 2.4.5.3 Scanning electron microscopy...... 164 2.4.5.4 Release of probucol from particles containing poloxamer188...... 168 2.4.5.5 Fourier transform infrared (FTIR) analysis ...... 174 2.4.6 Probucol, poloxamer 188, and probucol / poloxamer 188 compositions...... 181 2.4.6.1 Differential scanning calorimetry...... 181 2.4.6.2 Scanning electron microscopy...... 188 2.4.6.3 Release of probucol from particles containing poloxamer188...... 191 2.4.6.4 Fourier transform infrared (FTIR) analysis ...... 195 2.4.7 Probucol, poloxamer 403, and probucol / poloxamer 403 compositions...... 197 2.4.8 Summary: Comparison of probucol / poloxamer 407 and probucol / poloxamer 188 particles prepares by supercritical fluid processing...... 198

3. General summary...... 203

Bibliography...... 213

LIST OF TABLES

1.1 Critical points of various gases and liquids ...... 3434

1.2 Examples of industrial uses of supercritical fluids...... 3838

2.1 Materials ...... 6767

2.2 Equipment...... 7171

2.3 Thermal properties of lidocaine and poloxamer 407 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, n=2...... 8383

2.4 Comparison of fresh and stored (5 months, room temperature) thermal properties of lidocaine and poloxamer 407 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, n=2...... 9393

2.5 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of lidocaine and lidocaine/ poloxamer 407 binary mixtures...... 104104

2.6 FTIR analysis of lidocaine and compositions of lidocaine and poloxamer 407 ...... 109109

2.7 Thermal properties of lidocaine and poloxamer 188 processed

with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, n=2...... 116116

2.8 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of lidocaine/ poloxamer 188 binary mixtures...... 129129

2.9 FTIR analysis of lidocaine and compositions of lidocaine and poloxamer 188 ...... 132132

2.10 Thermal properties of lidocaine and poloxamer 403 processed with SCCO2 at 35 C, 7100 psi, and restrictor temperature of 20 C, n=2...... 137137 2.11 FTIR analysis of lidocaine and compositions of lidocaine and poloxamer 403 ...... 146146

2.12 Thermal properties of probucol and poloxamer 407 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, n=2...... 159159

2.13 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of probucol / poloxamer 407 binary mixtures...... 173173

2.14 Thermal properties of probucol and poloxamer 188 processed

with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, n=2...... 183183

2.15 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of probucol / poloxamer 188 binary mixtures...... 194194

LIST OF FIGURES

1.1 Hypothetical phase diagram for discontinuous solid solutions...... 1717

1.2 Phase diagram of a supercritical carbon dioxide ...... 3131

1.3 Schematic diagram of a supercritical fluid extraction system...... 3636

1.4 Comparison of steps involved with conventional processing versus supercritical fluid processing ...... 5050

2.1 Molecular structure of lidocaine ...... 6060

2.2 Molecular structure of probucol...... 6262

2.3 Molecular structure of poloxamers...... 6464

2.4 “Home made” chamber for collecting RESS processed particles...... 7070

2.5 Dissotest® dissolution system (Sotax, AG) ...... 7676

2.6 Representative DSC thermograms of lidocaine/poloxamer 407 compositions prepared by RESS processing...... 8282

2.7 Schematic representation of lidocaine and poloxamer 407 molecules...... 8787

2.8 Phase diagram of lidocaine / poloxamer 407 compositions ...... 8989

2.9 Phase diagram of lidocaine from lidocaine / poloxamer 407 compositions showing linear regression, r2 = 0.9756...... 9090

2.10 Representative DSC thermograms of lidocaine/poloxamer 407 stored compositions prepared by RESS processing...... 9292

2.11 Scanning electron photomicrographs of lidocaine and poloxamer 407 ...... 9494

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2.12 Scanning electron photomicrographs of 0.9812 mole fraction lidocaine prepared by RESS processing...... 9595

2.13 Scanning electron photomicrographs of lidocaine and poloxamer 407 compositions prepared by RESS processing ...... 9797

2.14 Release of lidocaine from lidocaine / poloxamer 407 particles prepared by RESS processing as functions of composition...... 9999

2.15 Apparent solubilities of lidocaine in the presence of poloxamer 407 in water, n=2...... 06106

2.16 FTIR spectra of lidocaine, poloxamer 407, and lidocaine / poloxamer 407 compositions ...... 08108

2.17 Schematic molecular structures of lidocaine and poloxamer showing hydrogen bonded interactions...... 10110

2.18 Schematic diagram of lidocaine dimer ...... 11111

2.19 Schematic molecular structures of lidocaine and a poloxamer showing hydrogen bonding as functions of lidocaine molecular orientation ...... 13113

2.20 Representative DSC thermograms of lidocaine / poloxamer 188 compositions prepared by RESS processing...... 15115

2.21 Phase diagram of lidocaine / poloxamer 188 compositions ...... 18118

2.22 Phase diagram of lidocaine from lidocaine / poloxamer 188 compositions showing linear regression, r2 = 0.9943 (blue) and r2 = 0.9342 (red)...... 20120

2.23 Scanning electron photomicrographs of poloxamer 188...... 22122

2.24 Scanning electron photomicrographs of lidocaine and poloxamer 188 compositions prepared by RESS processing ...... 23123

2.25 Release of lidocaine from lidocaine / poloxamer 188 particles prepared by RESS processing as functions of composition...... 25125

2.26 FTIR spectra of lidocaine, poloxamer 188, and lidocaine / poloxamer 188 compositions ...... 30130

2.27 Representative DSC thermograms of poloxamer 403...... 34134 xiii

2.28 Representative DSC thermograms of lidocaine / poloxamer 403 compositions prepared by RESS processing...... 36136

2.29 Phase diagram of lidocaine / poloxamer 403 compositions ...... 39139

2.30 Phase diagram of lidocaine from lidocaine / poloxamer 403 compositions showing linear regression, r2 = 0.9859 (blue) and r2 = 0.9523 (red)...... 41141

2.31 Scanning electron photomicrographs of lidocaine and poloxamer 403 compositions prepared by RESS processing ...... 42142

2.32 FTIR spectra of lidocaine, poloxamer 403, and lidocaine / poloxamer 403 compositions ...... 45145

2.33 Relationship between lidocaine mole fraction at zero enthalpy and total polyoxyethylene chain length of poloxamers 407, 188, and 403...... 49149

2.34 Relationship between lidocaine mole fraction at zero enthalpy and polyoxypropylene chain length of poloxamers 407, 188, and 403...... 50150

2.35 Solubilities of probucol in supercritical carbon dioxide at 7100 psi ...... 54154

2.36 Representative DSC thermogram of probucol as received...... 55155 . 2.37 Representative DSC thermogram of probucol after RESS processing ...... 56156

2.38 Representative DSC thermograms of probucol / poloxamer 407 compositions prepared by RESS processing...... 58158

2.39 Phase diagram of probucol / poloxamer 407 compositions...... 62162

2.40 Phase diagram of probucol from probucol / poloxamer 407 compositions showing linear regression, r2 = 0.9655...... 63163

2.41 Scanning electron photomicrographs of probucol...... 65165

2.42 Scanning electron photomicrographs of probucol and poloxamer 407 compositions prepared by RESS processing ...... 66166

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2.43 Scanning electron photomicrographs of probucol and poloxamer 407 compositions prepared by RESS processing ...... 67167

2.44 Release of probucol from probucol / poloxamer 407 particles prepared by RESS processing as functions of composition...... 69169

2.45 FTIR spectra of probucol and probucol after RESS processing ...... 75175

2.46 FTIR spectra of probucol, poloxamer 407, and probucol / poloxamer 407 compositions...... 76176

2.47 Schematic molecular structures of probucol and poloxamer showing hydrogen bonded interactions...... 78178

2.48 Schematic molecular structures of probucol and poloxamer showing hydrogen bonding as functions of probucol molecular orientation...... 79179

2.49 Representative DSC thermograms of probucol / poloxamer 188 compositions prepared by RESS processing...... 82182

2.50 Phase diagram of probucol / poloxamer 188 compositions...... 86186

2.51 Phase diagram of probucol from probucol / poloxamer 188 compositions showing linear regression, r2 = 0.9467...... 87187

2.52 Scanning electron photomicrographs of probucol and poloxamer 188 compositions prepared by RESS processing ...... 89189

2.53 Scanning electron photomicrographs of probucol and poloxamer 188 compositions prepared by RESS processing ...... 90190

2.54 Release of probucol from probucol / poloxamer 188 particles prepared by RESS processing as functions of composition...... 92192

2.55 FTIR spectra of probucol, poloxamer 188, and probucol / poloxamer 188 compositions...... 96196

2.56 Relationship between lidocaine and probucol mole fraction at zero enthalpy and total polyoxypropylene chain length of poloxamers 407, 188, and 403 ...... 00200

CHAPTER 1

INTRODUCTION

Concept Map

 The overall goal of this research was to develop a general method for preparing small particles of a relatively insoluble drug and a water-soluble component in the form of a solid solution. Solid solutions containing the relatively insoluble model drugs lidocaine or probucol were prepared using supercritical fluid technology and the physicochemical properties of these drug delivery systems were studied.

 The concepts of solid dispersions, including solid solutions, solubility, dissolution, and relevant analytical methods will be discussed with emphasis on the pharmaceutics of these systems.

 Supercritical fluid technology will be discussed with regard to its historical background, industrial applications, analytical and preparative methods, and applications in drug delivery.

1.1 Solubility, dissolution, and bioavailability

With the growing number of therapeutically active, but relatively insoluble compounds coming from drug discovery, significant pharmaceutical challenges have arisen with regard to ensuring that these substances are therapeutically effective. Relatively insoluble drugs, or sparingly soluble drugs as they are defined in the United States Pharmacopeia (1), require thirty to one hundred parts of solvent for every part of solute in order to dissolve. Solubility, dissolution rate, and bioavailability are all related properties that characterize a particular compound and describe its potential for therapeutic efficacy (2). Solubility is defined as the concentration of a dissolved compound in a solvent when the solution is saturated and the compound is in equilibrium with the solvent at a given temperature and pressure (2, 3). This property is also referred to as intrinsic solubility because it is an inherent property of a compound and is a function only of temperature (2). Some compounds, however, can exist in different energy states and crystalline forms besides their stable form and are thus referred to as having metastable polymorphs. The solubility of such compounds is measured at a metastable, or dynamic equilibrium, and therefore represents an apparent solubility, while the intrinsic solubility is obtained when a true equilibrium is achieved. Apparent changes in equilibrium, such as those created by a metastable compound, reflect changes in the rate at which equilibrium is achieved (4). Compounds that are in a metastable state may take days or months to convert to their stable form, at which time their apparent

solubility will decrease to its intrinsic value (3). Consequently, the apparent solubility of a compound can improve giving the appearance that more drug has dissolved in a given solution, but the intrinsic solubility will remain the same (3).

The solubility of a drug is important because relatively insoluble compounds have limited aqueous solubilities, which can lead to inadequate absorption and subsequent delivery of the active compound (3). For orally administered drugs, the absorbable form of the active compound must first be in solution in the gastrointestinal tract, preferentially at a site where absorption is greatest, before it can be absorbed and transported to the site of action.

Prodrugs present a different case in that they must first dissociate or convert to the parent drug, or to a more absorbable form of the drug, either in solution orin vivo before being absorbed from the gastrointestinal tract (5). Therefore, the dissolution rate of a drug, or rate in which the drug goes into solution, is an important parameter for determining how much drug is available for absorption

(5). If a drug is difficult to get into solution, then the amount of drug available for absorption may be small and thereby present difficulties in achieving the desired therapeutic effect (3).

Dissolution is the process by which drug molecules leave the surface of a solid and is the rate-limiting step for oral absorption of water-insoluble or relatively insoluble drugs. The rate of solution, or dissolution rate, is a kinetic parameter and is defined as “the amount of drug that goes into solution as a

function of time under standardized conditions (6).” In 1897, Noyes and Whitney proposed the following relationship to describe the dissolution rate, Dr, of a solute

(7):

Dr = DA(Cs-C) (1.1) hV

where D = diffusion coefficient,

A = surface area,

Cs = concentration at particle surface,

C = bulk concentration,

h = diffusion layer,

V = volume of dissolution medium.

When the solute is removed from the dissolution medium at a faster rate than it enters into solution then the concentration of drug in the dissolution medium, C, approaches zero and (Cs – C) can be approximated to Cs, which represents drug solubility (S). The resulting equation indicates a direct relationship between the dissolution rate and solubility as shown below:

Dr = DAS (1.2) hV

The dissolution rate of a compound is usually determined from the slope of a plot of concentration vs. time where the concentrations of drug in the

dissolution medium are plotted at given times (6). Concentration-time profiles provide direct information about how much drug has been released at a given time with respect to the total dose. In addition to providing the dissolution rate, these plots depict changes in the dissolution rate as functions of time and can be used to describe the mechanism by which a drug is dissolving, such as solubilization and/or wetting effects that could result from the method used to prepare the drug (8). For example, Farren (9) studied the dissolution profiles of lidocaine from compressed and molded tablets consisting of lidocaine and a polyethylene glycol. Percolation theory was applied to describe the mechanism by which lidocaine was released from these systems. In general, it was found that the release of lidocaine from molded tablets prepared from a fused mixture was slower than from compressed tablets (9). It was determined that the release of lidocaine from the molded tablets was not governed by percolation theory since randomly placed infinite clusters did not form throughout the tablet at any compositional ratio (9). Conversely, the release of lidocaine from the compressed tablets could be described by percolation theory since randomly placed infinite clusters of particles of lidocaine and polyethylene glycol formed above their respective percolation thresholds. As a result, lidocaine percolated throughout the tablet matrix. Dissolution of lidocaine was facilitated by rapid dissolution of the polyethylene glycol along its infinite clusters, thereby enhancing contact of the dissolution medium with the infinite clusters of particles of the relatively insoluble drug.

Bioavailability is a property that can be correlated with the dissolution rate of a drug (10-12). Bioavailability refers to the “rate and extent to which an active drug ingredient or therapeutic moiety is absorbed from a drug product and becomes available at the site of action (11).” As the dissolution rate of a compound increases, the amount of drug in solution increases thus increasing the amount of drug available for absorption, which may in turn lead to improved bioavailability. Variations in the bioavailability of a drug, however, can lead to inadequate and/or inconsistent therapeutic effects (11). For example, low bioavailability can result in low therapeutic plasma concentrations or in maintenance of therapeutic concentrations for only short periods of time (11).

Inherent physicochemical properties, such as solubility, are often responsible for the low bioavailability of a drug (12), e.g., a drug may have a low bioavailability because its rate of solution and/or solubility is low. As a result, many of these drugs, such as the antiepileptic drug phenytoin, are used in their salt forms, which are effective at increasing the dissolution rate of drug in gastric fluids and subsequently the bioavailability of a drug (13). Solubility related low bioavailability problems could occur for drugs with an aqueous solubility of less than 1 % w/v (10 mg mL-1) (14, 15). Since the intrinsic solubility of a compound cannot change, improvements in bioavailability must come through improving the rate of solution.

Equations 1.1 and 1.2 indicate that an increase in surface area, i.e., a decrease in particle size, would result in a higher dissolution rate. Small particles

have higher specific surface areas and higher surface curvatures compared with larger particles (4). As a result, the escaping tendency, or the ability of molecules to escape from the surface of small particles, tends to increase with decreasing particle size and is greater for smaller particles than larger particles

(4).

Techniques of particle size reduction, such as micronization, have achieved success in increasing dissolution rates (5). The importance of particle size reduction was first recognized in 1939 with phenothiazine (16) where reduction in particle size gave an increased toxicity to moth larvae. The particle size reduction of griseofulvin (from about 10 μm to 2.7 μm) is another example of the effect of particle size on dissolution rate and bioavailability of a drug (17).

Atkinson, et al. (17), showed an approximate doubling in the amount of griseofulvin absorbed in humans as a result of this reduction in particle size.

Duncan, et al., also demonstrated that half the dose of micronized griseofulvin could achieve similar blood levels as unmicronized griseofulvin (a dose of 0.25 g vs. 0.5 g of griseofulvin) (18). Other drugs whose bioavailabilities have been enhanced by particle size reduction include: sulfadiazine, tolbutamide, aspirin, and digoxin (19-21).

More recently supercritical fluid technology (see section 1.3) has been used as a method for producing small particles of drug to enhance dissolution rates. For example, using the method for producing particles from gas saturated

solutions (PGSS), the mean particle size of nifedipine was decreased from 50 μm to 15-30 μm, resulting in a dissolution rate 4 times greater than from the unprocessed nifedipine (22). Similarly, Ye (23) reported the first method for enhancing the dissolution rate of ropivacaine from small particles produced by the rapid expansion of supercritical solutions (RESS) method. Aggregates of small particles of ropivacaine tend to form due to electrostatic attraction thereby reducing their effective surface areas exposed to the dissolution medium and minimizing any increase in dissolution rate. Ye (23) however produced small particles with less tendency to aggregate, and those aggregates that did form were much smaller, i.e., ~1 μm vs. ~10 μm, causing little or no effect on dissolution rate.

In the case of hydrophobic drugs however, some techniques alone may not provide the necessary dissolution rate enhancement required to achieve the desired bioavailability (5). It is generally the case that, as drug particle size is reduced, dissolution rate increases as a result of an increase in the total surface area of a given amount of drug that is exposed to the dissolution medium.

However, this assumes that the surface of the drug can be easily wetted. If the drug particles are so hydrophobic that wetting is a problem, then the effective surface area exposed to the dissolution medium is essentially reduced, thereby affecting the dissolution rate of the drug. Felodipine is an example of a hydrophobic drug that exhibited no appreciable change in dissolution rate as a result of micronization (22). Supercritical fluid processing of felodipine reduced

the mean particle size from 60 μm to 42 μm and increased the specific surface area of the particles from 0.33 m2g-1 to 1.33 m2g-1. However, due to the poor wettability of felodipine particles, only 0.26 mg and 0.29 mg of unprocessed and micronized drug, respectively, dissolved after 1 hour (22). Similar results were seen by Ye with lidocaine (23). Dissolution rates of RESS processed lidocaine increased only ~7-9 % and were found to be insignificant compared to unprocessed lidocaine even though the particle size was reduced ~100 fold.

Therefore, it was concluded that poor wettability of the hydrophobic surfaces of lidocaine and the formation of lidocaine aggregates contributed to the lack of an enhanced dissolution rate.

An alternative to reducing particle size for improving dissolution rate would be to use a polymorph of the drug if such exists. As mentioned earlier, drugs that exist in more that one energy state or crystalline form are termed polymorphs (5).

The polymorphic form with the lowest energy is the most stable form, and has the highest melting point and a solubility equal to its intrinsic solubility in a particular solvent (usually water) under equilibrium conditions (24). Other polymorphic forms, however, are less stable, i.e., metastable, and have higher energy states that will eventually return to that of the stable form as a function of time depending on the energy differential. An example of a drug with polymorphic forms that have different rates of solution is phenylbutazone. Tuladhar, et al.

(25) prepared and characterized five different polymorphic forms of phenylbutazone and found each to have a different dissolution rate. Amorphous

forms of a drug can also have higher rates of solution than their corresponding crystalline forms (5). For example, Mullins and Macek (26) showed that the amorphous form of novobiocin has a higher dissolution rate than the crystalline form. However, the amorphous form of novobiocin in aqueous suspensions slowly converted to its more thermodynamically stable form with an accompanying loss of therapeutic effectiveness.

Salt forms of a drug are often used to increase dissolution rates (5). For compounds that are weak electrolytes, dissolution rate will be influenced by the solubility of the drug and the pH of the diffusion layer surrounding drug particles

(5). Consequently, certain conditions such as those created by salts can facilitate an enhanced rate of solution by creating an environment where dissolution of the drug is favored. For example, in gastric fluid, which has a pH generally of 1-3, the dissolution rate of a weak acid drug is relatively low. By changing a weak acid from a free acid to a strong alkali salt form, such as the sodium or potassium salt, the pH of the diffusion layer would increase to pH 5-6 compared to the bulk gastric fluid pH of 1-3 (5). As a result of the neutralizing action of the strong alkali cations, dissolution of the drug will occur at a faster rate than it would for the weak acid form in an acidic environment. Precipitation of the free acid will then occur when the ionized drug has diffused out of the diffusion layer into the lower pH bulk gastrointestinal fluid thereby creating a saturated solution of free acid in the gastrointestinal fluid (5). As a result of the saturated solution, the free acid will precipitate in the gastrointestinal fluid as fine particles.

These fine particles will have large specific surface areas, which will enhance the dissolution rate of the free acid, the preferentially absorbable form of the drug.

This method of enhancing the dissolution rate of a drug has been illustrated by

Nelson, et al. (27), who found that the sodium salt of tolbutamide produced a very rapid decrease in blood sugar level due to a rapid rate of absorption compared to the base form of tolbutamide. The latter had a slower rate of decrease of blood sugar levels resulting from a slower dissolution rate and hence slower absorption of the drug.

Another approach toward increasing the dissolution rate of a drug is to form a binary mixture consisting of two components, one of which could be a water-soluble carrier (28). Faster dissolution rates of relatively insoluble drugs can be achieved by formulating a drug with a water-soluble carrier where dissolution of the carrier exposes particles of the drug. As a result, the available surface area for dissolution is increased and enhanced dissolution of drug is facilitated (28). Naproxen is an example of a poorly water-soluble drug (0.025 mg mL-1) that exhibits low bioavailability after oral administration (29). Binary mixtures of naproxen and the water-soluble carrier chitosan were prepared by mixing, milling, kneading, and coevaporation and in each case found to enhance the dissolution efficiency (i.e., calculated from the area under the dissolution curve at time t using the trapezoid rule, expressed as the percentage of the area of the rectangle described at 100 % dissolution in the same time) of naproxen by

8 times, with milling giving the best dissolution profile (29). Improved wettability,

reduced aggregation, and increased effective surface area contributed to the enhanced drug dissolution. The mechanisms by which the dissolution rates of relatively insoluble drugs are enhanced through the use of binary mixtures are discussed in detail in section 1.2.4, which describes the dissolution of solid dispersions, which are examples of binary mixtures that can enhance the dissolution rate of a drug (29).

1.2 Solid dispersions

In 1961, Sekiguchi and Obi were the first to propose that solid dispersions could be used to increase rates of drug dissolution and absorption (30). A eutectic mixture of a relatively insoluble drug, sulfathiazole, and a water-soluble inert carrier, urea, was used as a model to demonstrate this concept. It was hypothesized that upon exposure to aqueous fluids the soluble component in the eutectic mixture would rapidly dissolve and the drug would be released into the fluid as fine, dispersed particles with a high surface area (30). Sekiguchi and Obi showed that simple mixtures of sulfathiazole and urea did not enhance the absorption of sulfathiazole, but that a eutectic mixture of the two resulted in blood levels of sulfathiazole reaching its maximum sooner, i.e., 3 hoursvs . 5 hours

(30). Based on this it can be assumed that the fastest rate of solution would occur if the drug were dispersed molecularly in the soluble carrier, i.e., as a solid solution (8).

1.2.1 Definitions

Chiou and Riegelman define a solid dispersion as “one or more active ingredients in an inert carrier or matrix at solid state prepared by melting (fusion), solvent, or melting-solvent method (28).” However, a dispersion of a drug in a solid carrier prepared by mechanical mixing is not considered to be a solid dispersion. Solid dispersions can therefore be categorized by the method of preparation or by the physical state of the dispersed phase in the dispersion.

Listed below are types of solid dispersions (28):

1. Simple eutectic mixtures

2. Solid solutions

3. Glasses and glass suspensions

4. Amorphous precipitations in a crystalline carrier

5. Compound or complex formations

6. Combinations of any of the above

A simple eutectic mixture is achieved when a binary mixture is cooled below its eutectic temperature, i.e., the lowest temperature at which the mixture exists as a liquid (8). Both components must be completely miscible in the liquid state, but have limited solubility in the solid state. According to the United States

Pharmacopeia (1), the term miscible refers to “a substance that yields a homogeneous mixture when mixed in any proportion with the designated

solvent.” Solid eutectic mixtures are prepared by sufficient rapid cooling of a co- melt to ensure a physical, or complete, mixture of the two components as they simultaneously crystallize. Examples of simple eutectic mixtures include paracetamol-urea (31) and griseofulvin-succinic acid (32). Griseofulvin was determined to have limited solubility in the solid state, i.e., <1% in succinic acid

(32). It is therefore considered to be a simple eutectic mixture rather than a true solid solution since griseofulvin is not completely soluble in succinic acid in the solid state. Likewise, griseofulvin or tolbutamide in PEG 2000 are monotectic systems, which are defined as having a eutectic point that appears to converge with the melting point of the pure material, and therefore displays no solid solubility (33).

A solid solution refers to a one-phase system in which a solid solute is molecularly dispersed in a solid solvent (28). Solid solutions are comparable to liquid solutions in that they consist of one phase regardless of the number of components involved. In a solid solution, the particle size of the drug is at its minimum, i.e., a molecule, which is desirable when considering the dissolution rates and bioavailability of relatively insoluble compounds. Relatively insoluble drugs that are molecularly dispersed in a water-soluble component would therefore have the greatest effective surface area possible upon dissolution of the water-soluble component. This would optimize dissolution rate and, it can be assumed, the bioavailability of a drug. Kanig (34) explored the use of fused mannitol as a solvent in which solid materials can be dispersed, i.e., a

dispersant, to produce solid solutions. Using mannitol and phenobarbitol or sulfanilamide, Kanig suggested that homogeneous solutions could be formed in which one solid dissolves in another to form a solid solution (34). For all two- component systems, some solid-state solubility can be predicted (35). The degree of solubility however is usually small enough to be considered negligible.

Goldberg, et al. (36), in 1965 suggested, “For practical purposes, solubility of greater than 5 % of one component in another can be considered to be a solid solution.” However, with enhanced sensitivity of instruments, such as differential scanning calorimetry and x-ray diffraction, solid solutions can now be detected below the 5 % level (28). It is likely that many solid dispersions consist of regions of molecularly dispersed drug, i.e., partial solid solutions, which would also contribute to an increased dissolution rate (37). Although formation of solid solutions has been reported, i.e., Goldberg, Gibaldi, and Kanig (36, 37), there is a lack of agreement as to their exact compositions and the degrees of solid solution formation. For example, in 1965 and 1966, Goldberg, et al. (36, 37), reported large portions of solid solutions of sulphathiazole or chloramphenicol in urea. It was later found by Chiou, et al. (1971), that actually only 15 % sulfathiazole (38) and 2.5 % chloramphenicol were soluble in urea (39).

Solid solutions can be classified according to their miscibility (solubility) as continuous or discontinuous, or by the way in which the solute molecules are arranged in the solid solvent as substitutional, interstitial, or amorphous (8).

Continuous solid solutions require the components be miscible in all proportions.

Therefore, the bond strength between the two components in a binary mixture would be greater than that of the bond strength between the individual components (8). In discontinuous solid solutions the solubility of one component in the other is limited, but greater solubility can occur at extreme concentrations

(see Figure 1.1). Goldberg, et al. (36) adapted Figure 1.1 from Daniels, et al.

(40) to describe solid solutions because of its similarity to the phase diagram of a eutectic mixture. The regions α and β in Figure 1.1 represent concentrations in which solid B dissolves in solid A to form the solid solution α and A dissolves in B to form the solid solution β. All other areas of the phase diagram represent concentrations where partial miscibility exists and the solid solution is discontinuous, as shown by the hash marks. In the region labeled α + β, two solid solutions of α and β coexist in equilibrium. At the eutectic composition E, solid solutions of α and β simultaneously precipitate at a fixed ratio. In regions shown by α + liquid solution and β + liquid solution, there exists a liquid melt of the two components, as well as a certain degree of solid solubility whose compositions are given by the liquidus and solidus curves. For example, if a system is cooled to a point a (see Figure 1.1), then the compositions b and c are in equilibrium and represent the composition containing solid and liquid solutions, respectively. Lastly, the solvus curves define the limit of solid solubility under equilibrium conditions.

Substitutional solid solutions exist when the solute molecule can substitute for solvent molecules in a crystal lattice (28). This is possible when the

Temperature

liquidus Temperature Temperature

solidus b a c solidus

solvus

Figure 1.1 Hypothetical phase diagram for discontinuous solid solutions. The hatched region showing where solid solutions are discontinuous. (modified from Ref. 36).

molecules differ in size by less than 15 %, according to the Hume-Rothery rule

(41, 42). This value was determined based on the solubility comparison of copper atoms in a nickel crystal lattice. The atoms in a copper crystal have an apparent diameter of 2.551 Å, which is approximately two percent larger than nickel (2.487 Å). This small difference in size presents only a small distortion of the crystal lattice as the copper atom enters a nickel crystal lattice, and is a common example used to describe continuous solid solubility. In interstitial solid solutions, the solute molecules are small enough to occupy the interstitial spaces between the solvent molecules in the crystal lattice. In this case the solute molecules should have a molecular diameter no greater than 0.59 of that of the solvent molecule and a molecular volume of less than 20 % of the solvent (28,

41). These values were determined based on the apparent diameters of carbon and iron atoms. Although the original criteria reported for the formation of solid solutions are based on the sizes of atoms in alloy systems, the same principles and criteria are applied to more complex systems such as those presented herein where molecules of one drug may exhibit solid solubility in another substance. Therefore, the size of the solute molecule is critical in order for it to fit into the interstitial spaces of the solvent molecules and thereby determine the type of solid solution that forms. Solute and solvent molecules that do not fit these size constraints are not likely to form solid solutions. In addition to substitutional and interstitial solid solutions, amorphous solid solutions may form

in which the solute molecules are dispersed molecularly, but randomly within the amorphous solvent, as opposed to an interstitial solid solution where the molecules have a more structured arrangement (8).

A glass solution exists as a homogeneous solid system in which a solute is dissolved in a glassy solvent (28). Polymers, such as polyethylene glycols, are examples of glassy solvents in that their chemical bonds differ significantly in length and no one temperature exists at which all of the bonds weaken simultaneously (43). As a result, a glassy solvent is also amorphous and is characterized by a change in heat capacity, during which the structure of the glassy material relaxes to a lower energy state as it is heated beyond its glass transition temperature, Tg. Below the glass transition temperature, materials become rigid and brittle, while above it they are elastic. In a glass solution consisting of a relatively insoluble drug and a glass-forming carrier, such as polyvinylpyrrolidone or citric acid, dissolution of the drug would be expected to occur rapidly as the carrier quickly dissolves. This is because the lattice energy of the glassy solvent is less than that of a crystalline solid solvent in a solid solution, and therefore in general the dissolution rate of a drug from an amorphous or glass solution should theoretically be greater than from a solid solution (28).

In simple eutectic mixtures, the drug and the carrier simultaneously crystallize from a melt or solution where they show complete liquid miscibility and

negligible solid-solid solubility (44). However, sometimes the drug may precipitate as an amorphous form in the crystalline carrier forming anamorphous precipitate in a crystalline carrier (28). An amorphous drug is a higher energy form since it has a less stable structure and is more prone to losing a molecule, compared to a crystalline drug which is in a lower energy state since most of the bond energies in its structure are satisfied. Consequently, an amorphous drug in a crystalline carrier would be expected to have a faster dissolution rate than a crystalline drug since their structural energies allow them to readily enter into solution. For example, the amorphous nature of sulfathiazole dispersed in crystalline urea is believed to be the contributing factor for increasing its oral absorption (30). Kuhnert-Brandstatter (45) showed in a survey that 67 % of steroids, 43 % of sulfonamides, and 63 % of barbiturates exhibit polymorphism.

Complex formation between the drug and a carrier can occur during the preparation of solid dispersions (28). As a result, drug dissolution rates can increase or decrease depending on the relative solubility of the complex and the ability of the water-soluble carrier to facilitate dissolution (28). The formation of a poorly soluble complex could decrease the dissolution rate of a drug by preventing the dissolution of the water-soluble component, which draws the dissolution medium to drug surfaces as it dissolves. Alternatively, a more water- soluble complex would be expected to dissolve more rapidly and have enhanced rates of solution. For example, the formation of an insoluble complex between

phenobarbital and propylene glycol 4000 or 6000 has been shown to reduce the dissolution rate of phenobarbital and therefore its subsequent permeation through the gastrointestinal membrane of rats (46).

Lastly, there is always the possibility that the solid dispersion contains more than one of the classes of dispersions discussed above (28).

Consequently, several mechanisms may contribute to enhanced dissolution rates, such as in the formation of a complex between the drug and carrier in which the drug is in its amorphous form.

1.2.2 Methods of preparation

Solid dispersions can be conventionally prepared using one of three methods: melting (fusion), solvent, or melting-solvent (28). In themelting method a physical mixture of the drug and carrier are directly heated until melted. The melt is then cooled and solidified rapidly using a suitable method such as in an ice bath or snap-cooling on stainless steel plates. Consequently, the degree of solid dispersion formed, or amount of drug completely dispersed in the carrier, depends on the degree of supersaturation and rate of cooling (47). The final solid is then crushed, pulverized, and sieved to the desired particle size. The advantages to using this method are that it is relatively simple and inexpensive.

However, in order to prepare solid dispersions by this method, the components must be completely miscible in the molten state. It may also be difficult to

completely solidify the mixture because the solidification temperature will affect the crystallization rate, crystal size, and hardness of the dispersion, which could lead to the formation of an unmanageable solid dispersion that could be tacky, hard, or too soft (47). Situations like these would require alternative methods to formulate the drug into a suitable dosage form which may require the use of organic solvents. In addition, many drugs and carriers can evaporate or decompose at the high temperatures required by this method. Evaporation can be avoided if the mixture is heated in a sealed container. However, care must be taken to avoid a dangerous pressurization of the container.

In the solvent method, a physical mixture of the components is dissolved in a common solvent, followed by evaporation of the solvent (28). Therefore, in many cases, temperatures near the boiling point of the solvent are necessary to evaporate the solvent. The terms coprecipitate and coevaporate should not be confused when considering solid dispersions prepared by the solvent method

(47). Coprecipitates are formed when a drug and polymer are dissolved in a solvent and precipitated by the addition of a nonsolvent (48). Therefore, the correct term to describe dispersions prepared by the solvent method is coevaporate. The disadvantages of this method are in finding a suitable common solvent; the costs of the solvents, which are often required in large quantities; and the difficulty of completely removing the solvent. For example, in order to prepare 5 grams of a 10 % griseofulvin dispersion in polyethylene glycol

6000, 500 ml of ethanol are required (49). Certain solvents can also plasticize

polymeric carriers such as polyvinylpyrrolidone (47). Also, the rate of solvent removal can affect the particle size and hence physicochemical properties, such as the melting points and crystal structure, of the solid dispersion. For example, rapid solvent removal would result in short crystal growth times with many nuclei forming in solution from which drug crystals will grow. The greater the number of nuclei, the smaller will be the particle size (16). Therefore, in some cases where an enhanced dissolution rate is needed, methods of preparation where the solvent is rapidly removed are favored because many small particles form that may readily dissolve upon coming in contact with the dissolution medium due to their large effective surface areas.

The melting-solvent method is a combination of the first two methods.

The solid dispersion is prepared by first dissolving the drug in a suitable solvent and then incorporating the solution into the melt of the carrier (28). This method avoids applying high temperatures directly to the drug as long as the melting point of the carrier is low. Many of the same disadvantages listed above also apply to this method, such as the use of high temperatures to melt the carrier, which could decompose the drug upon adding the drug solution, and the need to obtain adequate solvent removal (28).

1.2.3 Characterization of solid dispersions

Among the various methods available for the characterization of solid dispersions, the most commonly used are thermal analyses, such as differential scanning calorimetry or differential thermal analysis; x-ray diffraction; infrared spectroscopy; and dissolution studies (8). Dissolution studies often provide quantitative information about solid dispersions, like dissolution rates and the amount of drug released at a given time, and will be discussed in section 1.2.4.

Differential scanning calorimetry (DSC) records events in the form of a thermogram, which indicates the absorption or evolution of energy as endotherms or exotherms, respectively, as functions of temperature or time (50).

Generally, samples are placed in aluminum pans and heat is applied at pre- determined rates over a defined temperature range according to an analysis protocol. Simultaneously, a reference pan, or empty aluminum pan is heated in the same oven. During an endothermic event heat is absorbed by the system and the differential heat change is recorded as an endotherm. Conversely, during an exothermic event heat is released and the difference in temperature between the sample and reference is recorded as an exotherm on a DSC thermogram. The enthalpy of fusion, or the energy required to break a crystal lattice structure during melting, is calculated by the DSC from the area of the endotherm. The enthalpy of fusion can provide information about the presence of an interaction between components and/or quantitative changes such as the degree of crystallinity in a substance (50). The melting points and enthalpies

provided by DSC for multicomponent systems can also be plotted as phase diagrams from which eutectic systems and solid solutions can be identified. For eutectic systems, a single endotherm will appear in the thermogram at the lowest temperature at which the system can exist in the liquid phase (50). DSC can also be used to determine whether a drug is in an amorphous form since it would lack a sharp endotherm and instead generates a broad endotherm due to insufficient crystal energies necessary to produce a strong thermal event.

Other methods of thermal analysis used to characterize solid dispersions include hot stage microscopy and differential thermal analysis (DTA). In hot stage microscopy, the sample is studied by brightfield or more commonly polarized light microscopy using a hot stage, where melting points and other thermal events can be determined visually. Differential thermal analysis, like

DSC, also provides information about the thermal events of a system (50).

Similar to DSC, DTA measures the melting point of a substance over a given temperature range. DTA however is more sensitive to changes in melting points and is very likely to record onsets of melting, or early melting of less stable structures, as multiple sharp peaks. Unlike DSC, DTA provides qualitative data and does not generally provide information about the energies involved, such as the enthalpy of fusion. Nevertheless, DTA provides general information about the melting of a substance and phase transitions that can be useful in the characterization of solid dispersions (50).

X-ray diffraction is also used to characterize solid dispersions (8). In this method an x-ray beam, or incidence beam, is scattered from the layer of atoms at the surface of the sample. According to Bragg’s Law, the intensity and angle at which x-rays are diffracted varies depending on the wavelength used and the structure of the sample (51, 52). The diffraction pattern created by the reflection of x-rays gives information about the spacing and crystal structure within a sample. Therefore, substances with different crystalline structures will have different diffraction patterns. Substances that lack crystal structure, such as amorphous substances, will not produce an x-ray diffraction pattern and therefore cannot be identified using this method. X-ray diffraction also cannot distinguish between solid solutions and amorphous precipitates because both lack the diffraction patterns of the crystalline drug.

Based on the molecular structure and functional groups involved, compounds have a characteristic infrared spectrum from which they can be identified. Infrared spectroscopy can detect the energy differences that exist between various vibrational and rotational states of functional groups such as O-

H, N-H, C=O, and CH3. Through the use of Fourier transform infrared spectroscopy, the quality of the spectra is enhanced due to a high signal-to-noise ratio and a high wavelength accuracy and precision, which can be used to detect weak bonding (53). In addition, the spectra of particular compounds can be compared to other spectra to determine changes in structural bonding since the possibility that two compounds will have the same spectra is exceedingly small.

These changes appear as peak shifts, new peaks, or as the disappearance of peaks representing functional groups that are sensitive to changes in crystallinity

(8). As a result, complex formation between a drug and carrier can be detected

(8).

1.2.4 Dissolution of drug from solid dispersions

The purpose for using solid dispersions as drug delivery systems is to enhance the dissolution rates of relatively insoluble drugs and thereby improve their bioavailability. This can be achieved through several mechanisms, namely increasing the specific surface area by reducing the particle size of the drug, improving the wettability of the solid particles by the dissolution medium, increasing the energy state of the drug, and/or by increasing the apparent solubility of the drug.

The formation of solid dispersions often results in a reduction in the particle size of a drug within a solid matrix which itself may be in the form of small particles. The formation of large drug crystals may be inhibited by the presence of a carrier in a binary mixture as it is precipitated resulting in small crystals of drug dispersed within the carrier. As a result, the drug particles will have high specific surface areas and therefore potentially enhanced dissolution rates.

Furthermore, the inclusion of water-soluble carriers in the formation of solid dispersions can also serve as an additional factor contributing to the dissolution

rate of a drug. As the water-soluble carrier dissolves, dissolution medium is drawn to the surfaces of the drug particles while simultaneously exposing greater surface areas of drug (10). The presence of the water-soluble carrier can therefore promote the dissolution of drug from finite clusters of drug particles by creating an environment where the drug is readily exposed to the dissolution medium (10). In the case where infinite clusters of drug exist, dissolution of drug is described by percolation theory where dissolution of the water-soluble carrier will expose even greater drug surface areas while also creating channels for the dissolution medium to enter and further dissolve the drug (10).

Hydrophobic small particles can create problems due to poor wettability

(28). Wetting of the drug is an important step toward dissolution and can be impaired by the hydrophobicity of the drug. If a drug is so hydrophobic that wetting is a problem then the dissolution medium cannot physically interact with the drug particles, resulting in an apparent reduction in its effective surface area.

Solid dispersions can provide a means for improving drug wettability (28).

Surface active agents such as Tweens® and poloxamers can act in a dual role in solid dispersions: as water-soluble carriers and as wetting agents to lower the solid-liquid interfacial tension (54). They reduce the solid/liquid contact angle and improve wettability by displacing air (54). Contact angle refers to the angle obtained in the liquid when a drop of the liquid comes into contact with a solid

(55). Typically, contact angles of less than 90° indicate wetting (56). As a result of their ability to improve wetting, surfactants have been shown to enhance the

dissolution rates of drugs. For example, Solvang and Finholt (57) showed that the addition of the nonionic surfactant polysorbate 80 sprayed onto the surface of phenacetin particles increased the dissolution rate of the drug by more than 3- fold.

Ford (47) suggests that the major controlling factors in the release of drug from a solid dispersion is the ratio of drug-to-carrier and their physical structures within the dispersion. In this context, an increased dissolution rate of drug from drug/water-soluble carrier solid dispersions can be attributed to reduction of drug particle size within the water-soluble matrix as the solid dispersion is formed (47).

Dispersions such as solid solutions and glass solutions would therefore be expected to have the fastest dissolution rates since the particle size of the drug is reduced to its minimum, i.e., to the molecular level. During dissolution, the water-soluble carrier dissolves leaving the drug molecularly dispersed in solution.

This occurs because the drug has no crystal structure in the solid solution, i.e., no drug-drug bonds, and therefore the energy required to dissociate a crystalline structure before the drug can dissolve is not necessary (8). However, once the drug is in solution, precipitation can occur due to supersaturation. In some cases, drug precipitation can be prevented or inhibited by the carrier (28). If a drug does precipitate, it could precipitate as a metastable polymorph with a higher rate of solution compared to its parent form as previously mentioned in section 1.2.1.

1.3 Supercritical fluid technology

1.3.1 Background

In 1822, Baron Charles Cagniard de la Tour was the first to show that there is a critical temperature above which a substance can exist only as a fluid and not as either a liquid or a gas (58). He performed experiments using substances in both the liquid and vapor states and heated them in a sealed cannon, which was rocked back and forth. He found that at a certain temperature, called the critical temperature, splashing stopped and that the substance did not condense or evaporate. In the following years the critical points of various substances (e.g., carbon dioxide and water) were characterized and their behavior in this region was studied. An important property also studied was “critical opalescence,” light scattering due to density fluctuations (59).

Critical opalescence occurs in the region near the critical point and has since been used as a method to determine the critical values of supercritical fluids.

The term supercritical fluid refers to a substance that exists above its critical pressure and temperature (60). A phase diagram showing the region in which a supercritical fluid occurs is given in Figure 1.2 (61). Supercritical fluids have properties of both gases and liquids, which can be controlled by regulating pressure and temperature (61). They have densities comparable to liquids, which can be altered, e.g., increasing pressure increases the density of the fluid

Melting Line Supercritical Fluid

Solid

Boiling Line CP Pressure, bar bar Pressure,

Liquid TP Pc = 73.8 bar

Gas

Temperature, °C Sublimation Tc = 31.06 °C Line

Figure 1.2 Phase diagram of carbon dioxide. CP = critical point, TP = triple point, Pc = critical pressure, Tc = critical temperature (modified from Ref 61).

and allows it to mimic the solvating power of various liquid solvents (62). For example, carbon dioxide can be forced to assume solvent properties equivalent to conventional solvents such as benzene, chloroform, acetone and toluene, which are not generally useful in the pharmaceutical industry due to their toxicities (62). Unlike conventional solvents, most supercritical fluids, such as carbon dioxide and water, do not have additional problems related to emissions and disposal. The disposal of solvents such as benzene and methanol can be toxic and costly, whereas carbon dioxide and water are natural to the environment. In some cases, depending on the device used, the supercritical fluid can be recycled for future uses (61).

In addition to behaving like liquids, supercritical fluids are unique in that they also appear to behave as gases, which are compressible and can fill a container (62). The diffusivity and viscosity of a supercritical fluid are also similar to those of gases (62). As a result, supercritical fluid extractions can occur at much faster rates than with conventional solvents (62). Supercritical fluids have solute diffusivities between 10-4 cm2 s-1 and 10-13 cm2 s-1 compared to

10-5 cm2 s-1 for conventional solvents and viscosities near 10-4 N s m-2 compared to 10-3 N s m-2 for conventional solvents (62). For example, benzoic acid had values of diffusivity of 6 x 109 m2 s-1 in supercritical carbon dioxide compared to

1.8 x 109 m2 s-1 in methanol. The viscosity of supercritical carbon dioxide at

200 atm and 55 °C is 1.00 x 107 m2 s-1 compared to 6.91 x 107 m2 s-1 for methanol. This means that supercritical fluids tend to have higher rates of mass

transfer than conventional solvents. The low viscosities also allow for better flow properties, in addition to a low, or essentially zero, value of surface tension, which in turn allows the supercritical fluid to penetrate porous solid matrices during extraction (62). These properties also facilitate the use of supercritical fluids for chromatographic purposes (63). The gas and liquid-like properties of a supercritical fluid as the mobile phase makes analyte separation, for example of alkane mixtures, more selective due to the stronger interactions between the mobile and stationary phases.

Although many solvents can exist in the supercritical state, carbon dioxide is the most commonly used because of its relatively low critical temperature and pressure. Other solvents, such as ammonia, argon, and water are limited in their use due to their high critical values. High temperatures, for example, cannot be used to process thermally labile substances. Table 1.1 lists the critical values of some supercritical fluids. Several supercritical fluids, such as carbon dioxide, are generally considered to be inert at reduced temperatures and pressures and somewhat environmentally friendly (62). In addition, carbon dioxide is widely available, inexpensive, and has low toxicity and reactivity. In its supercritical state, it is primarily used for the extraction of nonpolar and slightly polar substances from organic matrices (62). Unfortunately, the number of pharmaceutical compounds that can be processed with carbon dioxide is limited due to their polarity. Attempts to process the more polar compounds led to the use of modifiers added to the supercritical fluid. Modifiers, also known as co-

Substance Critical Temperature Critical Pressure (°C) (atm)

CO2 31.1 73.8 N2O 36.6 72.4 NH3 132.5 113.5 Xe 16.7 58.4 Ar 150.9 48.0 H2O 374.1 221.2 CCl2F2 96.3 49.7 C2H6 32.4 48.8 CH3OH 240.1 80.9

Table 1.1 Critical points of various gases and liquids (from Ref. 62).

solvents, are intended to increase the polarity of supercritical carbon dioxide, which would in turn enhance the solubility of both polar and nonpolar compounds in the supercritical fluid (62). Methanol, acetone, and ethanol are the most commonly used modifiers and are typically added to the supercritical fluid in concentrations of 10 % v/v or less (64-66). The potential for interactions to occur between the modifier and the solute must be considered when using modifiers.

For example, methanol can act as either a Lewis acid or Lewis base and interact with functional groups on the solute (62). Acid-base interactions should also be considered in systems where carbon dioxide can combine with water to form carbonic acid and lower pH (67).

The basic configuration of a supercritical fluid extraction system is depicted in Figure 1.3. The syringe pump is initially filled with the gas of choice after the pump has been cooled by a water jacket that encases the pump while refrigerated water is circulated through it. For carbon dioxide, the pump is cooled to -5 °C in order to achieve a liquid state to allow more quantitative filling of the pump. As the pump is heated and/or the volume in the syringe pump reduced, the pressure in the syringe increases. Although not necessary, the pump temperature may also be increased to render the gas in its supercritical state.

Otherwise, a supercritical state will be achieved as the gas passes through a heated coil upon being pumped into the extraction cell, or sample cartridge, of known volume containing a known amount of the sample. Surrounding the cell is

Check Valve Sample Cartridge

Restrictor

Extraction Unit CO2 Cylinder Syringe Pump Collection Chamber

Figure 1.3 Schematic diagram of a supercritical fluid extraction system.

a heated oven that maintains supercritical conditions. Exiting the extraction unit is a heated restrictor, or capillary nozzle, which is inserted into a collection chamber. This chamber is covered to prevent particles from escaping during the expansion process, which occurs as the supercritical solution exits the capillary restrictor and returns to atmospheric pressure, while enabling the carbon dioxide to be released into the atmosphere.

1.3.2 Applications of supercritical fluid technology

The first industrial uses of supercritical fluids can be traced back to the

1950s. Researchers at the Max Planck Institute for Kohlenforschung studied the applications of supercritical fluids in the food, petroleum, and chemical industries.

An example of this work was the license for the supercritical carbon dioxide process for the decaffeination of coffee issued to the HAG AG Corporation in

1976 (62). This led to the construction of facilities for the extraction of active components from hops and for the decaffeination of tea. The first use of this process in the United States was in 1988 when Maxwell House opened its coffee decaffeination plant (62). Other industries later began to develop new applications for the use of supercritical fluids, such as in waste refineries of the petroleum industry (62). The nuclear industry has also applied supercritical fluid technology for the recovery of waste products (68). Some industrial uses of supercritical fluids are given in Table 1.2.

Year Operator Materials Processed

1978 HAG AG Co. Caffeine from coffee

1982 SKW/Trotsberg Hops Fuji Flavor Tobacco

1984 Barth and Co. Hops Natural Care Byproducts Hops and red peppers

1986 CEA Aromas and pharmaceuticals

1988 Nippon Tobacco Takeda Acetone residue from antibiotics CAL-Pfizer Aromas

1989 Clean Harbor Waste waters Ensco, Inc. Solid wastes

1990 Jacobs Suchard Coffee Raps and Co. Spices Pitt-Des Moines Hops

1991 Texaco Refinery wastes

1993 Agrisana Pharmaceuticals from botanicals Bioland Bone US Air Force Aircraft gyroscopic components

1994 AT&T Fiber optic rods

1996 Nutrasweet Co. NuEgg, fat and cholesterol from eggs

1998 Union Carbide Device for automobile painting

Table 1.2 Examples of industrial uses of supercritical fluids (from Ref. 62).

Other areas where supercritical fluid technology has been used include reaction and separation processes and the pharmaceutical industry.

Supercritical carbon dioxide has been used as a nonaqueous solvent medium for enzymatic reactions to stabilize enzymes against conformational changes that may lead to deactivation (69). The kinetic resolution of enantiomers by lipase- catalyzed reactions in supercritical carbon dioxide is another example of the use of supercritical fluid technology with potential commercial application (69).

Supercritical fluids have also been used as alternative solvents in certain liquid chromatography methods as a result of their ability to decrease analysis time, increase sample throughput, and decrease liquid waste (60). In 1983, Hewlett-

Packard offered the first commercial supercritical fluid chromatography instrumentation and a number of applications were developed with it for the separation of ubiquinone from bacterial cell extracts (70), polycyclic aromatic hydrocarbons (71), and oligomers (72). Applications using supercritical chromatography linked to mass spectrometry have also been studied (73).

Coupling supercritical fluid chromatography to FTIR is also attractive because it can provide qualitative information about the analytes (73). One of the advantages of using supercritical fluid chromatography was that it could use both gas- and liquid chromatography detectors. For example, since carbon dioxide is in the mobile phase, a universal flame ionization detector can be useful in certain applications. UV and diode array detectors were also used in early applications of supercritical fluid chromatography (59).

The use of supercritical fluids to produce particles of materials used in the pharmaceutical, nutraceutical, cosmetic, and chemical industries has been reviewed by Perrut (74). Using methods such as the rapid expansion of supercritical solutions and the gas anti-solvent method (see section 1.3.3), supercritical fluids have primarily been used to reduce and control particle size.

As a result of the research being done in this area, various patents are on record showing the use of supercritical fluids for pharmaceutical purposes. For example, a hand held device has been patented for the delivery of fine aerosol particles through oral or nasal passages to the lungs (74). Using this device, a supercritical solution containing vitamin E was expanded to atmospheric pressure using the RESS method and mixed with a large quantity of oxygen to form an aerosol. Vitamin E in the form of micron-sized droplets was then directly administered to the lungs of rats (58). Another patent where supercritical fluids were applied for pharmaceutical purposes includes the formation of polymeric microcapsules. Mishima (75) claimed the formation of microcapsules from a suspension of an active drug in a supercritical solution containing the polymer using the RESS method. Microcapsules produced by this method include 3- hydroxyflavone with Eudragit® E100 or PEG 6000. Supercritical fluids have also been used to form suspensions of sub-micron particles of water-insoluble drugs.

In a patent submitted by Godinas, et al. (76), RESS processing lead to the suspension of fenofibrate with a mean particle size of 200 nm in Lipoid 80® and

Tween 80® in water. Additional applications of the use of supercritical fluid technology in drug delivery are discussed in section 1.3.4.

1.3.3 Supercritical fluid processing methods

1.3.3.1 Rapid expansion of supercritical solutions (RESS)

RESS was first described by Hannay and Hogarth (77). They observed,

“When the solid is precipitated by suddenly reducing pressure, it is crystalline, and may be brought down as a ‘snow’ in the gas, or on the glass as a ‘frost’…”

(77). In 1981, however it was Krukonis (78) who recognized that this phenomenon could be used as “a way of tailoring the size and size distribution of difficult-to-comminute organic materials.” The RESS concept is relatively simple and consists of saturating a supercritical fluid, typically supercritical carbon dioxide (SCCO2), with one or more substances and depressurizing the solution through a heated capillary nozzle in order to precipitate small particles of the substance or substances. No organic solvents are included in this process and the operating conditions are typically mild due to the low critical values of CO2, i.e., Tc=31.1 °C, Pc=73.8 bar (69). The complete removal of SCCO2 and rapid and homogenous nucleation of the substance(s) occurs at supersonic velocities

(10-8- 10-5s) resulting in the formation of small particles, fibers, or films depending on the material (79). This process therefore is superior to traditional crystallization methods because the crystal growth time is drastically reduced

(69). Traditional crystallization methods can take minutes to hours for solvent removal to occur. Particle size distributions created by methods such as the solvent method or by spray drying are often large due to unavoidable Oswald

ripening as larger particles grow at the expense of smaller particles (16).

However, during RESS, the solvent, i.e., SCCO2, is rapidly removed thereby eliminating the effects of Oswald ripening during crystallization. RESS also allows for complete removal of the solvent (79). As the system is depressurized, the carbon dioxide is released into the atmosphere or into a recycling system by its instantaneous conversion to a gas. Other attractive features of RESS are the ability to recompress and reuse the supercritical fluid (79), adjust the solvent strength of the supercritical fluid so that it can function in the same manner as other organic solvents, and manipulate particle morphology by varying the diameter of the restrictor (capillary) nozzle or by varying the temperature of the nozzle, both of which can alter the rate of flow (80). However, there are limitations to the RESS method due to the types of drugs that cannot be dissolved in SCCO2, such as polar substances. Some of these problems can be circumvented through the use of a modifier or co-solvent (62). As mentioned earlier, methanol and ethanol are typically used to increase the polarity of SCCO2 and thereby enhance the solubility of more polar compounds in SCCO2. Co- solvents, which are filled into a pump separate from the carbon dioxide, are mixed with the SCCO2 prior to entering the sample cartridge.

1.3.3.2 Gas anti-solvent method (GAS)

Unlike the RESS method, in the gas anti-solvent method, the supercritical fluid is used as an anti-solvent to cause the substance dissolved in a liquid

solvent to precipitate (81). Typically substances that cannot be processed with

SCCO2 alone or that are sensitive to mechanical handling, such as peptides and proteins, are processed by this method (81). The substance or drug of interest is first dissolved in an organic solvent and then brought into contact with a supercritical fluid, typically SCCO2. Upon such contact, there is a sudden reduction in solvent density that produces a decrease in solvent strength and in turn a reduction in the solubility of the drug in the organic solvent (69). As the organic solvent partitions into the supercritical fluid, drug concentration increases leading to supersaturation followed by the precipitation of particles (78). Unlike

RESS, trace amounts of organic solvent can remain in the particles (69).

Therefore, it may be difficult to find an appropriate organic solvent that can be removed sufficiently from the drug particles. Furthermore, because GAS processing requires that the drug initially be dissolved in an organic solvent this method is no longer environmentally “green” or “clean”, which is unique to supercritical fluid processing with SCCO2, specifically as in the RESS process.

Materials processed by GAS must be tested to ensure that residual solvent levels are at or below acceptable levels prescribed by regulatory agencies (81).

1.3.3.3 Other methods utilizing supercritical fluids

RESS and GAS are the primary methods designed to process pharmaceuticals via supercritical fluid technologies. Other methods have been developed which use the same principles, but are designed differently. For

example, the SAS method, or supercritical anti-solvent method, involves spraying an organic solution containing the drug through a nozzle as fine droplets into supercritical carbon dioxide (81). This method has also been termed the aerosol spray extraction system (ASES) (69). The PGSS method, or particles from gas- saturated solutions, differs from the RESS method in that the supercritical fluid is dissolved in a molten drug or mixture of a drug and a carrier without the need to incorporate organic solvents (74, 23). As in the RESS method, the gas-saturated solution is allowed to expand and particles precipitate. Hanna and York (82) have developed a technique called the SEDS method, which is claimed to have better control of particle size distribution. Solution enhanced dispersion by supercritical fluids (SEDS), forms particles as a supercritical solution containing the solute exits a nozzle. A solution containing the drug and carrier enters a coaxial nozzle and is mixed with the antisolvent carbon dioxide under supercritical conditions. The supercritical carbon dioxide extracts the solvent from the solution causing the concentration of drug and carrier in the droplets to increase and eventually precipitate into a pressurized vessel (83). Particle size can be controlled by the rate of flow of the solution containing the solute and the supercritical fluid.

1.3.4 Applications of supercritical fluid technology in drug delivery

The initial applications of supercritical fluid processing in pharmaceuticals was in the micronization of compounds such as lecithin (78), progesterone (84),

and nifedipine (85) to produce small particles with uniform size distributions.

Using the RESS method, Krukonis reported a lecithin particle size of 1 μm and a progesterone particle size of 2-5 μm (78, 84). Nifedipine particle size was approximately 1-3 μm (85). Furthermore, Debenedetti (86) has developed applications for supercritical fluids, such as in the formation of microparticulate protein powders; the processing of polymers, e.g., poly (L-lactic acid), poly (D, L- lactic acid), and poly (glycolic acid) using the RESS method; and in the formation of microspheres of polymers containing the anti-cholesterol drug lovastatin (81).

The objective of these studies was the formation of small particles of potent peptides and proteins that could be incorporated into injectable drug loaded microspheres without the use of harsh processing conditions that can produce low yields (i.e., spray-drying), broad particle size distributions (i.e., lyophilization), and denaturation (i.e., milling and precipitation with organic solvents) (86). Using the GAS method, Debenedetti produced two types of insulin particles: microspheres that were less than 1 μm in size and needles of approximately 5

μm in length and 1 μm in width (81). In addition, microspheres of poly (D, L-lactic acid) embedded with lovastatin needles were also produced using the RESS method (81). These microspheres contained single and multiple lovastatin needles protruding from oval shaped polymer particles. Nucleation and growth of the drug occurred first which then functioned as nucleation sites for the polymer.

Supercritical fluid processing has also been used to produce submicron (400-700 nm) suspensions of water-insoluble drugs (87). Young (87) and colleagues produced a stable suspension of cyclosporine by the RESS process by spraying

the supercritical solution into an aqueous solution of Tween 80® (87). The surfactant served to impede particle growth and agglomeration. Concentrations as high as 38 mg mL-1 of cyclosporine were achieved in a 5.0 % w/w Tween 80® solution and 6.20 mg mL-1 in 1.0 % w/w Tween 80® (87). More recently supercritical fluid processing has been used to form solid dispersions and solid solutions. For example, solid dispersions of nifedipine (88), felodipine (22), and carbamazepine (48) have been produced when processed with PEG 4000 using the PGSS and GAS methods. These solid dispersions consistently showed better dissolution rates than those of the drug processed alone. For example, the amounts of drug dissolved in 1 hour from solid dispersions of nifedipine and

PEG 4000 were an average of 9 times greater compared to that of unprocessed nifedipine (22). Moneghini, et al. (89), also reported an increased rate and extent of dissolution of carbamazepine from solid dispersions containing PEG 4000 prepared using the GAS method, i.e., 10 % of unprocessed carbamazepine was released after 10 minutes compared to 100 % release from a 1:11 system with

PEG 4000. Such improvements were attributed to reduction in particle size, better shape of the crystals, and increased wettability when the polymer dissolved. Unprocessed carbamazepine was characterized as well-defined prisms, while processed drug resulted in needle-like structures that formed a light voluminous powder. These needle-like structures expose greater surface areas from which the drug can come into contact with the dissolution medium and thus dissolve. Juppo, et al. (83), formed solid dispersions and solid solutions using the SEDS method from 2,6-dimethyl-8-(2-ethyl-6-methylbenzylamino)-3-

hydroxymethylimidazo-[1,2-a]pyridine mesylate and Eudragit® E100 or mannitol.

The ability to form a solid solution appeared to be dependent on the materials in question. For instance, solid dispersions of drug and mannitol did not form solid solutions since the drug was found to be amorphous, did not give an endotherm at concentrations below 50 % drug, and strong interactions between the drug and carrier could be identified using FTIR. Solid solutions of drug and Eudragit®

E100, however, were said to be formed since the particles resembled the processed polymer particles and no endotherms for the drug could be detected.

SEM photomicrographs of SEDS processed Eudragit® E100 did not show any well-defined particles due to its low glass transition temperature, Tg = 50°C.

Mixtures of drug and Eudragit® E100 also failed to show well-defined particles of either the drug or of Eudragit® E100, suggesting a homogeneous solid solution of the two. Juppo, et al. (83), also reported an interaction between the hydroxyl group of the drug and carbonyl group of Eudragit® E100. Sethia and Squillante

(48) characterized carbamazepine solid dispersions in PEG 8000 alone and in combination with Glucire 44/14 (a saturated polyglycolized glyceride) and/or vitamin E TPGS (d-α-tocopheryl PEG 1000 succinate) processed by the GAS method. Solid dispersions containing carbamazepine and PEG 8000 showed the best dissolution profile with >90% of the drug dissolving in 10 minutes. Solid dispersions containing high drug concentrations resulted in larger carbamazepine crystals with lesser enhancement in dissolution rate and therefore drug/

carrier ratios of 1:5 yielded the best dissolution profiles. Supercritical fluid processing also resulted in polymorphic conversion of carbamazepine from form

III to form I.

Ye (23) reported the formation of solid dispersions of lidocaine with PEG

8000 and of lidocaine with ropivacaine by the RESS method. An increase in the dissolution rate of lidocaine was found from solid dispersions containing 10-80 % w/w of lidocaine with PEG 8000, compared to that of lidocaine alone. This was attributed to an improvement in wetting of the lidocaine particles by the incorporation of the water-soluble carrier. In the case of lidocaine and ropivacaine solid dispersions, Ye reported enhanced rates of release of lidocaine where lidocaine functioned as the more soluble and more rapidly released component and ropivacaine as the more sustained release component.

Lidocaine reached 100 % release in about 5 hours at concentrations of 20 % w/w lidocaine compared to 3 hours for concentrations containing 50 % and 80 % w/w lidocaine. Furthermore, Ye found that the rate of release of ropivacaine was 6 times faster from solid dispersions than from ground (milled) particles at concentrations of 80 % w/w lidocaine. This was explained by the apparent formation of a solid solution of lidocaine and ropivacaine, in which the particle size of ropivacaine was reduced to the molecular level. Thermal analysis of lidocaine and ropivacaine systems processed using the RESS method also suggested the formation of a solid solution at lidocaine concentrations above 60

% w/w. Thermograms of these systems had a single endotherm about 62 °C at

lidocaine concentrations above 60 % w/w, which is below the melting point of lidocaine, 68 °C, suggesting a new structure formed by the two components, i.e., a solid solution. Van Nijlen, et al. (90), also found an improvement in the dissolution rate of the anti-malarial drug artemisinin from solid dispersions containing PVPK25 (polyvinylpyrrolidone, Kollidon® K25) prepared using the

RESS method. There was an increase in the amounts of drug released after 60 minutes from 5 mg for the unprocessed drug to 15 mg for solid dispersions with

PVPK25 (90).

One of the advantages of supercritical fluid processing is the feasibility of scaling-up the process (91). The ability to use supercritical fluid processing on a larger scale would create a potentially general process that could be used for many pharmaceutical substances. Furthermore, since the food industry already employs supercritical fluid technology, it may be feasible to apply many of the same engineering aspects that are in place to the pharmaceutical industry (92).

Compared to conventional drug processing, such as crystallization, milling, spray-drying, and lyophilization, supercritical fluid processing involves fewer steps, which could reduce the cost of production (see Figure 1.4). In many conventional processes a drug must first be crystallized from a solvent, harvested, dried, and/or reduced to micron-sized particles for production, whereas with supercritical fluid processing, several of these steps are eliminated

(91). In terms of material and energy costs, supercritical fluid processing is comparable to single-stage spray drying (66). High yields of certain products that

a) Drug Solvent

Crystallization

Harvesting

Drying

Sieving

Milling

Micron-sized product

b) Drug Co-solvent Supercritical fluid (depending on the method)

Micron-sized product

Figure 1.4 Comparison of conventional processing versus supercritical fluid processing (adapted from Ref 91).

are virtually free of solvent are also possible (91). For example, Bradford Particle

Design has the resources to produce one ton of cGMP material annually suggesting the potential to generate larger quantities of pharmaceutical substances that are comparable to those quantities produced by the food industry (92). Furthermore, the low cost of carbon dioxide (69), and the ability to recycle it and other supercritical fluids can reduce solvent cost.

1.4 Objectives

The overall objective of this research was to form small particles consisting of solid solutions of relatively insoluble drugs and a water-soluble excipient using supercritical fluid processing as a general method. In this context, the specific objectives were:

 To use supercritical carbon dioxide (SCCO2) via the rapid expansion of

supercritical solutions (RESS) method to precipitate small particles of a

drug and carrier in the form of a solid solution.

 To characterize the physical and chemical properties of these solid

systems.

 To identify potential interactions between the drug and carrier as a result

of RESS processing.

 To determine the mechanism under which solid solutions form.

CHAPTER 2

CHARACTERIZATION OF DRUG / SURFACTANT SYSTEMS CONSISTING

OF A RELATIVLEY INSOLUBLE DRUG AND VARIOUS POLOXAMERS

FORMED BY RESS PROCESSING

Concept Map

 Small particles of solid solutions of a relatively insoluble drug, lidocaine or probucol, and a surfactant, poloxamer 407,188, or 403, were prepared using supercritical fluid technology.

 Thermal analysis of these particles indicated the formation of solid solutions coexisting with excess drug at compositions above 0.9456 mole fraction of lidocaine and 0.9043 mole fraction of probucol. On the other hand, DSC thermograms indicated the formation of complete solid solutions in the absence of excess drug below these compositions.

 Apparent dissolution rates of lidocaine and probucol from particles containing poloxamer 407 or 188 were enhanced compared to that from particles of lidocaine or probucol alone.

 Poloxamers appear to hydrogen bond with lidocaine and probucol, via

the polyoxyethylene segments, as functions of increasing poloxamer concentration.

 Solid solutions consisting of poloxamer 407 or 188 have similar

physicochemical properties as a result of similarities in their polyoxyethylene chain lengths. Since poloxamer 403 has shorter

polyoxyethylene chains, the formation of a solid solution in the absence of excess drug did not occur.

2.1 Introduction

A challenge in dealing with relatively insoluble drugs is to obtain a sufficient rate of solution to maximize bioavailability to ensure optimum therapeutic levels of the drug. Often simply reducing the particle size of a drug is not sufficient to adequately enhance the dissolution rate of a drug due to the formation of increased hydrophobic surface area, which can result in wetting problems that can limit the ability of the drug to go into solution (5). In such cases, solid dispersions, including solid solutions, of a relatively insoluble drug and a more hydrophilic substance may serve as a means of enhancing the dissolution rate of the drug. The more hydrophilic component would be expected to dissolve at a faster rate than the relatively insoluble drug, which in turn would enhance the effective area of contact of the dissolution medium with the relatively insoluble drug thereby facilitating its dissolution. Binary mixtures of nifedipine with poloxamer 407 in the form of solid dispersions have been shown to increase the release of the poorly soluble drug compared to solid dispersion of nifedipine and PEG 6000, PEG 4000, or hydroxypropyl-β-cyclodextrin (93). These solid dispersions, however, were formed using traditional methods such as the melting and solvent methods. While these methods are appropriate for some drugs, they may not be optimal or even appropriate for many drugs. For example, some drugs are thermally sensitive and could decompose at the temperatures needed to form a solid dispersion using the melting method. Furthermore, in the solvent method the need to use a solvent in which both the drug and the water-soluble

component are miscible is often difficult to obtain, as is the eventual complete removal of the solvent. Consequently, a better method is necessary to create a general process for making solid dispersions, including solid solutions of a relatively insoluble drug in a water-soluble component (carrier). To address this problem supercritical fluid processing of relatively insoluble drug/surfactant systems by the RESS method is proposed as a means of producing small particles of solid solutions. Examples of such systems could include lidocaine or probucol as the relatively insoluble component, with a hydrophilic block copolymer surfactant, i.e., poloxamer 407, 188, or 403.

Therefore, mixtures of lidocaine, or probucol, and a poloxamer were dissolved in supercritical carbon dioxide and small particles coevaporated upon subsequent expansion of the supercritical solution. During this expansion step it is hypothesized that the molecular arrangement of the solute/solvent system is retained upon removal of the supercritical fluid as a solid solution, which may be in equilibrium with excess solid solvent.

Lidocaine and probucol have low aqueous solubilities that make them good models for relatively insoluble drugs. Consequently, they have been used to prepare solid dispersions consisting of lidocaine or probucol and a water- soluble component. For example, lidocaine has been used as the model drug in solid dispersions with polyethylene glycol (PEG) 8000 using the fusion method

(94). Although dissolution studies were not performed for these systems, Roy

(94) reported the presence of an interaction between lidocaine and the oxyethylene linkage of PEG 8000, which could potentially influence the dissolution rate of lidocaine. Expanding on this work, Ye (23) prepared solid dispersions of lidocaine and PEG 8000 using supercritical fluid technology, specifically the method of rapid expansion of supercritical solutions. Dissolution studies showed an enhanced rate of release of lidocaine from the PEG 8000 systems. Ye reported that the formation of eutectic mixtures and an increase of the amorphous character of PEG 8000 were the contributing factors for enhancing the dissolution rate of lidocaine (23).

Previous attempts have also been made to formulate probucol into solid dispersions with the goal of improving its dissolution rate (95, 96). For example, probucol has been formulated into solid dispersions with polyvinylpyrrolidone

(PVP) (95, 96), polyacrylic acid (PAA) (96), and polyoxyethylene (POE) (96) by the solvent method and/or by compression moulding. Broman, et al. (96) showed that the release of probucol depended mainly on the properties of the polymer, rather than the drug in the solid dispersion. It was shown that the greatest extent of drug release was obtained for a blend of probucol and polyoxyethylene with nearly 80 % probucol released after 29 hours from samples prepared by compression moulding. It was also found that probucol was amorphous in PVP, and mainly crystalline in PAA or POE. Since amorphous probucol can form as a result of melting the crystalline drug and cooling, it was

concluded that the drug was initially amorphous in all systems upon melting.

Polyvinylpyrrolidone, however, was able to inhibit recrystallization by interacting with the drug, while PAA and POE were not.

Poloxamers have also been used to enhance the dissolution rates of drugs from various drug delivery systems, including solid dispersions (93, 97).

For example, Rouchotas, et al. (97) studied the dissolution properties of phenylbutazone treated with poloxamer 407 by an absorption method onto the drug surface and in solid dispersions with the same poloxamer. It was shown that the release of phenylbutazone after 140 minutes was 16.7 % from particles of untreated phenylbutazone, 71.4 % for the drug from solid dispersions, and

85.6 % from particles of surface treated phenylbutazone.

In addition to forming small particles of solid solutions of the relatively insoluble drugs lidocaine or probucol with a poloxamer via supercritical fluid processing to enhance drug release, an underlying goal of this research is to investigate the formation of solid solutions. Solid solutions are considered to be a potential method for maximizing the dissolution rates of relatively insoluble drugs. This is because in solid solutions formed by RESS processing, drug is molecularly dispersed and would be released as such during dissolution.

Therefore, an understanding of the conditions and mechanism(s) by which a solid solution forms would be necessary to create drug delivery systems with enhanced dissolution rates.

2.2 Purpose of the study

The specific aims of this study were therefore:

 To use the rapid expansion of supercritical solutions (RESS)

method to coevaporate (coprecipitate1) small particles of a relatively

insoluble drug, i.e., lidocaine or probucol, and a hydrophilic

surfactant, a poloxamer, in the form of a solid solution.

 To characterize the physical and chemical properties of these solid

solutions.

 To study the potential of these two-component systems for

enhancing the dissolution rates of lidocaine and probucol.

 To identify potential drug-surfactant interactions in solid solutions

formed by RESS processing

 To determine the effect of poloxamers, specifically their

polyoxyethylene and polyoxypropylene chains, on the formation of

solid solutions, as well as their ability to enhance the dissolution

rates of lidocaine and probucol.

1 A coprecipitate is obtained by simultaneous precipitation of two or more substances dissolved in a solvent through the addition of a nonsolvent or change in physical parameters. A coevaporate is formed when the solvent is removed to obtain dry, solid particles. In supercritical fluid methods, such as the gas antisolvent method, small particles are precipitated upon the addition of supercritical carbon dioxide as an antisolvent, or nonsolvent. In this context, the term coprecipitate would be appropriate. However, in RESS processing, small particles of drug form as the supercritical carbon dioxide evaporates during expansion. Therefore, the correct term in this case would be to use coevaporate. 58

2.3 Experimental

2.3.1 Materials

2.3.1.1 Lidocaine

Lidocaine belongs to a family of local anesthetics that “halt the neuronal traffic along an axon in a predictable and reversible manner, leaving the nerve none the worse for its brief period of rest (98).” Löfgren was the first to synthesize lidocaine from a series of aniline derivatives in 1943 (99). Lidocaine is a white, crystalline powder with a melting point of 68-69°C. The molecular formula is C14H22N2O, and molecular weight 234.34 (100) (see Figure 2.1).

Lidocaine is soluble in alcohol, chloroform, benzene, and ether, and practically insoluble in water (1). According to the USP, lidocaine is categorized as

“sparingly soluble” in water, i.e., from 30 to 100 parts of solvent are required for 1 part of solute (1). Lidocaine was chosen as a model compound because of its low aqueous solubility of 0.004 g mL-1 at 25 °C, which makes it a good representative of relatively insoluble drugs (100). Lidocaine is also soluble in the nonpolar supercritical carbon dioxide. The solubility of lidocaine is 36.2 ± 0.45 % w/w at supercritical conditions of 75 °C and 7100 psi (23) and forms small particles when processed using the RESS method (23).

CH3 CH3 O

C N CH3 N C H H2

CH3

Figure 2.1 Molecular structure of lidocaine.

2.3.1.2 Probucol

Probucol, 4,4’-[(1-methylethylidene)bis(thio)]bis[2,6-bis(1,1- dimethylethyl)]phenol, is a water-insoluble drug with potent cholesterol-lowering activity (95) (Figure 2.2). Probucol acts by lowering the blood levels of both low density lipoproteins (LDL) and high density lipoproteins (HDL). It is a white, odorless crystalline powder with the molecular formula C31H48O2S2 and molecular weight of 516.82 (101). The original form (Form I) of probucol has a melting point of 125 °C. However, a stable polymorph of probucol with a melting point of

116 °C (Form II) has also been reported (96, 101 and 102). Bostanian (101), for example, prepared Form II of probucol by dissolving it in absolute ethanol, followed by evaporation of the ethanol. The melting points of probucol were determined to be 127.9 °C for Form I and 119.4 °C for Form II. Probucol is freely soluble in chloroform, benzene, ether, acetone, ethanol, and methanol, and practically insoluble in water (96). The solubility of probucol in water is

5 ng mL-1 at 25 °C (99). Because of its poor water solubility, absorption from the gastrointestinal tract is less than 10% of an oral dose (102). As such, attempts have been made to improve its dissolution rate in hopes of improving its bioavailability (95, 96).

(H3C)3C C(CH3)3

CH3

HO S C S OH

CH3

(H3C)3C C(CH3)3

Figure 2.2 Molecular structure of probucol.

2.3.1.3 Poloxamers 407, 188, and 403

Poloxamers, also known as Pluronic Polyols®, are a family of nonionic, A-

B-A block copolymer surfactants (103) that consist of a polyoxypropylene (POP) block sandwiched between two polyoxyethylene (POE) blocks (Figure 2.3).

Within the poloxamer family, the ratios and weights of ethylene oxide and propylene oxide vary thereby creating different structures, physical forms and surfactant characteristics (103). The number assigned to a particular poloxamer provides information about the poloxamer being used (103, 104). The first two numbers following the name poloxamers correspond to the approximate average molecular weight of the polyoxypropylene portion when multiplied by 100. The last number, multiplied by 10, corresponds to the percentage by weight of the polyoxyethylene portion. Using these criteria, poloxamer 407 has an approximate average molecular weight of the polyoxypropylene portion of 4000, and a 70 % w/w polyoxyethylene portion. However, using the trade name

Pluronic Polyols®, the first number, or the first two numbers in a three-digit number, when multiplied by 300, also represents the approximate average molecular weight of the polyoxypropylene portion. The last number represents the weight percent of the polyoxyethylene portion when multiplied by 10.

Poloxamer 407, or Pluronic Polyol F127® in this case, would have an approximate average polyoxypropylene molecular weight of 3600, and a

70 % w/w polyoxyethylene portion. The trade name Pluronic® also designates a letter to describe the physical form of the block copolymer. The letters ‘L’, ‘P’, and ‘F’ stand for liquid, paste, and flake, respectively (104). Poloxamers with 63

a. CH3

HO-(CH2CH2O)X-(CH2CHO)Y-(CH2CH2O)X-H

POE POP POE

Poloxamer POE (X) POP (Y) 407 101 56 188 80 27 403 43 56

b. POE POE

POE

POP POP POP

Poloxamer 407 Poloxamer 188 Poloxamer 403

Figure 2.3 Molecular structure of poloxamers. a., basic structure and b., schematic comparison of poloxamers 407, 188, and 403 showing the approximate relative sizes of the polyoxyethylene and polyoxypropylene moieties.

longer POE chains tend to be flakes, while they become more fluid as the POE chain is shortened. This is because weak interactions occurring along the longer

POE chains provide rigidity to the structure, whereas shorter chains lack such rigidity and the compounds are more fluid (104). Poloxamers 407 and 188 are solid flakes at ambient conditions, have melting points ranging from 52-57 ºC, and are freely soluble in ethanol and water (103). Poloxamer 403 is a semi-solid at ambient conditions and melts at about 37 °C. The dual properties of the poloxamers, i.e., the hydrophilicity of the POE segment and hydrophobicity of the

POP segment, are ideal for the purposes of this study. The hydrophobic properties of polyoxypropylene allow adequate solubility of a poloxamer in supercritical carbon dioxide, while the hydrophilic chains draw water to the surfaces of particles of the relatively insoluble drug, while lowering interfacial tension and improving wetting, thereby facilitating dissolution of the drug. In addition, since the POE and POP chain lengths of poloxamers 407, 188, and 403 vary (see Figure 2.3 b), they can be used to study the effect of chain length on the formation of solid solutions, as well as potential drug-poloxamer bonding.

Figure 2.3 b does not represent the definitive structural orientation of the poloxamer molecule, but rather one of an infinite number of orientations that are possible with respect to the orientation of the polyoxyethylene chains to the polyoxypropylene chains, such as a linear structure.

Poloxamers tend to be of relatively high purity (104), and are primarily used in the pharmaceutical industry as emulsifying, solubilizing, and stabilizing

agents (103). In this context, they have been used in ointments, suppositories, dental products, eye drop formulations, and gels (105-107). Studies show that in general the toxicity of poloxamers is low and decreases as the molecular weight and the amount of ethylene oxide present increases (103). Select poloxamers, including poloxamers 407 and 188, have also been approved by the FDA for pharmaceutical and medical applications (103). For example, Schmolka (108), used Pluronic Polyol F-127®, which has reverse thermal gelling properties, to prepare a synthetic skin for the treatment of burns. When poured onto the surface of a burn, the solution of Pluronic Polyol F-127® will congeal and form an artificial barrier that can be removed by irrigation with water since it is water- soluble.

A list of materials is given in Table 2.1.

2.3.2 Equipment

An Isco® SFX 220 supercritical fluid extraction system was used for RESS processing (see Figure 1.3) (109). The system consists of a temperature controlled syringe pump (heated by circulating thermostatted water) which compresses supercritical fluid chromatographic grade carbon dioxide. The carbon dioxide has a purity of 99.995 % and the cylinder valve was equipped with a dip tube to draw liquid carbon dioxide (110). The temperature of the

Materials Source

Lidocaine Astra USA Inc., Westborough, MA

Probucol Sigma, USA St. Louis, MO

Poloxamer 407 and 188 Sigma, USA St. Louis, MO

Poloxamer 403 BASF Corp., Mount Olive, NJ

Table 2.1 Materials.

syringe pump was approximately 5 °C when filling to ensure that most of the carbon dioxide was in the liquid state. This allows more experiments to be performed without the need to refill the pump after each run. Since the carbon dioxide will pass through a heating coil before entering the extraction chamber containing the sample to be processed, it is not necessary to raise the pump temperature to that necessary to produce a supercritical state.

The extraction unit itself is directly connected to the pump and is temperature controlled. The extraction unit holds a 2 mL stainless steel or 10 mL disposable/reusable sample cartridge made of the high temperature resistant crystalline polymer Vectra® that holds the sample to be processed. The unit can be programmed to allow the sample to equilibrate with supercritical carbon dioxide for up to 16.5 minutes, the maximum time according to the instrument protocol (109). Exiting the extraction unit is a heated capillary restrictor with an approximate flow rate of supercritical carbon dioxide of 1.5 mL min-1 at 5000 psi and 80 C (111). The flow rates were determined by the instrument manufacturer from nitrogen gas flow rather than the flow of decompressed carbon dioxide. The restrictor has an inner diameter of approximately 500 μm that facilitates the coevaporation of small particles.

Particles form as the supercritical carbon dioxide solution explosively decompresses to atmospheric pressure and thereby evaporates as it leaves the

restrictor, and are collected in a “home made” collection chamber (see Figure

2.4). The cylindrical “U” shaped collection chamber is lined with a removable aluminum foil collection surface. Additional equipment is listed in Table 2.2.

2.3.3 Methods of particle formation

2.3.3.1 Formation of drug/poloxamer 407, 188, or 403 particles by

supercritical fluid processing

Lidocaine, probucol, poloxamers 407, 188, or 403, and various concentrations of lidocaine or probucol with poloxamer 407, 188, or 403 containing drug mole fractions of 0.9936, 0.9812, or 0.9456 were placed in the sample cartridge and inserted into the extraction unit and processed with supercritical carbon dioxide according to the RESS method. Extraction conditions were as follows: 75 C, 7100 psi, equilibration time of 16.5 minutes, and restrictor temperature of ~ 40 C for drug / poloxamer 407 and drug/ poloxamer 188 systems; and 35 C, 7100 psi, equilibration time of 16.5 minutes, and restrictor temperature of ~ 20 C for drug/poloxamer 403 systems.

Extraction temperatures at or above the melting points of the poloxamers, i.e.,

~52-57 °C for poloxamers 407 and 188 and ~35 °C for poloxamer 403, were used to promote the dissolution of poloxamer in the supercritical carbon dioxide.

Temperatures near the melting point tend to weaken the intramolecular forces, thereby facilitating the dissolution of the substance in the supercritical carbon

Figure 2.4 “Home made” chamber for collecting RESS processed particles. Hash marks indicate aluminum foil lining (against the walls and bottom surfaces of the container).

Equipment Manufacturer Supercritical Extraction System Syringe Pump, Model 260D Isco. Inc., Extraction Unit, Model SFX 220 Lincoln, NE Controller, SFX 200 Heated Restrictor Restrictor Temperature Controller HPLC System Software, System Gold® Beckman Instruments, Solvent Pump, Model 110B San Ramon, CA Injector, 200 µL loop Analog Interface Module 406, Model 338 Scanning detector module, Model 166 Computer, PS/2 Model 30 IBM Corporation, Armonk, NY

PRP-1, 10μm Column, 250x4.1 mm Hamilton Company, Reno, NV Differential Scanning Calorimeter Model TA 4300 Mettler - Toledo, Columbus, OH Dissotest Dissolution Apparatus Dissolution Module CE- 6 Sotax AG, Basel, Switzerland Dissopump CY-6 Sotax AG, Basel, Switzerland FTIR Spetrophotometer ProtégéTM FTIR Spectrophotometer Nicolet Instrument Corporation, ProtégéTM 460 Optical Bench Madison, WI

Model 129 KBr Die Assembly Spectra-Tech®, Inc., Stamford, CT

Model “C” Carver® Hydraulic Laboratory Press Fred Carver, Inc. Menomonee Falls, WI Scanning Electron Microscope Model XL-30 Philips, New York Miscellaneous Mettler UMP Microbalance Mettler Instruments, Corp., Mettler AE 240 Balance Highstown, NJ pH meter, Model 72 Beckman Instruments, Fullerton, CA

Table 2.2 Equipment.

dioxide (23). This is because at higher temperatures solute molecules are then able to overcome the energy barrier of solute-solute interactions and leave the interface between the solid and solvent. Higher temperatures, as suggested by

Broman, et al. (96), also promote mobility of the polyoxyethylene chains of poloxamer, which can increase the probability of contact and bonding with the drug. Furthermore, for these systems the restrictor temperature was set just below the melting points of lidocaine or probucol and poloxamers 407, 188, or

403 to prevent melting during expansion, while maintaining fluidity in the restrictor to prevent clogging.

During RESS processing the drug and poloxamer 407, 188, or 403 are in solution in the supercritical carbon dioxide. As the solution is allowed to expand, the carbon dioxide is released into the atmosphere at supersonic velocities and the drug and poloxamer coevaporate simultaneously forming small particles.

Since the expansion process occurs almost instantaneously, it is suspected that the molecular arrangement of the drug and poloxamer in the supercritical solution is retained and forms a solid solution upon expansion.

RESS processed samples were collected for analysis, while unused particles were stored in closed glass vials with plastic screw top lids at room temperature for approximately five months for reproducibility and stability studies.

2.3.4 Methods of analysis

2.3.4.1 Differential scanning calorimetry (DSC)

Approximately 5 mg of particles were collected after RESS processing and weighed in aluminum DSC pans using a Mettler UM3 microbalance. The pans were sealed and pierced with a small hole to prevent pressurization. A Mettler

TA 4300 thermal analysis system was used to analyze samples by heating a sample at a specified rate within a temperature range determined according to the melting point of the substance. For the purposes of this work, samples were analyzed according to the following stepwise protocol (50):

First Heating: 20 C to 100 C or 150 C, at 2 C min-1;

Cooling: 100 C or 150 C to 0 C, at -2 C min-1;

Second Heating: 0 C to 100 C or 150 C, at 2 C min-1.

Based on the melting points of lidocaine and probucol (68 C and 127 C, respectively), samples containing lidocaine were heated to 100 C, while those containing probucol were heated to 150 C. An end value for heating that is higher than the melting point was selected so that the thermal event could be completely recorded and the baseline could return to normal. The lower starting temperature ensures a thorough evaluation of potential thermal events. Lastly, the heating and cooling rates were determined to provide the best accuracy while

maintaining reasonable times for sample analysis. A heating and cooling rate of

2 C min-1 was chosen versus the more commonly used 5 and 10 C min-1 because slower heating rates provide more accurate measurements due to higher detector sensitivity to thermal events (50).

DSC thermograms describe the endo- and exothermal events that take place as functions of temperature and rate of heating (50). The melting point and enthalpy of fusion for each endotherm was determined using Mettler GraphWare

TA72PS.2 software, and used to generate phase diagrams in terms of mole fractions as functions of temperature. Samples of lidocaine, poloxamer and combinations thereof that were stored for approximately five months in sealed glass vials at room temperature were also analyzed according to the same protocol to study the stability and reproducibility of the mixtures.

2.3.4.2 Scanning electron microscopy (SEM)

Photomicrographs of samples processed by RESS were taken by scanning electron microscopy (SEM) using a Philips XL-30 scanning electron microscope. SEM was used to examine the surface characteristics of the particles generated. Preparation of samples for imaging consisted of placing the particles on a carbon-based adhesive and gold coating the sample using a

sputtering technique. The interior structures of particles were also examined. To do this, fractured samples were prepared for imaging by randomly chopping the particles with a razor blade prior to mounting and gold coating.

2.3.4.3 Dissolution of drug from particles consisting of drug and

poloxamer

Dissolution studies utilized the flow-through method (Apparatus 4, USP

23). A Dissotest® dissolution system (Sotax, AG) which includes a dissolution module (Model CE-6) and a multiple-piston pump (Model CY-6) was used. The dissolution cells have volumes of approximately 15 mL and an inner diameter of

22.6 mm, which taper to a cone at the bottom and are mounted inside cylindrical thermostatted chambers (see Figure 2.5) (112). In the flow through method, dissolution medium enters the bottom of the cell and exits at the top through a filter head containing a 2.4 cm Whatman glass microfiber filter. It is assumed therefore that sink conditions are maintained since fresh dissolution medium is constantly being pumped through the vessel at 10 mL min-1 (24). Based on the

Noyes-Whitney equation (see Equation 1.1), (Cs-C) can be approximated to Cs, or the solubility, i.e., saturation solubility, of the drug at a particle surface, if drug concentration in the bulk dissolution medium (C) is maintained at a low value, i.e., less than 10 % of the value of Cs, and can therefore be considered to be negligible. Given the rate at which fresh dissolution medium is entering the

Figure 2.5 Dissotest® dissolution system (Sotax, AG). Key: a, thermostatted water bath, b, mounted “flow-through” cell, c, filterhead, and d, clamping device (from ref. 112, with permission).

dissolution cell, this approximation is reasonable for the present work. Contained in the cell are glass beads (1 mm diameter, as specified in the USP) (1), which produces a laminar flow of dissolution medium over the sample (112), thereby reducing turbulence in the cell. The bottom of the cell contains a ruby bead that acts as a check valve preventing disruption of the bed of glass beads and loss of beads as dissolution medium enters the cell.

The volume of glass beads placed in the dissolution cell was divided into thirds, with the middle third consisting of glass beads mixed with small particles of RESS processed lidocaine, probucol, or particles containing lidocaine or probucol and poloxamer 407, 188 or 403. In the case of lidocaine, approximately

100 mg of particles containing lidocaine or particles consisting of lidocaine and poloxamer 407, 188 or 403 was used. However, approximately 20 mg of probucol or particles consisting of probucol and poloxamer 407, 188, or 403 was used. Probucol absorbs strongly at 242 nm using HPLC and a smaller sample size was necessary to generate peak areas representing concentrations comparable to those determined for the standard curve.

Dissolution rates were measured at 37 C in deionized, distilled water

(pH~6.1), or in the case of probucol a 50:50 mixture of water and ethanol at flow rates of approximately 10 mL min-1 for two or three hours. A dissolution time of three hours was used for probucol due to the slower release of the more water- insoluble drug, compared to lidocaine (two hours). Bostanian (101) determined a

50:50 mixture of water and ethanol to be the best dissolution medium for measuring the dissolution rate of probucol. Therefore, ethanol was also added to the dissolution medium to enhance the apparent solubility of probucol. Fractions of the dissolution medium were collected every 5 minutes for the first half hour where changes in the initial dissolution rate are most critical and every 15 minutes thereafter. The amounts of drug released were measured by HPLC.

Analyses of the drug in solution were performed with a Beckman HPLC system using Beckman System Gold® software version 3.11. The mobile phase for the analysis of lidocaine H+ consisted of 47 % formate buffer (pH~3.3) / 53 % methanol at a flow rate of 1.5 mL min-1. Probucol analysis was performed using a mobile phase consisting of 85 % acetonitrile/15 % water at a flow rate of 1.0 mL min-1. A reversed phase Hamilton PRP-1 column and 200 μL injection loop was used.

2.3.4.4 Aqueous solubility of lidocaine

The following study was performed to determine the relationship between surfactant concentration and the apparent solubility of lidocaine in its presence.

The aqueous solubility of lidocaine in the presence of poloxamer 407 was determined at concentrations of 0, 4, 6, 8, and 12 % w/w of poloxamer in water, all of which are above the critical micelle concentration of poloxamer 407, i.e.,

0.08 % w/w at 30 °C (113). Excess amounts of lidocaine, or equal amounts of

excess lidocaine and poloxamer 407 were weighed and placed into screw- capped glass test tubes. Deionized, distilled water (50 mL) was added and the test tubes were sealed and placed on a rotator at ~41 rpm in a thermostated water bath at 30 °C (Sustained Release Apparatus, VanKel). After 48 hours the tubes containing a saturated solution in equilibrium with an excess amount of drug were allowed to stand motionless for an additional 24 hours allowing the undissolved drug to settle to the bottom of the container (23). The apparent solubility of lidocaine was determined by withdrawing a portion of the supernate, which was then diluted with mobile phase and quantified by HPLC.

2.3.4.5 Fourier transform infrared (FTIR) spectroscopy

Thin films containing potassium bromide (KBr) and lidocaine, probucol, poloxamer 407, 188, or 403, and various amounts of lidocaine or probucol, and poloxamer 407, 188, or 403 were prepared using a Spectra-Tech, Inc., Model

129 KBr Die assembly mounted on a Model “C” Carver hydraulic press. Prior to preparing the films, KBr was dried in an oven overnight at 55 °C to remove any moisture. To prepare the films, approximately 150 mg of KBr was mixed with approximately 1.5 mg of a sample (collected after supercritical fluid processing) in an agate mortar and pestle. The mixture was then transferred to the KBr die assembly and compressed at an applied load of 5,000 pounds for 5 minutes.

The above-mentioned compositions and compression requirements produced transparent films with measurable FTIR spectra. All films were prepared in

duplicate from separate batches of processed samples and scanned the same day on a Nicolet ProtégéTM 460 FT-IR spectrophotometer using Nicolet OMNIC software. The films were mounted on a Spectra-Tech magnetic film holder and scanned according to the instrument specifications. The mean spectrum for each sample was generated. A background scan was also performed before each sample and was automatically subtracted by the software.

2.3.4.6 Solubility of lidocaine, probucol, or poloxamer in supercritical

carbon dioxide

The solubility of lidocaine in supercritical carbon dioxide was previously determined by Ye (23) as 36.2 ± 0.45 % w/w at 75 °C and 7100 psi. The solubility of poloxamers 407, 188, and 403 in supercritical carbon dioxide, however, could not be determined since an accurate and reliable method of analysis could not be found for poloxamer.

A similar experimental design, such as that used for lidocaine, was used for measuring the solubility of probucol in supercritical carbon dioxide compared to lidocaine (23). Approximately 300 mg of probucol was loaded into a sample cartridge and inserted into the extraction chamber. The sample was allowed to equilibrate in the supercritical carbon dioxide for 16.5 minutes, the maximum time according to the instrument protocol (109). Following equilibration, 2 mL of supercritical carbon dioxide with dissolved probucol was collected by immersing

the open end of the restrictor in 10 mL of a 50:50 ethanol:water mixture contained in a glass vial (23, 101). The solution was further diluted with the

50:50 ethanol:water mixture as necessary for HPLC analysis. The solubilities were calculated in terms of % w/w probucol at a pressure of 7100 psi and temperatures of 35, 55, or 75 °C.

2.4 Results and Discussion

2.4.1 Lidocaine, poloxamer 407, and lidocaine/poloxamer 407

compositions

2.4.1.1 Differential scanning calorimetry

DSC of particles of lidocaine and poloxamer 407 processed by the RESS method indicated melting points at 68.3 C and 56.1 C, respectively (see

Figures 2.6 a and b). These experimental values are in agreement with the literature and indicate that the thermal properties of either compound are not altered under the specified conditions used during supercritical fluid processing, i.e., 75 C, 7100 psi, and a restrictor temperature of 40 C (1, 73). Melting points and enthalpies of fusion of lidocaine and lidocaine/poloxamer mixtures are summarized in Table 2.3. Lidocaine was expected to have a greater enthalpy of fusion than poloxamer 407 since it is known to be an aggressive crystallizer (99).

Lidocaine / Poloxamer 407 May 2002

a. b.

c. d. e.

Figure 2.6 Representative DSC thermograms of lidocaine/poloxamer 407 compositions prepared by RESS processing. Key: a., lidocaine (purple); b., poloxamer 407 (black); c., lidocaine MF=0.9936 (blue); d., lidocaine MF=0.9812 (red); and e., lidocaine MF=0.9456 (green).

% (w/w) of Mole Fraction Melting Point, C Enthalpy, J g-1 Lidocaine of Lidocaine Lidocaine Poloxamer Lidocaine Poloxamer 407 407 100 1.0000 68.3±0.00 - 71.6±0.42 - 80 0.9952 67.5±0.28 49.4±0.21 51.8±5.94 21.0±5.73 75 0.9936 68.0±0.00 50.1±0.21 56.9±2.55 13.6±0.64 60 0.9874 65.7±0.28 50.4±0.07 26.8±0.28 48.1±1.34 50 0.9812 65.2±1.34 50.3±0.07 24.3±9.33 50.7±12.3 40 0.9720 57.3±0.00 49.8±0.28 0.30±0.28 82.9±5.02 25 0.9456 - 50.9±0.35 0.00±0.00 99.0±1.84 0 0 - 56.0±0.21 - 115±2.26

Table 2.3 Thermal properties of lidocaine and poloxamer 407 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, (n = 2).

However, poloxamer 407 has instead a larger enthalpy than lidocaine, i.e., 114.7

J g-1 and 71.6 J g-1 respectively, apparently due to strong interactions between its polyoxyethylene chains.

Representative thermograms for particles of lidocaine/poloxamer 407 containing 0.9936, 0.9812, and 0.9456 mole fraction lidocaine are also shown in

Figure 2.6. All concentrations were analyzed in duplicate using separate batches of processed samples. The resulting thermograms are similar and the two thermograms appear as averages in Table 2.3. At concentrations containing

0.9936 mole fraction lidocaine a large endotherm appears near the melting point of lidocaine (68 C) with a smaller endotherm at 50.1 C. This smaller endotherm is assumed to be that of the poloxamer although it does not correspond with the melting point of poloxamer alone (56 C). This is because lidocaine is acting as an impurity in the system, which would create instability in the structural energy of the poloxamer thereby shifting the endotherm to a lower melting point (50).

Varying lidocaine concentration has little effect on the value of the depressed melting point of poloxamer 407. As the lidocaine concentration decreases and that of poloxamer 407 increases, the endotherm representative of lidocaine begins to decrease and then disappear while the endotherm for poloxamer 407 becomes larger. The latter endotherm does not shift to a higher or lower melting point until 0.9720 mole fraction where the melting point decreases from 65.2 °C at 0.9812 mole fraction to 57.3 °C. At this mole fraction it can be assumed that greater amounts of lidocaine are bonding with poloxamer 407, and that

poloxamer 407 is acting as an impurity in the lidocaine, which reduces its melting point. Distinct endotherms for lidocaine and poloxamer 407 can be assigned for concentrations containing up to 0.9720 mole fraction lidocaine (endotherm not shown). At lidocaine concentrations below 0.9720 mole fraction lidocaine only a single endotherm appears with a melting point that corresponds to that of poloxamer 407. For example, at 0.9456 mole fraction lidocaine there is a single endotherm at 50.9 C (see Figure 2.6 e). The disappearance of the lidocaine endotherm at this concentration, as well as all subsequent endotherms for lidocaine, suggests that lidocaine may be dissolving in poloxamer 407, forming a solid solution. In order for an endotherm to appear there needs to be sufficient intra-molecular interactions, i.e., lidocaine-lidocaine interactions, to be detected within the limits of DSC. It can be concluded therefore that lidocaine is molecularly dispersed within the poloxamer resulting in no detectable endotherm for lidocaine-lidocaine interactions even though its mole fraction is significantly large.

In this context, it is interesting to note that at concentrations containing

0.9456 mole fraction lidocaine there is a large number of lidocaine molecules for every poloxamer 407 (16:1). However, each poloxamer 407 molecule has approximately 258 polyoxyethylene and polyoxypropylene segments to which lidocaine can potentially hydrogen bond. In support of this, FTIR studies discussed later will show that lidocaine primarily hydrogen bonds with the polyoxyethylene segments of poloxamer 407. Based on the calculated mole

fractions, the large number of lidocaine molecules bonding with a poloxamer 407 molecule could create a solid solution as illustrated in the hypothetical model shown in Figure 2.7. This is in agreement with the DSC data, which indicates that below 0.9720 mole fraction lidocaine all of the lidocaine is associated with the poloxamer 407 molecules since there is no endotherm for lidocaine-lidocaine interactions. Conversely, above 0.9720 mole fraction lidocaine the presence of an endotherm for lidocaine indicates that solid solutions of lidocaine in poloxamer

407 exist in equilibrium with excess crystalline lidocaine dispersed in the solid matrix. This agrees with the concept of partial solid solutions, which states that regions of solid solutions can exist in a matrix (36). The idea that a solid solution coexists with excess lidocaine becomes more evident as the proportion of lidocaine increases in the system and the endotherm for lidocaine becomes more defined.

In terms of enthalpy, differences in the enthalpies between the different lidocaine/poloxamer 407 systems can be attributed to the ratio of lidocaine to poloxamer 407. The intensity (or area) of an endotherm, the enthalpy, is dependent on concentration, whereby changes in the amount of lidocaine or poloxamer 407 present in a system would result in differences in the intensities of the endotherms. For example, at lidocaine mole fraction 0.9936 the enthalpy of the endotherm assigned to lidocaine is 56.9 J g-1 compared to 24.3 J g-1 for lidocaine mole fraction 0.9812 (see Table 2.3). Further reductions in the

L L L L L L L P

POE L O

L P L L L L L L L

Figure 2.7 Schematic representation of lidocaine/poloxamer 407 solid solution showing location of lidocaine (L) and poloxamer 407 (red and blue) molecules.

amounts of lidocaine would result in progressive decreases in the areas of the endotherms for lidocaine, leading ultimately to the disappearance of the endotherm and the apparent formation of a solid solution.

Figure 2.8 is a phase diagram of enthalpies for lidocaine and poloxamer

407 as functions of mole fraction of lidocaine. The enthalpies of lidocaine decrease as the amounts of lidocaine decrease, while the enthalpies of poloxamer 407 increase. This relationship corresponds with decreases in the amounts of excess lidocaine due to increased bonding to poloxamer 407. From the enthalpies in Table 2.3 it was determined by dividing the enthalpy at a particular concentration by that of the enthalpy of the pure compound, that 0.4 % lidocaine remains in excess at 0.9720 mole fraction lidocaine, compared to

72.3 % at 0.9952 mole fraction. Conversely, the percent of poloxamer 407 increases from 18.3 % at 0.9952 mole fraction lidocaine to 86.1 % at 0.9456 mole fraction. Furthermore, a solid solution apparently forms between 0.9720 and 0.9456 mole fraction lidocaine, i.e., the range within which zero enthalpy for lidocaine is attained. Linear regression of the enthalpies of lidocaine (r2 =

0.9756) predicts that the formation of a solid solution should occur at 0.9732 mole fraction lidocaine (see Figure 2.9). Therefore, at mole fractions less than

0.9732, solid solutions of lidocaine in poloxamer 407 would form. Conversely, at higher mole fractions solid solutions of lidocaine in poloxamer 407 would coexist in equilibrium with excess lidocaine. In addition, samples of RESS processed

140 140 Lidocaine Poloxamer 407 120 Experimental Total Enthalpy 120

100 100

80 80

60 60 Enthalpy (J/g)

40 40

20 20

0 0 0.04 0.08 0.92 0.960.00 1.00 Mole Fraction Lidocaine

Figure 2.8 Phase diagram of lidocaine/poloxamer 407 compositions (1st DSC heating step, data from Table 2.3).

80 80

60 60

40 40

20 20 Enthalpy (J/g) of Lidocaine of (J/g) Enthalpy

0 0 0.04 0.08 0.92 0.960.00 1.00

Mole Fraction Lidocaine

Figure 2.9 Phase diagram of lidocaine from lidocaine/poloxamer 407 compositions showing linear regression, r2 = 0.9756 (data from Table 2.3).

lidocaine and poloxamer 407 stored for five months at room temperature (~ 24

°C) exhibited similar thermal behavior as did “fresh” particles studied within an hour after processing (see Figure 2.10 and Table 2.4).

2.4.1.2 Scanning electron microscopy

Lidocaine and poloxamer 407 have different physical properties.

Lidocaine is an aggressive crystallizer and forms solid particles with rough edges as shown in Figure 2.11 a. The needle-like crystal structure typical of lidocaine appears to have been disrupted by the explosive expansion of supercritical carbon dioxide in the last step of the RESS process, resulting in what could be interpreted as disrupted or discontinuous crystal layers. Poloxamer 407 not processed by RESS, on the other hand, has smooth surfaces enclosing spherical structures (see Figure 2.11 b). Their globular appearance could be the result of preparation by spray drying in which droplets of the substance form and are air- dried.

By contrast, SEM images of particles consisting of lidocaine and poloxamer 407 show a distinct morphology after supercritical fluid processing in that many of the discrete particles have small invaginations or holes on their surfaces (Figures 2.12 a and b). Since the system would be in the form of a liquid droplet just prior to loss of carbon dioxide, interfacial tension would cause the formation of a spherical or near-spherical particle with relatively smooth

Lidocaine / Poloxamer 407 Stored Samples Oct 2002

a. b. c. d. e.

Figure 2.10 Representative DSC thermograms of lidocaine/poloxamer 407 stored compositions prepared by RESS processing. Key: a., lidocaine (purple); b., poloxamer 407 (black); c., lidocaine MF=0.9936 (blue); d., lidocaine MF=0.9812 (red); and e., lidocaine MF=0.9456 (green). (see Figure 2.6 for comparison)

Mole Melting Point, C Fraction of Lidocaine Poloxamer 407 Lidocaine Fresh Stored Fresh Stored 1.0000 68.3±0.00 68.2±0.00 - - 0.9936 68.0±0.00 67.8±0.42 50.1±0.21 50.2±0.07 0.9812 65.2±1.34 65.4±1.13 50.3±0.07 51.0±0.07 0.9456 - - 50.9±0.35 50.9±0.00 0 - - 56.0±0.21 56.1±0.00

Mole Enthalpy, J g-1 Fraction of Lidocaine Poloxamer 407 Lidocaine Fresh Stored Fresh Stored 1.0000 71.6±0.42 72.1±0.00 - - 0.9936 56.9±2.55 53.1±1.91 13.6±0.64 20.1±2.97 0.9812 24.3±9.33 22.6±8.56 50.7±12.3 55.1±10.9 0.9456 0.00±0.00 0.00±0.00 99.0±1.84 107±1.41 0 - 0.00±0.00 115±2.26 122±2.90

Table 2.4 Comparison of fresh (1 hour) and stored (5 months, room temperature) thermal properties of lidocaine and poloxamer 407 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, (n = 2).

Figure 2.11 Scanning electron photomicrographs of lidocaine and poloxamer 407. Key: a., lidocaine prepared by RESS processing; b., poloxamer 407 as received. 94

Figure 2.12 Scanning electron photomicrographs of lidocaine/poloxamer 407 particles containing 0.9812 mole fraction lidocaine prepared by RESS processing.

surfaces. The holes could result from carbon dioxide escaping the forming particle during expansion. The small invaginations, or holes may prove to be advantageous with regard to dissolution of the drug since such irregularities would enhance the apparent surface area of a particle.

Images of fractured particles show that the holes continue throughout the interior of the particles and lead to large void spaces (Figures 2.13 a-f). The interior surfaces are porous and contain cavities formed by the expanding carbon dioxide. Fractured samples show that separate crystals of lidocaine do not form within the poloxamer particles and that lidocaine and poloxamer form homogeneous, or near homogeneous, particles. At lidocaine mole fractions

0.9456 and 0.9812, the surface texture of the particles is smooth, as shown in

Figures 2.13 a-d, apparently due to the presence of poloxamer 407. While some surfaces may appear to have few holes, the interiors for the most part remain porous after the expansion of carbon dioxide (Figure 2.13 a.). In Figure 2.13 b, the particles appear to be very interconnected, i.e., sponge-like, and have cavities and/or channels from which carbon dioxide escaped. Figures 2.13 c and d show several smooth spheres along with larger fractured spheres that have porous interiors, again probably due to the expansion of carbon dioxide. In addition, Figures 2.13 c and d represent particles consisting of solid solutions of lidocaine in poloxamer 407 coexisting with excess lidocaine. In these particles, the darker region in the wall of the particles (see circled area in Figure 2.13 c) may represent excess crystalline lidocaine, which would be expected to be more

a. b.

c. d.

e. f.

Figure 2.13 Scanning electron photomicrographs of lidocaine and poloxamer 407 compositions prepared by RESS processing. Key: a. and b., lidocaine MF=0.9456; c. and d., lidocaine MF=0.9812; e. and f., lidocaine MF=0.9936.

textured than a solid solution. On the other hand, the outer edges of the wall could be that of a solid solution since the surfaces of the particles appear smooth and homogeneous. Such an appearance would result from interfacial forces at the liquid/air interface before and as the spherical structures were being formed by coevaporation.

More rough edge surfaces begin to appear at lidocaine mole fraction

0.9936 (Figures 2.13 e and f), which may be characteristic of lidocaine (with the exception of the web-like structure). Despite the high concentration of lidocaine, porosity of the particles is retained as a result of the supercritical fluid processing and the presence of a small amount of poloxamer.

2.4.1.3 Release of lidocaine from particles containing poloxamer 407

The release of lidocaine from lidocaine/poloxamer 407 particles prepared by RESS processing is shown in Figure 2.14. Release of lidocaine alone approaches complete release, i.e., 100 %, and would be expected to be complete if the experiment was run to longer times. In Figure 2.14, it can be seen that the addition of poloxamer 407 enhances the apparent dissolution rates of lidocaine from the three binary mixtures compared to that of lidocaine alone.

This effect is however not rank-ordered with increasing poloxamer 407 concentration. The release curves also indicate that the initial rates of release of lidocaine (within the first 5-10 minutes) are enhanced regardless of the

120

100

40 %Release Lidocaine of Lidocaine MF = 0.9456 20 Lidocaine MF = 0.9812 Lidocaine MF = 0.9936 Lidocaine MF = 1.0000 0 0 20 40 60 80 100 120 140 Time (minutes)

Figure 2.14 Release of lidocaine from lidocaine/poloxamer 407 particles prepared by RESS processing as functions of composition. (Total weight of each sample = 100 mg, n = 3).

poloxamer 407 concentrations. This rapid release of drug may be attributed to the presence of small particles in the samples collected during the first 10-15 minutes of the dissolution process as indicated by cloudy white samples. Even though the filter which is incorporated into the dissolution apparatus has a pore size of 2.7 µm, smaller particles could have passed through the filter giving a sample which was unsuitable for HPLC analysis at these early time points. As a result, these small particles would scatter light in the flow cell during detection giving an apparent increase in absorption and hence an apparent increase in initial rates of release. Also contributing to the increases in dissolution rates may be particle morphology, the structure of which (see Figure 2.13) in the presence of poloxamer 407 would facilitate penetration of the dissolution medium to a greater extent than that of lidocaine alone. Furthermore, since poloxamer 407 is a water-soluble surface-active agent, it is likely that it would increase wetting by reducing the contact angle of the dissolution medium at the solid/liquid interface and thereby improve the dissolution rates of lidocaine.

At lidocaine mole fraction 0.9456 the dissolution profile reached zero slope below 100 % release, i.e., ~90 %. The apparent “loss” of lidocaine at this concentration, which is a solid solution, could in theory be due to micellar solubilization of lidocaine. Poloxamer 407 forms micelles due to its low critical micelle concentration, i.e., 0.08 % w/w at 30 °C. As a result of micelle formation, it is probable that a portion of the lidocaine is sequestered in the micelle and thereby evades HPLC detection, resulting in an apparent loss of lidocaine.

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Although micelles can scatter light in the flow cell, the apparent loss of lidocaine due to micelle solubilization would appear to more than compensate for the apparent increase in absorption due to the scattering of light. On the other hand, at lidocaine mole fractions 0.9812 and 0.9936, the dissolution profiles exceed

100 % lidocaine release. Based on the DSC data where endotherms for both lidocaine and poloxamer 407 are present, unlike that for lidocaine mole fraction of 0.9456, the molecular model proposed in section 3.4.1.1 and Figure 2.7 suggests that solid solutions of lidocaine and poloxamer 407 exist along with crystalline lidocaine. Similar to that of lidocaine mole fraction 0.9456, poloxamer

407 would form micelles and lidocaine would be solubilized as it dissolves from the solid solution. Simultaneously, the crystalline lidocaine may be dissolving to form a supersaturated solution from which precipitation would occur resulting in numerous small particles of lidocaine. The precipitated lidocaine would in turn scatter light in the dissolution medium in the flow cell and result in an apparent increase in absorption. The overall result would be an apparent increase in the amount of drug being detected, which in this case would give dissolution profiles that exceed 100 % release of lidocaine. Therefore, the fundamental difference in the release of lidocaine from compositions containing lidocaine mole fraction

0.9456 to that of lidocaine mole fractions 0.9812 and 0.9936 is that at lidocaine mole fraction 0.9456 there is only one source of free drug available to go into solution for HPLC detection, which results from the dissociation of lidocaine from the solid solution. As previously mentioned, a portion of this drug may evade detection by way of micellar solubilization. At the higher concentrations, i.e.,

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lidocaine mole fractions 0.9812 and 0.9456, the dissociation of lidocaine from the solid solution and the presence of crystalline lidocaine provide two sources of free drug that can enter into solution for HPLC detection. Unlike lidocaine mole fraction 0.9456, the precipitation of crystalline lidocaine from a supersaturated solution and light scattering during detection would result in apparent increases in the amount of lidocaine released.

A comparison of the dissolution profiles was performed using the “Model

Independent Approach Using a Similarity Factor” as described in the Center for

Drug Evaluation and Research (CDER) Guidance for Industry: Dissolution

Testing of Immediate Release Solid Oral Dosage Forms (114). This document describes a method used to compare dissolution profiles when there are component and composition changes in immediate release solid oral dosage forms. The model independent approach uses a difference factor (f1) which calculates the percent difference between the two curves at each time point and is a measurement of the relative error between the two curves (114). This equation is as follows:

n n f1 = { [Σt=1 |Rt – Tt| ] / [ Σt=1 Rt ] }*100 (2.1)

where n is the number of time points, Rt is the reference dissolution value at time t, and Tt is the test dissolution value at time t. In addition, the similarity factor (f2)

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measures the similarity between two curves in terms of percent release through a logarithmic reciprocal square root transformation of the sum of the squared error

(114).

n 2 -0.5 f2 = 50 * log { [ 1+(1/n) Σt=1 (Rt – Tt) ] * 100 (2.2)

As a general approach, the following suggestions are recommended when determining the difference and similarity factors. These include using the mean dissolution values from both curves at each time interval, dissolution measurements should be made under the same conditions and time points, and only one measurement should be considered after 85% release of both products.

Furthermore, for curves to be considered similar and/or equivalent, 1f values should be close to 0 (0-15) and f2 values greater than 50 (50-100) (114).

Table 2.5 gives the calculated f1 and f2 values for lidocaine and lidocaine/poloxamer 407 binary mixtures based on their respective release data.

From these values it can be concluded that only the release profiles for lidocaine from particles containing poloxamer 407 are similar, having 1f values of 6.7 for lidocaine mole fractions 0.9936 and 0.9456 and 12.0 for lidocaine mole fraction

0.9812 and 0.9456. In addition, their f2 values suggest similarity at 67.1 for lidocaine mole fractions 0.9936 and 0.9456, and at 54.7 for lidocaine mole fractions 0.9812 and 0.9456. Lastly, all three compositions are different and not comparable to the release profile of lidocaine alone since their 1f values are

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Lidocaine Mole Fraction f1 f2 1.0000 0.9936 0.9812 0.9456 x x 112.5 24.45 x x 205.0 20.77 x x 81.09 25.27 x x 23.05 48.88 x x 6.719 67.07 x x 12.04 54.71

Table 2.5 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of lidocaine and lidocaine/ poloxamer 407 binary mixtures.

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greater than 15 and their f2 values are less than 50 (see Table 2.5). This evaluation therefore suggests that poloxamer 407 contributes to the release mechanism by which lidocaine is released from the particles of binary mixtures containing lidocaine and poloxamer 407 created using the RESS method.

2.4.1.4 Solubility of lidocaine in the presence of poloxamer 407

The aqueous solubility of lidocaine and of lidocaine in the presence of poloxamer 407 was measured to determine the effect of poloxamer 407 on the aqueous solubility of lidocaine. The aqueous solubility of lidocaine alone was determined to be 3.35 mg mL-1. Figure 2.15 shows that as the concentration of poloxamer 407 increases, the solubility of lidocaine also increases linearly with a slope of 0.7289 and r2 value of 0.9940. Since poloxamer 407 has a relatively low critical micelle concentration (cmc), i.e., 0.08 % w/w at 30 C, the study was performed using poloxamer 407 concentrations greater than the cmc (113).

Therefore the concentrations of lidocaine in solution in the presence of the poloxamer are apparent solubilities, not true solubilities due to micellar solubilization.

2.4.1.5 Fourier transform infrared (FTIR) analysis

The FTIR spectra of particles for lidocaine and poloxamer 407 and concentrations thereof, all of which were prepared by RESS processing are

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12 ) ) -1 ) )

-1 10

4 Conc. of Lidocaine (mg ml

Conc. of Licocaine (mg L (mg of Licocaine Conc. 2

0 0 2 4 6 8 10 12 14

% w/w of Poloxamer 407 in water

Figure 2.15 Apparent solubilities of lidocaine in the presence of poloxamer 407 in water (r2 = 0.9920), n = 2.

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shown in Figure 2.16, and their characteristic wavenumbers are given in Table

2.6. As the amount of lidocaine increases, its characteristic absorption bands appear and become more intense, while those of poloxamer 407 gradually decrease in intensity. For example, the characteristic amine (3249 cm-1), C=O

(1663 cm-1), and amide (1492 cm-1) absorption bands of lidocaine gradually become less intense as poloxamer concentration increases. There are small shifts at the wavenumbers corresponding to -C-C-, -C=C-, and –C-O-C- stretches

(see Table 2.6), which may be due to changes in bonding at the neighboring amine group as shown in Figure 2.17. The shift in N-H stretching from 3249 cm-1 for lidocaine to 3257cm-1 for the 0.9456 mole fraction lidocaine system may be due to lidocaine bonding with poloxamer 407. Lidocaine is known to hydrogen bond with itself, i.e., to form a dimer in both the solid state and in solution (see

Figure 2.18) (115 and 116). If lidocaine dissolves in poloxamer 407, forming a new hydrogen bond, then the overall number of lidocaine-lidocaine hydrogen bonds will be reduced. This would result in a change at a site of hydrogen bonding, i.e., a shift in N-H stretching. It has been found that nonionic surfactants such as poloxamer 407 favor hydrogen bonding of the ether oxygen with hydroxyl groups of polymers such as Carbopol® (117, 118). Kanis, et al.

(117) indicated that a shift in the free hydroxyl groups of Carbopol® from 3550 cm–1 to 3490 cm–1 is the result of the POE/Carbopol® interaction, i.e., a OH···O-C hydrogen bond is formed. Barreiro-Iglesias, et al. (118) also reported a shift in the carbonyl stretch of the carboxylic group of Carbopol® from 1700 cm–1 to 1730 cm–1 as poloxamer 407 concentration increased, indicating a reduction of internal

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Figure 2.16 FTIR spectra of lidocaine, poloxamer 407, and lidocaine/poloxamer 407 compositions.

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Description Lidocaine Lidocaine Lidocaine Lidocaine MF=1.0000 MF=0.9936 MF=0.9812 MF=0.9456 N-H 3249.83 3249.83 (0) 3249.83 (0) 3257.98 (8.15) stretching -CH, -CH2, - 2968.52 2968.52 (0) 2968.52 (0) CH3 2801.36 2797.28 (4.08) 2797.28 (4.08) stretching amide I 1663.87 1663.87 (0) 1663.87 (0) 1663.87 (0)

amide II 1492.64 1496.72 (4.08) 1496.72 (4.08) 1496.72 (4.08) 1382.56 1288.78 1207.25 1207.25 (0) aromatic 762.85 762.85 (0) 762.85 (0) 762.85 (0) -C-H- Poloxamer 407 MF=1.0000 -CH 2886.98 2886.98 (0) 2886.98 (0) -C-C- 1464.1 1464.1 (0) 1341.79 1341.79 (0) 1341.79 (0) 1280.63 1280.63 (0) 1280.63 (0) -C-O-C- 1105.32 1109.4 (4.08) 1109.4 (4.08) stretch 962.63 962.63 (0) 962.63 (0) 840.32 840.32 (0) 840.32 (0)

Table 2.6 FTIR analysis of lidocaine and compositions of lidocaine and poloxamer 407. Differences in wavenumber from lidocaine and poloxamer 407 are indicated in parenthesis.

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CH3 CH3 CH3 CH3 O O

C N CH3 C N CH3 N C N C H2 H2 H3C CH3 H H CH3 H H2 2 H2 C O H H2 C H H2 2 O CH C O 2 C C O C H C C O C C HO C O H H2 C O H2 2 C O C H2 H H2 H2 2 H x H3C CH3 H H C H x y 2 3 N C H2 N CH3 C N C N CH3 C O CH3 O CH3 CH3 CH3

blue = lidocaine black = poloxamer

Figure 2.17 Schematic molecular structures of lidocaine and poloxamer showing hydrogen bonded interactions.

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H3C

H3C N H3C CH2

C O N

CH3 H H N O H3C C

H2C

CH3 N CH3

CH3

Figure 2.18 Schematic diagram of lidocaine dimer (from ref. 115).

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hydrogen bonding and the formation of new bonds. It may be possible therefore for hydrogen bonding to occur between the lidocaine amine group and the ether oxygen of the polyoxyethylene segment of poloxamer 407 (see Figure 2.17).

Figure 2.17 shows representative sites of hydrogen bonding between the amine moiety of lidocaine and the ether oxygen of a poloxamer. Repeated units of the polyoxyethylene segments can allow additional lidocaine molecules to hydrogen bond. Furthermore, in three dimensions the lidocaine molecules can rotate and orient themselves so as to minimize steric hindrances (see Figure 2.19). For example, in Figures 2.19 a and d lidocaine is rotated ~ 90 ° and is perpendicular to the poloxamer chain. This orientation would allow several lidocaine molecules to align themselves side by side and bond to poloxamer. Alternatively, lidocaine may be oriented parallel to the poloxamer chain as shown in Figure 2.19 c. This configuration suggests that steric properties do not interfere with lidocaine molecules hydrogen bonded to the polyoxyethylene segment next to a polyoxypropylene segment, at least not when the poloxamer is extended linearly.

However, rotation of lidocaine ~180 ° from the orientation shown in Figure

2.19 a to the configuration shown in Figure 2.19 b would result in interference of the methyl group of the polyoxypropylene segment with the methyl group from the aromatic ring of lidocaine as shown by the circle. Due to the close proximity of the methyl group of the polyoxypropylene segment, hydrogen bonding between lidocaine and the polyoxypropylene segment is therefore less likely to occur because of steric hindrance.

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a. b.

c. d.

red = oxygen blue = nitrogen grey = carbon white = hydrogen

Figure 2.19 Schematic molecular structures of lidocaine and a poloxamer showing hydrogen bonding as functions of lidocaine molecular orientation. Key: a., rotated lidocaine perpendicular to poloxamer chain; b., less favorable lidocaine orientation; c., favorable lidocaine orientation; d., rotated lidocaine perpendicular to poloxamer chain.

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2.4.2 Lidocaine, poloxamer 188, and lidocaine/poloxamer 188

compositions

2.4.2.1 Differential scanning calorimetry

Poloxamer 188 differs from poloxamer 407 in that it contains polyoxypropylene segments that are about half the length of those in poloxamer

407, i.e., 27 versus 56 repeat units, and a polyoxyethylene segment that is smaller in length, 80 and 101 units, respectively (see Figure 2.3). The purpose for studying lidocaine and poloxamer 188 compositions is to determine what effect the length of the polyoxypropylene segment has on potential intermolecular interactions and therefore on the formation of solid solutions.

DSC of these particulate systems was similar to that of poloxamer 407 and lidocaine/poloxamer 407 systems (compare Figures 2.6 and 2.20). Like poloxamer 407, poloxamer 188 has a melting point between 52-57 °C (Figure

2.20 b). A summary of the thermal properties of lidocaine and poloxamer 188 compositions is given in Table 2.7. Figure 2.20 shows representative endotherms for the various lidocaine/poloxamer 188 systems, which were analyzed in duplicate by using particles from separately processed batches prepared by the RESS method. In the presence of lidocaine, the melting point of poloxamer 188 is depressed from 53 °C to 48 °C, but remains essentially constant over the compositional range, indicating that lidocaine is functioning as

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Oct. 2002 Lidocaine / Poloxamer 188 a.

b. c. d.

Figure 2.20 Representative DSC thermograms of lidocaine / poloxamer 188 compositions prepared by RESS processing. Key: a., lidocaine (purple); b., poloxamer 188 (black); c., lidocaine MF=0.9936 (blue); d., lidocaine MF=0.9812 (red); and e., lidocaine MF=0.9456 (green).

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Mole Fraction Melting Point, °C Enthalpy, J g-1 of Lidocaine Lidocaine Poloxamer Lidocaine Poloxamer 188 188 1.0000 68.3±0.00 - 71.6±0.42 0.00±0.00 0.9952 68.3±0.07 47.9±0.00 60.7±0.92 10.8±2.40 0.9936 68.1±0.14 48.0±0.28 58.5±0.14 13.9±0.78 0.9874 67.8±0.21 48.4±0.00 48.1±0.14 27.3±0.07 0.9812 65.4±0.64 48.8±0.28 26.6±4.38 56.5±5.80 0.9720 60.1±1.13 48.7±0.07 9.90±0.71 77.9±2.40 0.9456 - 48.7±0.42 0.00±0.00 113±6.93 0 - 53.1±0.28 0.00±0.00 129±2.40

Table 2.7 Thermal properties of lidocaine and poloxamer 188 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, (n = 2).

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an impurity in the systems (50) (Figures 2.20 c-e). On the other hand, the melting point and enthalpy of lidocaine gradually decrease as its mole fraction decreases, i.e., the concentration of poloxamer 188 increases. For systems with a lidocaine mole fraction of 0.9936, there are two endotherms near 68 °C and 48

°C with enthalpies of approximately 48 J g-1 and 27 J g-1, respectively, which represent lidocaine and poloxamer 188 (Figure 2.20 c). As the mole fraction of lidocaine decreases upon the addition of increasing amounts of poloxamer 188, the endotherm for lidocaine progressively decreases in magnitude while the endotherm for poloxamer 188 becomes larger (see Figures 2.20 c and d). At lidocaine mole fraction 0.9456 there is only one endotherm present near 48 °C with an enthalpy of 113 J g-1, which is therefore assigned to poloxamer 188

(Figure 2.20 e), suggesting the formation of a solid solution.

Figure 2.21 is a phase diagram of the enthalpies for lidocaine and poloxamer 188 for lidocaine / poloxamer 188 systems as functions of lidocaine mole fraction. Similar to Figure 2.8 for lidocaine / poloxamer 407 systems (see section 2.4.1.1), Figure 2.21 shows that changes in enthalpies are dependent on concentration, whereas increases in the amounts of poloxamer 188 result in progressively larger enthalpies due to the presence of increasing amounts of poloxamer 188. Conversely, reductions in the amounts of lidocaine result in corresponding decreases in enthalpies, or in the amounts of excess lidocaine, leading to the disappearance of the endotherms, indicating the apparent formation of a solid solution. For example, from the enthalpies in Table 2.7 it was

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140 140

120 120

100 100

80 80

60 60 Enthalpy (J/g)

40 40

Lidocaine 20 Poloxamer 188 20 Experimental Total Enthalpy

0 0 0.04 0.08 0.92 0.960.00 1.00 Mole Fraction Lidocaine

Figure 2.21 Phase diagram of lidocaine / poloxamer 188 compositions (1st DSC heating step, data from Table 2.7)

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determined that 13.8 % lidocaine is in excess at 0.9720 mole fraction lidocaine, compared to 84.8 % at 0.9952 mole fraction. Conversely, the percent of poloxamer 188 increases from 8.3 % at 0.9952 mole fraction to 87.6 % at 0.9456 mole fraction. Linear regressions of the enthalpies of lidocaine indicate a linear relationship with decreases in excess lidocaine (see Figure 2.22). Table 2.7 indicates that solid solution formation should occur between 0.9720 and 0.9456 mole fraction lidocaine. By including the zero enthalpy obtained for 0.9456 mole fraction lidocaine, the linear regression (r2 = 0.9342) shown in Figure 2.22 (red line) predicts solid solution formation at 0.9552 mole fraction lidocaine. However, if the zero enthalpy point is eliminated, the prediction would be that of the linear regression shown by the blue line in Figure 2.22, (r2 = 0.9943) and the solid solution should form at 0.9676 mole fraction lidocaine. Based on this information, the phase diagram in Figure 2.21 therefore indicates that solid solutions of lidocaine in poloxamer 188 exist in the region below 0.9676 mole fraction lidocaine. Conversely, at higher mole fractions a solid solution of lidocaine coexists in equilibrium with excess crystalline lidocaine dispersed within the matrix.

Since the thermal analyses of particles of lidocaine/poloxamer 407 and lidocaine/poloxamer 188 are similar, it can be concluded that changing the length of the polyoxypropylene segment of the poloxamers has no apparent effect on the thermal properties of lidocaine. The overall magnitude of the

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80 80

60 60

40 40

20 20 Enthalpy (J/g) of Lidocaine

0 0 0.04 0.08 0.92 0.960.00 1.00 Mole Fraction Lidocaine

Figure 2.22 Phase diagram of lidocaine from lidocaine / poloxamer 188 compositions showing linear regressions, r2 = 0.9943 (blue) and r2 = 0.9342 (red) (data from Table 2.7).

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potential bonding between lidocaine and poloxamers are therefore not dependent on the length of the polyoxypropylene segment, but on the length of the polyoxyethylene segments, where hydrogen bonding primarily occurs.

2.4.2.2 Scanning electron microscopy

After RESS processing, poloxamer 188 forms aggregates of spherical structures with small invaginations on the surface (see Figure 2.23). Fractured particles show cavities in their interiors resulting from the expansion of supercritical carbon dioxide as it returns to atmospheric pressure at the end of the RESS process. After RESS processing, compositions containing poloxamer

188 and lidocaine show similar features (see Figure 2.24) to those systems containing poloxamer 407 as shown in Figures 2.12 and 2.13. The morphological appearance of particles containing lidocaine mole fractions 0.9456 and 0.9812 indicate that the presence of poloxamer 188 produces the same structures as did poloxamer 407, i.e., spherical shapes, smooth surfaces with holes, and cavities in the interiors (Figures 2.24 a-d). Fractured particles also do not show the presence of lidocaine crystals within the interior structures and it may be assumed therefore that the particles consist of homogeneous, or near homogeneous, mixtures of lidocaine and poloxamer 188. As the concentration of lidocaine increases, the appearance of the particles becomes rough and textured

(Figures 2.24 e-f) similar to that of lidocaine in Figure 2.11 a. These latter

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a. b.

c. d.

Figure 2.23 Scanning electron photomicrographs of poloxamer 188. Key: a., poloxamer 188 as received; b., fractured particles of poloxamer 188 as received; c., poloxamer 188 particles prepared by RESS processing; d., fractured particles of poloxamer 188 after RESS processing.

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a. b.

c. d.

e. f.

Figure 2.24 Scanning electron photomicrographs of lidocaine and poloxamer 188 compositions prepared by RESS processing. Key: a. and b., lidocaine MF=0.9456; c. and d., lidocaine MF=0.9812; e. and f., lidocaine MF=0.9936.

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particles are less spherical in shape and instead of pores on the surface have a sponge-like structure, reflecting the influence of lidocaine on the physical structure of the particles.

2.4.2.3 Release of lidocaine from particles containing poloxamer 188

The release of lidocaine from lidocaine/poloxamer 188 particles prepared by RESS processing is shown in Figure 2.25. As in the case of the lidocaine/poloxamer 407 systems, the dissolution rate of lidocaine processed with poloxamer 188 is enhanced compared to that of lidocaine alone. For the first ~8 minutes the release profiles of lidocaine and of the lidocaine/poloxamer

188 systems were similar with approximately 35 % release of lidocaine. Beyond this time mark, plots of the release of lidocaine (except for MF=0.9456) undergo a gradual decrease in slope. The water-soluble poloxamer 188 likewise facilitated an increased area of contact between lidocaine and the dissolution medium through a reduction in interfacial tension and contact angle, thereby increasing the dissolution rate. Release of lidocaine was most efficient from particles containing lidocaine mole fraction 0.9812, as ~100 % release was achieved within 30 minutes. At 0.9936 mole fraction lidocaine, ~100 % release occurred after approximately 45 minutes. This delay was probably due to the smaller amount of poloxamer 188 present in the system. Alternatively, lidocaine release from particles containing the largest amount of poloxamer, i.e., lidocaine

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120

100

40 %Release Lidocaine of Lidocaine MF = 0.9456 20 Lidocaine MF = 0.9812 Lidocaine MF = 0.9936 Lidocaine MF = 1.0000 0 0 20 40 60 80 100 120 140

Time (minutes)

Figure 2.25 Release of lidocaine from lidocaine / poloxamer 188 particles prepared by RESS processing as functions of composition. (Total weight of each sample = 100 mg, n =3).

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mole fraction 0.9456, was about 79 % when zero slope was attained. Similar to the lidocaine/poloxamer 407 systems, the apparent loss of lidocaine at this lower concentration may be the result of a portion of the lidocaine being solubilized by poloxamer 188 micelles, which in turn causes the lidocaine to evade HPLC detection.

It should be noted that there are several differences in the release of lidocaine from particles of lidocaine and poloxamer 188 compared to lidocaine and poloxamer 407 (see Figures 2.14 and 2.25). For the 0.9456 mole fraction lidocaine/poloxamer 188 solid solution systems ~79 % of the lidocaine was released as shown in Figure 2.25, whereas for the lidocaine/poloxamer 407

~90 % release was attained (see Figure 2.14). The difference in the amount of lidocaine released between the two systems may be due to a different micellar structure. As previously mentioned, the length of the polyoxypropylene segment of poloxamer 188 is approximately half that of poloxamer 407. The shorter polyoxypropylene segment would allow the longer polyoxyethylene chains to be in closer proximity to each other, resulting in stronger chain-chain interactions.

As a result, it is probable that poloxamer 188 is packing more tightly within the micellar structure than would poloxamer 407 at this mole fraction. This tighter packing could create a denser barrier to HPLC detection than would be expected with poloxamer 407, resulting in an even further decrease in the apparent amount of lidocaine released. Furthermore, tighter packing of the poloxamer 188 would create a higher density of the hydrophobic polyoxypropylene segments

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within the interior of the micelle, producing a more favorable environment for the solubilization of the hydrophobic lidocaine. Therefore, solubilization of lidocaine would be more efficient in micelles of poloxamer 188 than in poloxamer 407.

An apparent increase in micellar solubilization of lidocaine would also result in a decrease in the amount of lidocaine released from the 0.9936 and

0.9812 mole fraction lidocaine systems compared to that from poloxamer 407 systems. It was previously proposed that precipitation of lidocaine from supersaturated solutions of lidocaine resulting from dissolution of these particulate systems caused scattering of light in the flow cell producing false values of absorption. Due to the apparent greater efficiency of solubilization in the poloxamer 188 systems, less drug would be precipitated. Therefore, the amount of light scattering due to lidocaine precipitation in the poloxamer 188 systems would not be as effective in increasing the apparent release of lidocaine above 100 %. Lastly, the decrease in the initial slope of the release of lidocaine from the poloxamer 188 systems compared to that of poloxamer 407 systems would reflect smaller amounts of free lidocaine in solution due to a potential increase in micellar solubilization.

As in the previous case, a comparison of the dissolution profiles were performed using the “Model Independent Approach Using a Similarity Factor” as described in the Center for Drug Evaluation and Research (CDER)Guidance for

Industry: Dissolution Testing of Immediate Release Solid Oral Dosage Forms

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(114). Using equations 2.1 and 2.2 (see section 2.4.1.3) the difference factors

(f1) and similartity factors (f2) were calculated and quantitative comparison of the release curves was obtained (114). Table 2.8 gives the calculated f1 and f2 values for the various binary mixtures of lidocaine and poloxamer 188 based on their respective release data. These samples were collected using different time points compared to that of lidocaine alone and therefore cannot be compared to the release profile for lidocaine according to the model independent approach.

However, a comparison of the release profiles for the lidocaine/poloxamer 188 binary mixtures indicates that the release profiles for lidocaine mole fraction

0.9936 and 0.9456 are similar and meet both f1 and f2 criteria, i.e., f1 ~ 0-15 and f2 ~ 50-100, for having similar release profiles. This similarity however only extends for the first 15 minutes since beyond this time point the percent release for lidocaine from lidocaine mole fraction 0.9936 is greater than 85% and additional time points cannot be considered in equations 2.1 and 2.2. Similarly, at the other compositions only 2 or 3 time points could be used due to their rapid release rates. Therefore, due to a lack of usable data an adequate evaluation of the similarities between the release profiles cannot be made.

2.4.2.4 Fourier transform infrared (FTIR) analysis

The FTIR spectra of particles of lidocaine, poloxamer 188 and various concentrations thereof, all of which were prepared by RESS processing are shown in Figure 2.26, and their characteristic wavenumbers are given in

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Lidocaine Mole Fraction f1 f2 1.0000 0.9936 0.9812 0.9456 x x 27.63 47.42 x x 10.19 60.12 x x 15.48 52.12

Table 2.8 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of lidocaine/ poloxamer 188 binary mixtures.

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Figure 2.26 FTIR spectra of lidocaine, poloxamer 188, and lidocaine/poloxamer 188 compositions.

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Table 2.9. Since both poloxamers 407 and 188 consist of the same functional groups, similar FTIR spectra were found. In addition, since they both have approximately the same number of polyoxyethylene units, the spectra of lidocaine and poloxamer 188 at various concentrations mimicked those of lidocaine and poloxamer 407 (see Figure 2.16 and Table 2.6 for comparison). As the amounts of poloxamer 188 increased, its characteristic absorption bands at, for example ~2886 cm-1 and 1105 cm-1, become more intense while those of lidocaine begin to decrease. A similar shift was found at the N-H stretch for lidocaine, from 3249 cm-1 to 3257 cm-1, likewise indicating that this is a possible site for hydrogen bonding via the ether oxygen of the polyoxyethylene segment

(see Figure 2.17).

2.4.3 Lidocaine, poloxamer 403, and lidocaine/poloxamer 403

compositions

2.4.3.1 Differential scanning calorimetry

Unlike poloxamers 407 and 188, poloxamer 403 is a semi-solid at room temperature. It has the same number of polyoxypropylene units and half the number of polyoxyethylene units as poloxamer 407 (see Figure 2.3). Compared to poloxamer 188, poloxamer 403 also has half the number of polyoxyethylene units and approximately twice the number of polyoxypropylene units. DSC of

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Description Lidocaine Lidocaine Lidocaine Lidocaine MF=1.0000 MF=0.9936 MF=0.9812 MF=0.9456 N-H 3249.83 3249.83 (0) 3249.83 (0) 3257.98 (8.15) stretching -CH, -CH2, - 2968.52 2968.52 (0) 2968.52 (0) 2968.52 (0) CH3 2801.36 2797.28 (4.08) 2797.28 (4.08) stretching amide I 1663.87 1663.87 (0) 1663.87 (0) 1663.87 (0)

amide II 1492.64 1496.72 (4.08) 1496.72 (4.08) 1496.72 (4.08) 1382.56 1382.56 (0) 1288.78 1288.79 (0.01) 1207.25 1207.25 (0) aromatic 762.85 762.85 (0) 762.85 (0) 762.85 (0) -C-H- Poloxamer 188 MF=1.0000 -CH 2886.98 2886.98 (0) 2886.98 (0) -C-C- 1464.1 1464.1 (0) 1341.79 1341.79 (0) 1341.79 (0) 1280.63 1280.63 (0) 1280.63 (0) 1239.86 1239.86 (0) 1146.09 1146.09 (0) 1146.09 (0) -C-O-C- 1105.32 1109.4 (4.08) 1109.4 (4.08) stretch 1056.4 1056.4 (0) 962.63 962.63 (0) 962.63 (0) 840.32 840.32 (0) 840.32 (0)

Table 2.9 FTIR analysis of lidocaine and compositions of lidocaine and poloxamer 188. Differences in wavenumber from lidocaine and poloxamer 188 are indicated in parenthesis.

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poloxamer 403 as received indicated a primary melting point at approximately

34 °C. The broader endotherm and lower enthalpy of fusion compared to both poloxamers 407 and 188 (44.1 J g-1 vs. 114.7 J g-1 and 128.9 J g-1) (see Figures

2.27, 2.6 a, and 2.20 a) can be attributed to weaker chain-chain interactions due to the shorter polyoxyethylene segments, which also increases structural fluidity making poloxamer 403 semi-solid at room temperature. Supercritical fluid processing produced a paste of poloxamer 403 with a melting point of 37 °C, which is higher than the melting point of unprocessed poloxamer 403 (34 °C)

(see Figure 2.27). DSC of poloxamer 403 as received shows a split broad endotherm. The lower temperature portion of the endotherm probably represents melting of less ordered structures and/or melting of shorter chain molecules, since poloxamer molecular weights are an average of various similar chain lengths. The lower melting endotherm disappears upon RESS processing suggesting that a volatile impurity was removed by RESS processing, and/or that the instantaneous precipitation of the particles induced the formation of more ordered structures of the polymers due to stronger chain-chain interactions (see

Figure 2.27). More ordered structures might be produced by way of equal and opposite forces as carbon dioxide expands. Upon expansion of carbon dioxide, equal and opposite forces could contribute to the compaction of the poloxamer

403 molecules during precipitation to form paste-like masses with more ordered structures than found in the unprocessed materials. Consequently, DSC analysis of RESS processed poloxamer 403 results in an endotherm with a single higher melting point.

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a. Poloxamer 403 as received

b. Poloxamer 403 after RESS processing

Figure 2.27 Representative DSC thermograms of poloxamer 403. Key: a., poloxamer 403 as received; b., poloxamer 403 after RESS processing.

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Representative endotherms for particles of various concentrations of lidocaine and poloxamer 403 processed with supercritical carbon dioxide are shown in Figure 2.28. Thermal properties for the lidocaine and poloxamer 403 compositions are summarized in Table 2.10. For lidocaine mole fraction 0.9936 the thermogram shows an endotherm for lidocaine at approximately 68 °C, and a small endotherm at ~34.9 °C for poloxamer 403 (Figure 2.28 d). As the amount of poloxamer 403 increases the areas of its endotherms become larger, such that the enthalpy increases from 2.8 J g-1 at lidocaine mole fraction 0.9936 to

15.9 J g-1 at lidocaine mole fraction 0.9456 (Figures 2.28 b-d). Simultaneously, the endotherm for lidocaine decreases in both size and in melting point while becoming broader. Unlike the thermograms for poloxamers 407 and 188 at lidocaine mole fraction 0.9456, which were solid solutions, there are endotherms for each component, specifically a melting point at 60.6 °C for lidocaine and

32.6 °C for poloxamer 403 (see Figure 2.28 b). At this mole fraction the endotherm for lidocaine has decreased significantly in magnitude and has shifted toward the endotherm for poloxamer 403, i.e., an enthalpy of 19.9 J g-1 compared to 56.6 J g-1 at 0.9936 mole fraction lidocaine. As previously discussed in section

2.4.1.1, differences in enthalpies can be attributed to differences in the amount of free lidocaine available for detection based on lidocaine-lidocaine interactions. In this context, since an endotherm for lidocaine exists at this mole fraction (0.9456 mole fraction lidocaine), it can be assumed that significant amounts of free lidocaine exist and that not all of it is bonding with poloxamer 403. In addition, at the point where the two endotherms meet there is a decrease in heat capacity,

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a. Poloxamer 403 from RESS b. Lidocaine / Poloxamer 403, MF=0.9456

c. Lidocaine / Poloxamer 403, d. Lidocaine / Poloxamer 403, MF=0.9812 MF=0.9936

Figure 2.28 Representative DSC thermograms of lidocaine/poloxamer 403 compositions prepared by RESS processing. Key: a., poloxamer 403 from RESS; b., lidocaine/poloxamer 403, MF=0.9456; c., lidocaine/poloxamer 403, MF=0.9812; d., lidocaine/poloxamer 403, MF=0.9936.

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Mole Fraction Melting Point, °C Enthalpy, J g-1 of Lidocaine Lidocaine Poloxamer Lidocaine Poloxamer 403 403 1.0000 68.3±0.00 - 71.6±0.42 0.00±0.00 0.9952 68.0±0.00 - 59.2±1.63 0.00±0.00 0.9936 67.8±0.07 34.9±0.00 56.6±1.13 2.80±0.57 0.9874 67.4±0.07 35.2±3.32 50.8±1.56 4.60±1.91 0.9812 66.8±0.00 34.1±1.13 44.2±0.57 7.50±1.70 0.9720 64.2±1.41 32.8±0.21 29.7±3.39 14.8±1.20 0.9456 61.9±1.84 33.3±0.92 19.9±4.60 15.9±2.76 0 - 37.2±0.00 0.00±0.00 44.1±2.19

Table 2.10 Thermal properties of lidocaine and poloxamer 403 processed with SCCO2 at 35 C, 7100 psi, and restrictor temperature of 20 C, (n = 2).

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or an incomplete return to baseline (see Figure 2.28), suggesting a merger or overlap of the two endotherms due to bonding between the two components as shown in Figure 2.17. Essentially, the DSC thermograms show that as the proportion of poloxamer 403 increases more lidocaine is bonding with it. The degree of hydrogen bonding is dependent on the length of the polyoxyethylene chain, where as lidocaine forms hydrogen bonds with the poloxamer. Since the polyoxyethylene segment of poloxamer 403 is approximately half the length of the polyoxyethylene segments of poloxamers 407 and 188, there are fewer sites for the lidocaine to hydrogen bond. Consequently, for there to be sufficient sites for lidocaine to hydrogen bond the amount of poloxamer 403 must increase.

Figure 2.29 is a phase diagram of the enthalpies for lidocaine and poloxamer 403 as functions of lidocaine mole fraction for lidocaine/poloxamer

403 systems. Similar to lidocaine/poloxamer 407 and lidocaine/poloxamer 188 systems, the enthalpies of poloxamer 403 and lidocaine follow the same trends.

The enthalpies of poloxamer 403 increase with increasing amounts of poloxamer

403, while that of lidocaine decrease due to the formation of solid solutions, which result in progressive decreases in the amounts of excess lidocaine.

However, unlike previous systems, the phase diagram for lidocaine/poloxamer

403 does not show a crossover between the enthalpies of lidocaine and poloxamer 403 (see Figure 2.29). In addition, based on the data in Table 2.10, there is no region suggestive of a solid solution, i.e., where only one endotherm appears in the DSC thermogram. However, linear regression of the enthalpies of

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80 80 Lidocaine Poloxamer 403 Experimental Total Enthalpy

60 60

40 40 Enthalpy (J/g) Enthalpy

20 20

0 0 0.04 0.08 0.92 0.960.00 1.00 Mole Fraction Lidocaine

Figure 2.29 Phase diagram of lidocaine / poloxamer 403 compositions (1st DSC heating, data from Table 2.10).

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lidocaine (red line) predicts the formation of a solid solution at 0.9371 mole fraction lidocaine (r2 = 0.9523) as shown in Figure 2.30. So as not to bias the linear regression by a single value, which may be an outlier, linear regression of the data was performed omitting the value at 0.9456 mole fraction lidocaine (blue line, r2 = 0.9859). This regression indicates that the formation of a solid solution should occur at 0.9538 mole fraction lidocaine.

2.4.3.2 Scanning electron microscopy

Since poloxamer 403 is a semi-solid, it was not possible to obtain scanning electron microscopy (SEM) photomicrographs. However, paste-like masses of lidocaine and poloxamer 403 formed by RESS processing could be studied by SEM. Such particles were found to have unique surface characteristics dependent on concentration (see Figures 2.31 a-f). For masses with the largest concentration of poloxamer 403, i.e., 0.9456 mole fraction lidocaine, the surfaces are generally smooth. However, on these surfaces smaller particles may be protruding from the interior or it may be that aggregates of small lidocaine particles may be coated with the poloxamer 403 (see Figures

2.31 a and b). As lidocaine concentration increases, the masses appear to grow as layers upon each other (see Figures 2.31 c and d). In addition, there are small cavities created by the layering effect as can be seen in Figures 2.31 e and f. These cavities however are different from those found in poloxamers 407 and

188

140

80 80

60 60

40 40

20 20 Enthalpy Lidocaine (J/g) of

0 0 0.0 1.0 Mole Fraction Lidocaine

Figure 2.30 Phase diagram of lidocaine from lidocaine / poloxamer 403 compositions showing linear regressions, r2 = 0.9859 (blue) and r2 = 0.9523 (red) (data from Table 2.10).

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a. b.

c. d.

e. f.

Figure 2.31 Scanning electron photomicrographs of lidocaine and poloxamer 403 compositions prepared by RESS processing. Key: a. and b., lidocaine MF=0.9456; c. and d., lidocaine MF=0.9812; e. and f., lidocaine MF=0.9936.

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systems, which resulted from the rapid expansion of supercritical carbon dioxide at the end of the RESS process. In this context, the expansion of supercritical critical carbon dioxide may not create the same types of pores because poloxamer 403 is a semi-solid and may flow to close the pores as a response to the shear force of the exiting supercritical carbon dioxide.

Upon comparison, the size of the masses of lidocaine/poloxamer 403 are larger than particles from lidocaine/poloxamer 407 and lidocaine/poloxamer 188 systems (see Figures 2.13 and 2.24). Particles produced by RESS processing of lidocaine and poloxamers 407 or 188 are discrete particles of approximately 10-

20 μm in size. Conversely, RESS processing of lidocaine and poloxamer 403 did not produce discrete particles, but rather masses with a paste-like consistency that increased with increasing amounts of poloxamer 403. Based on the SEM photomicrographs, these paste-like masses were at least 100 μm in size.

2.4.3.3 Release of lidocaine from particles containing poloxamer 403

Due to the semi-solid nature of poloxamer 403 and the paste-like consistency of the lidocaine/poloxamer 403 systems, dissolution studies did not produce results that can be evaluated in parallel to the other systems, i.e., the lidocaine/poloxamer 407 and lidocaine/poloxamer 188, since they were not particulate in nature. As such, these paste-like masses of lidocaine and poloxamer 403 have limited surface areas that present a mechanical obstacle to

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the dissolution medium. Furthermore, dissolution of lidocaine from these paste- like masses using the flow-through method would be erroneous since it would be more of an erosion process than dissolution.

2.4.3.4 Fourier transform infrared (FTIR) analysis

Figure 2.32 and Table 2.11 show the FTIR spectra and characteristic wavenumbers of lidocaine, poloxamer 403, and three compositions, all prepared by RESS processing. Since the poloxamers contain the same functional groups, similar absorption bands can be seen in the FTIR spectra for poloxamer 403 as in the case for poloxamers 407 and 188. However, unlike the other two poloxamers, poloxamer 403 has only half the number of polyoxyethylene segments available for potential bonding with lidocaine. As such, the FTIR spectra of lidocaine/poloxamer 403 compositions did not show significant shifts suggestive of an interaction. Instead, the spectra show few to no changes in the absorption bands characteristic of lidocaine. As the poloxamer concentration increased, only the absorption band near 1105 cm-1 becomes more intense. At the concentration containing the most poloxamer, i.e., MF=0.9456 lidocaine, the absorption band near 2886 cm-1 was not as strong as in the IR spectra of

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Figure 2.32 FTIR spectra of lidocaine, poloxamer 403, and lidocaine/poloxamer 403 compositions.

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Description Lidocaine Lidocaine Lidocaine Lidocaine MF=1.0000 MF=0.9936 MF=0.9812 MF=0.9456 N-H 3249.83 3249.83 (0) 3249.83 (0) 3249.83 (0) stretching -CH, -CH2, - 2968.52 2968.52 (0) 2968.52 (0) 2968.52 (0) CH3 2801.36 2801.36 (0) 2801.36 (0) 2801.36 (0) stretching amide I 1663.87 1663.87 (0) 1663.87 (0) 1663.87 (0)

amide II 1492.64 1492.64 (0) 1492.64 (0) 1496.72 (4.08) 1382.56 1382.56 (0) 1288.78 1288.79 (0.01) 1288.79 (0.01) 1288.79 (0.01) 1207.25 1207.25 (0) 1207.25 (0) 1207.25 (0) aromatic 762.85 762.85 (0) 762.85 (0) 762.85 (0) -C-H- Poloxamer 403 MF=1.0000 -CH 2886.98 2886.98 (0) 2886.98 (0) -C-C- 1464.1 1464.1 (0) 1341.79 1341.79 (0) 1341.79 (0) 1239.86 1239.86 (0) -C-O-C- 1105.32 1109.4 (4.08) 1109.4 (4.08) 1109.4 (4.08) stretch 1056.4 1056.4 (0) 962.63 962.63 (0) 962.63 (0) 840.32 840.32 (0) 840.32 (0)

Table 2.11 FTIR analysis of lidocaine and compositions of lidocaine and poloxamer 403. Differences in wavenumber from lidocaine and poloxamer 403 are indicated in parenthesis.

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poloxamers 407 and 188 (see Figures 2.16 and 2.26). Differences in polyoxyethylene chain length therefore can affect the degree of lidocaine bonding with the poloxamers.

2.4.4 Summary: Comparison of lidocaine/poloxamer 407,

lidocaine/poloxamer 188, and lidocaine / poloxamer 403 particles

prepared by supercritical fluid processing

Solid solutions of lidocaine and poloxamer coexist along with excess lidocaine at concentrations above 0.9732 mole fraction lidocaine for poloxamer

407 and 0.9676 mole fraction for poloxamer 188. In a differential scanning calorimetry thermogram, excess lidocaine gives an endotherm representative of lidocaine-lidocaine interactions. On the other hand, differential scanning calorimetry of various lidocaine and poloxamer 407 or 188 compositions indicate that solid solutions generally form at lidocaine mole fractions below 0.9732 or

0.9676, respectively. This is seen by a single endotherm representative of the poloxamer and the absence of an endotherm for lidocaine. Lidocaine hydrogen bonds with poloxamer primarily along the polyoxyethylene chain and is therefore cannot bond with another lidocaine to form a dimer. FTIR analyses of these systems also indicate the presence hydrogen bonding of the lidocaine amine and poloxamer. Solid solution formation is dependent on the availability of sufficient sites for bonding. For lidocaine/poloxamer 403 systems, the number of lidocaine molecules hydrogen bonding to the poloxamer is primarily a function of the length

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of the polyoxyethylene chain. Fewer lidocaine molecules are bound to poloxamer 403 than with poloxamer 407, which differ in the length of their polyoxyethylene chains, but have essentially the same polyoxypropylene moieties. In this context, comparison of poloxamers 407 and 188 indicate that virtually the same number of lidocaine molecules were bound, despite the difference in their polyoxypropylene chain lengths.

Figure 2.33 is a plot of the mole fractions of lidocaine at zero enthalpies, i.e., the concentration at which solid solutions form, as functions of the polyoxyethylene chain length for each poloxamer. As shown in Figure 2.33, a linear relationship (r2 = 0.9934) exists between the mole fraction at which a solid solution forms and polyoxyethylene chain length. As the polyoxyethylene chain lengths decrease, fewer sites would be available for bonding with lidocaine.

Therefore, the lidocaine mole fraction at which a solid solution forms decreases as the poloxamer polyoxyethylene chain length decreases. Conversely, Figure

2.34 does not show a linear relationship between the mole fractions at which solid solutions form and polyoxypropylene chain length. However, even though poloxamers 407 and 403 have similar polyoxypropylene chain lengths (56 units), the mole fraction at which a solid solution should occur is less for poloxamer 403 than for poloxamer 407 (see Figure 2.34). Since the mole fraction is a function of the overall poloxamer chain length, it can be presumed that the lower mole fraction of poloxamer 403 is due to the difference in polyoxyethylene chain length. This indicates that lidocaine primarily bonds with the polyoxyethylene

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1.00

0.95 Poloxamer Poloxamer 407 Poloxamer 188 403

0.90

MoleFraction Lidocaine 0.85

0.80 0 50 100 150 200 Total Polyoxyethylene Chain Length

Figure 2.33 Relationship between lidocaine mole fraction at zero enthalpy and total polyoxyethylene chain length of poloxamers 407, 188, and 403, (r2 = 0.9934).

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1.00

0.98 Poloxamer 407 Poloxamer 188 0.96

Poloxamer 403 0.94 Mole Lidocaine Fraction 0.92

0.90 0 20 40 60 80 100 120 Polyoxypropylene Chain Length

Figure 2.34 Relationship between lidocaine mole fraction at zero enthalpy and polyoxypropylene chain length of poloxamers 407, 188, and 403.

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segment of poloxamer to form solid solutions. Scanning electron microscopy of particles of RESS processed lidocaine and poloxamers 407 or 188 show distinct morphological characteristics between the various concentrations. For concentrations with the highest mole fraction of poloxamer, particles tend to have smooth surfaces, which is a characteristic of the poloxamers in general. As the proportion of lidocaine increases the surfaces become more textured and develop rougher edges similar to that of lidocaine alone. Many of the particles also have small surface pores, which can be attributed to the effects of the expansion of supercritical carbon dioxide. It is interesting to point out that very small differences in lidocaine mole fractions were used and yet significant changes can be seen in the structural characteristics of particles containing lidocaine and poloxamer 407 or 188. The exceptions to these physical characteristics are the concentrations of lidocaine and poloxamer 403. Since poloxamer 403 is a semi-solid, concentrations of RESS processed lidocaine and poloxamer 403 produced particle masses with a paste-like consistency that increased in apparent viscosity with poloxamer 403 concentration.

The release of lidocaine from particles consisting of RESS processed lidocaine and poloxamers 407 or 188 was significantly enhanced compared to that from lidocaine alone. Due to the low critical micelle concentration of the poloxamers, micellar solubilization of lidocaine would occur at all concentrations during the dissolution process. As a result, the release of lidocaine could show an apparent loss of lidocaine. This was apparent for the solid solution at 0.9456

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mole fraction lidocaine, where the release profile of lidocaine attained a steady state below 100 % release. At lidocaine mole fractions 0.9812 and 0.9936, solid solutions exist along with crystalline lidocaine. It appears that crystalline lidocaine may be dissolving to form a saturated solution from which small particles are precipitating. These small particles could result in the scattering of light in the flow cell during detection leading to false absorption values. In the case of lidocaine/poloxamer 407, this scattering would be sufficient to produce an apparent total release of lidocaine greater than 100 %. For lidocaine/poloxamer

188 systems, an apparent greater efficiency of the micellar solubilization of lidocaine resulted in fewer particles of precipitated lidocaine and about 100 % release of lidocaine. Furthermore, a quantitative comparison of the release profiles of lidocaine/poloxamer 407 to that of lidocaine alone indicate that the profiles are not similar or equivalent.

2.4.5 Probucol, poloxamer 407, and probucol / poloxamer 407

compositions

2.4.5.1 Solubility of probucol in supercritical carbon dioxide

The solubility of probucol in supercritical carbon dioxide was determined at

75, 55, and 35 °C, and 7100 psi. Of these temperatures, the solubility was determined to be greatest at 75 °C and 7100 psi with 2.11 ± 0.31 % w/w probucol dissolving in supercritical carbon dioxide. Decreasing the processing

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temperature lowered the solubility of probucol in supercritical carbon dioxide from

2.11 ± 0.31 % w/w at 75 °C, to 0.62 ± 0.18 % w/w at 55 °C, and 0.21 ± 0.04 % w/w at 35 °C (see Figure 2.35). At higher temperatures the intramolecular forces of probucol are weakened and facilitate drug dissolution in the supercritical carbon dioxide. Consequently, larger amounts of probucol would be expected to dissolve in supercritical carbon dioxide at higher temperatures. However, because of the large melting temperature difference between probucol and poloxamer (127 °C vs. 56 °C), 75 °C was used to facilitate simultaneous processing of both compounds. At processing conditions of 75 °C, 7100 psi, and a restrictor temperature of ~40 °C, small particles of probucol form. Therefore, these conditions were used to generate small particles of probucol and of probucol/poloxamer binary systems via the RESS method.

2.4.5.2 Differential scanning calorimetry (DSC)

DSC thermograms of unprocessed and RESS processed probucol are shown in Figures 2.36 and 2.37. Unprocessed probucol shows a single melting endotherm at 127.3 °C, which corresponds with literature values for the melting point of probucol (95, 101). The DSC thermogram of probucol after RESS processing (Figure 2.37) shows two endotherms at 126.6 °C and 117.4 °C that represent probucol and its polymorph, respectively (101). Approximately 92 % of the probucol was converted to its polymorphic form upon RESS processing.

Since precipitation of drug from the supercritical solution occurs almost

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3.0

2.5

2.0

1.5

1.0 Probucol(%Solubility w/w) 0.5

0.0 30 40 50 60 70 80 Temperature (oC)

Figure 2.35 Solubilities of probucol in supercritical carbon dioxide at 7100 psi.

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Figure 2.36 Representative DSC thermogram of probucol as received.

155

Figure 2.37 Representative DSC thermogram of probucol after RESS processing (75 °C, 7100 psi, and restrictor temperature of 40 °C).

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instantaneously using the RESS method, it is likely that the polymorph of probucol does not convert to its more stable form during the ultra short expansion time period of the supercritical carbon dioxide.

DSC thermograms for probucol/poloxamer 407 compositions are shown in

Figure 2.38, while the melting points and enthalpies of fusion are listed in Table

2.12. Figure 2.38 shows representative thermograms for probucol and probucol/poloxamer 407 systems (all samples were analyzed in duplicate using separately processed batches of each sample). The endotherms for poloxamer

407 and probucol alone are shown in Figures 2.38 a and b, respectively, and were taken from Figures 2.6 b and 2.37. Figures 2.38 c through e represent different probucol/poloxamer 407 systems based on probucol mole fractions. At

0.9812 mole fraction probucol (Figure 2.38 c) there are two distinct endotherms: for probucol (Form I) and its polymorph (Form II) at 127 °C and 117 °C, respectively. There is no endotherm for poloxamer 407 at this concentration, which is likely due to the small quantity of poloxamer 407 present in the system, i.e., below the detection limits of the DSC. Endotherms for poloxamer 407 appear at compositions containing 0.9456 mole fraction probucol and lower (see

Figures 2.38 d and e). These endotherms are shifted to a lower melting point

(~52 °C), due to the presence of probucol as an impurity in the systems. At mole fraction 0.9456 the endotherm for probucol appears near 99 °C and is reduced in area (i.e., enthalpy) compared to those at higher mole fractions. For example, the enthalpies for Forms I and II of probucol at mole fraction 0.9456 are

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Probucol and Poloxamer 407

a. b. c. d. e.

Figure 2.38 Representative DSC thermograms of probucol/poloxamer 407 compositions prepared by RESS processing. Key: a., poloxamer 407 (purple); b., probucol (black); c., probucol MF=0.9812 (blue); d., probucol MF=0.9456 (red); e., probucol MF=0.9043 (green).

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Mole Melting Point, °C Enthalpy, J g-1 Fraction Probucol Poloxamer Probucol Poloxamer of Form I Form II 407 Form I Form II 407 Probucol 1.0000 127.2±0.00 116.7±0.00 - 14.2±0.00 63.0±0.00 0.00 0.9952 127.0±0.35 116.7±0.14 - 10.0±7.07 61.8±3.75 0.00 0.9936 126.7±0.00 116.8±0.07 - 3.50±0.71 64.7±0.57 0.00 0.9874 126.9±0.57 116.9±0.14 - 11.4±11.7 60.4±8.20 0.00 0.9812 126.6±0.00 116.7±0.35 - 13.4±0.28 58.3±0.92 0.00 0.9720 126.7±0.00 117.0±0.57 - 2.10±3.00 65.7±1.41 0.00 0.9456 125.5±0.00 110.9±4.10 55.1±0.35 0.50±0.71 25.0±13.2 43.4±13.2 0.9220 - 99.30±4.10 55.1±0.14 0.00 10.3±0.99 71.9±4.81 0.9043 - - 54.9±0.07 0.00 0.00 86.8±1.34 0 - - 55.6±0.21 0.00 0.00 115±2.26

Table 2.12 Thermal properties of probucol and poloxamer 407 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, (n = 2).

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approximately 0.5 and 25.0 J g-1, respectively, compared to 10.0 and 61.8 J g-1 at probucol mole fraction 0.9952 (see Table 2.12). At probucol mole fraction 0.9043

(Figure 2.38 e) no endotherm for probucol appears, however, one endotherm does appear near 55 °C and is assigned to poloxamer 407. Since the mole fraction of probucol in this system is large, but no endotherm appears, probucol is apparently molecularly dispersed and therefore bonding with poloxamer 407 to form a solid solution. In order to form a solid solution it is necessary for one component to be molecularly dispersed and interacting with the other component rather than with itself.

At 0.9456 mole fraction probucol, it can be concluded therefore that probucol bonds with poloxamer 407 to form a solid solution which coexists with unbound (excess) probucol. The formation of a solid solution is apparent in the

DSC thermogram (Figure 2.38), which shows decreases in enthalpy with increasing poloxamer 407 concentration leading to solid solution formation. At

0.9456 mole fraction probucol (Figure 2.38 d), unbound probucol is shown by endotherms for probucol at the high temperature end of the thermogram, along with an endotherm for poloxamer 407. These endotherms suggest that bonding between probucol and poloxamer 407 is involved in the formation of the solid solution. Based on previous work with lidocaine/poloxamer systems, it can be assumed that this bonding also occurs via the polyoxyethylene segment of poloxamer 407. Broman, et al., reported similar endotherms for solid dispersions of probucol and polyoxyethylene at 50:50 percent compositions by weight (96).

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Endotherms at 65 °C and 118 °C were assigned to the melting of polyoxyethylene and probucol (Form II), likewise suggesting bonding between the polyoxyethylene and probucol.

Figure 2.39 is a phase diagram of enthalpies for probucol and poloxamer

407 as functions of probucol mole fraction for probucol/poloxamer 407 systems.

Similar to the phase diagram of the lidocaine/poloxamer 407 systems (see Figure

2.8), the phase diagram for probucol/poloxamer 407 shows an increase in enthalpy of Form II of probucol as the amounts of probucol increase, while the enthalpies of poloxamer 407 decrease. In the region below 0.9043 mole fraction probucol, one endotherm appeared for poloxamer 407 in the DSC thermograms

(see Figure 2.38), suggesting the formation of a solid solution of probucol in poloxamer 407. At higher mole fractions, solid solutions coexist with excess probucol. From the enthalpies in Table 2.12 it was determined that 16.3 % probucol (Form II) remains in excess at 0.9220 mole fraction probucol, compared to 98.1 % at 0.9952 mole fraction. Conversely, at 0.9952 mole fraction probucol no poloxamer was detected compared to 75.5 % poloxamer at 0.9043 mole fraction probucol. Linear regression of the enthalpies of probucol (r2 = 0.9655) indicates a linear relationship between the decrease in excess probucol due to increasing bonds with poloxamer 407 (see Figure 2.40). Figure 2.40 predicts that a solid solution should form at 0.8994 mole fraction probucol. Therefore, as shown in Figure 2.39, the region below 0.8994 mole fraction probucol should

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140 140 Probucol, Form I Probucol, Form II 120 Poloxamer 407 120 Experimental Total Enthalpy

100 100

80 80

60 60 Enthalpy (J/g)

40 40

20 20

0 0 0.04 0.08 0.92 0.960.00 1.00 Mole Fraction Probucol

Figure 2.39 Phase diagram of probucol/poloxamer 407 compositions (1st DSC heating step, data from Table 2.12).

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80 80

60 60

40 40

20 20 Enthalpy Probucol (J/g) of

0 0 0.04 0.08 0.92 0.960.00 1.00 Mole Fraction Probucol

Figure 2.40 Phase diagram of probucol from probucol/poloxamer 407 compositions showing linear regression in red, r2 = 0.9655 (data from Table 2.12).

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represent solid solutions of probucol in poloxamer 407, while above this mole fraction a solid solution of probucol would coexist in equilibrium with excess probucol.

2.4.5.3 Scanning electron microscopy

Particles of probucol are small and irregular in shape (see Figure 2.41), and the morphology of these particles does not change after RESS processing with supercritical carbon dioxide. RESS processing of probucol does however reduce the particle size of the drug significantly as can be seen by the differences in magnification in Figures 2.41 a and b, i.e., 650xvs . 2500x. In contrast, poloxamer 407 is globular and near spherical in appearance (see

Figure 2.13 b). SEM photomicrographs of particles containing mixtures of probucol and poloxamer 407 processed by the RESS method are shown in

Figures 2.42 and 2.43. At concentrations containing the largest amount of probucol (Figure 2.42) the particles appear small and irregular in shape much like that of probucol alone (see Figure 2.41). As a result, the particles have large apparent surface areas that can come into contact with the dissolution medium and contribute to the dissolution of probucol. As the amount of poloxamer increases, i.e., 0.9456 and 0.9043 mole fractions of probucol, the particles differ little in morphology and are also irregular in shape (see Figure 2.43). Probucol, therefore, appears to be the dominating structure for all concentrations of probucol and poloxamer 407 (compare Figures 2.41 and 2.43).

164

Figure 2.41 Scanning electron photomicrographs of probucol. Key: a., as received and b., after RESS processing.

165

a. b.

c. d.

Figure 2.42 Scanning electron photomicrographs of probucol and poloxamer 407 compositions prepared by RESS processing. Key: a. and b., probucol MF=0.9936; c. and d., probucol MF=0.9812.

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a. b.

c. d.

Figure 2.43 Scanning electron photomicrographs of probucol and poloxamer 407 compositions prepared by RESS processing. Key: a. and b., probucol MF=0.9456; c. and d., probucol MF=0.9043.

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2.4.5.4 Release of probucol from particles containing poloxamer 407

The release of probucol from particles of probucol and from particles of probucol/poloxamer 407 systems prepared by RESS processing is shown in

Figure 2.44. Due to their higher dissolution rates, release of probucol from RESS processed probucol and probucol/poloxamer 407 systems was greater than that from unprocessed probucol. Differences in the rates of release of probucol before and after RESS processing can be attributed to two factors. As shown in the SEM images (Figure 2.41), RESS processing reduced the particle size of probucol thereby creating an increase in the apparent surface area of the particles. This increase in apparent surface area results in a greater dissolution rate of probucol from the RESS processed material (see Figure 2.44). Also contributing to an enhanced dissolution rate of probucol after RESS processing could be the formation of a polymorph (Form II) of probucol. As shown by DSC,

RESS processing produced a lower melting form of probucol at 116 °C. This indicates that the polymorph is a less stable form which may readily dissolve during the dissolution process.

The dissolution rates of probucol from particles of probucol processed by

RESS processed with poloxamer 407 increase as functions of increasing poloxamer mole fraction compared to those of probucol alone. Unlike differences in dissolution rates found for RESS processed and unprocessed probucol where

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140 Probucol Probucol from RESS 120 Probucol MF=0.9936 Probucol MF=0.9812 Probucol MF=0.9456 100 Probucol MF=0.9043

40 %Release Probucol of

0 0 20 40 60 80 100 120 140 160 180 200 Time (minutes)

Figure 2.44 Release of probucol from probucol/poloxamer 407 particles prepared by RESS processing as functions of composition. (Total weight of each sample = 20 mg, n = 3).

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differences in particle size were seen, the particle sizes of these systems are similar. Therefore, particle size is not likely to be the major contributing factor for enhancing the dissolution rate of probucol, but rather it is dependent on the inherent properties of poloxamer 407. Poloxamer 407 is a water-soluble surfactant that will dissolve in the dissolution medium and reduce the contact angle at the solid/liquid interface thereby improving the wettability of probucol and enhancing its dissolution rate.

As the amount of poloxamer 407 increases, the release of probucol increases and approaches 100 % release. The initial rate of release of probucol is greatest during the first 45 minutes from particles containing 0.9043 mole fraction probucol, which is a solid solution. Beyond 45 minutes the rate of release of probucol from these particles begins to slow and approaches 90 % release after 180 minutes, while the other compositions (probucol mole fractions

0.9936, 0.9812, and 0.9456) continue to approach 100 % release. The apparent

“loss” of probucol at mole fraction 0.9043 could also be the result of micellar solubilization much like that in the lidocaine/poloxamer 407 systems discussed earlier. Poloxamer 407 has a critical micelle concentration of 0.08 % w/w at 30

°C. As a result, poloxamer 407 forms micelles where it is probable that a portion of the probucol is being solubilized and thereby evading HPLC detection. On the other hand, the slope of the release of probucol from the 0.9936 and 0.9812 mole fraction systems appears to increase beyond 180 minutes to give percents release of probucol above 100 %. At these mole fractions solid solutions of

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probucol and poloxamer 407 coexist in equilibrium with crystalline probucol.

Similar to lidocaine/poloxamer 407 systems at these concentrations, poloxamer

407 would form micelles as probucol dissociates from the solid solution and is solubilized by the poloxamer 407. Simultaneously, crystalline probucol would dissolve to form a supersaturated solution from which precipitation occurs.

These precipitated small particles scatter light in the dissolution medium in the flow cell resulting in an apparent increase in absorption. This apparent increase in absorption would be expected to over compensate for the apparent loss of probucol due to micellar solubilization, thereby giving dissolution profiles that exceed 100 % release of probucol.

A comparison of the dissolution profiles was performed using the “Model

Independent Approach Using a Similarity Factor” as described in the Center for

Drug Evaluation and Research (CDER) Guidance for Industry: Dissolution

Testing of Immediate Release Solid Oral Dosage Forms (114). As previously described (see section 2.4.1.3), equation 2.1 of the model independent approach uses a difference factor (f1) which calculates the percent difference between the two curves at each time point and is a measurement of the relative error between the two curves. In equation 2.2, the similarity factor (f2) measures the similarity between two curves in terms of percent release through a logarithmic reciprocal square root transformation of the sum of the squared error. For curves to be considered similar and/or equivalent, f1 values should be close to 0 (0-15) and f2 values greater than 50 (50-100) (114).

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Table 2.13 gives the calculated f1 and f2 values for unprocessed probucol,

RESS processed probucol, and probucol/poloxamer 407 binary mixtures based on their respective release data. Based on this comparison and the values obtained it can be concluded that the release profiles for RESS processed probucol and probucol mole fraction 0.9936 are similar, having an f1 value of 12.4 for RESS processed probucol and probucol mole fraction 0.9936. Figure 2.44 shows that this similarity clearly occurs in the first 60 minutes of dissolution and is likely due to the high concentration of probucol compared to poloxamer 407.

Also considered similar are the release profiles for probucol mole fractions

0.9456 and 0.9043. The f1 value for this comparison is 10.0 and the f2 value is

61.8. Likewise, because the difference in probucol concentration is small the difference in release profile is unlikely to be different, especially at these higher concentrations of poloxamer 407 which contribute significantly toward facilitating the release of drug from the particles. The remaining comparisons indicate that the release profiles are different and not comparable to each other. Furthermore, none of the release profiles are similar to that of unprocessed probucol suggesting that both RESS processing of probucol or processing of probucol/poloxamer 407 compositions are proven methods of enhancing the rate of release of probucol.

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Probucol Mole Fraction f1 f2 1.0000 1.0000 0.9936 0.9812 0.9456 0.9043 (Unprocessed) (RESS Processed)

x x 113.1 38.78 x x 197.5 44.82 x x 398.9 18.31 x x 594.2 16.21 x x 648.0 14.83 x x 12.40 77.57 x x 113.4 27.15 x x 180.1 22.78 x x 201.8 20.74 x x 97.22 37.77 x x 174.9 26.66 x x 210.4 22.86 x x 28.87 45.55 x x 38.85 37.98 x x 10.03 61.77

Table 2.13 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of probucol / poloxamer 407 binary mixtures.

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2.4.5.5 Fourier transform infrared (FTIR) analysis

The FTIR spectra of probucol before and after RESS processing are shown in Figure 2.45. Similar to DSC, the spectra of probucol after RESS processing shows the conversion of probucol to its polymorphic form (Form II) through the appearance of a new absorption band at 3543 cm-1. In DSC a new endotherm due to the polymorph, Form II, appeared. Bostanian (101) described this absorption band as representing the phenolic group bending vibration for probucol. A small absorption band at the same wavenumber is also seen in the spectra of unprocessed probucol and suggests that probucol as received is contaminated with a small amount of its polymorph. Other characteristic

-1 absorption bands of probucol include O-H stretching at 3624 cm and CH3 (C-H stretching) at 2997 cm-1.

Figure 2.46 compares the FTIR spectra of RESS processed probucol and poloxamer 407 at various probucol/poloxamer 407 compositions. As the amount of poloxamer 407 in the probucol/poloxamer 407 systems increases, the characteristic absorption bands of probucol weaken in intensity. For example, at probucol mole fraction 0.9043, the characteristic O-H absorption bands are reduced in size and intensity compared to the spectra of the probucol mole fraction 0.9936 system. The characteristic poloxamer absorption bands, such as the C-H stretch (2891 cm-1), C-O-C stretch (1109 cm-1), and p-disubstituted aromatic stretch (840 cm-1) also become more intense as the proportion of

174

Figure 2.45 FTIR spectra of probucol and probucol after RESS processing.

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Figure 2.46 FTIR spectra of probucol, poloxamer 407, and probucol/ poloxamer 407 compositions.

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poloxamer 407 increases. No significant shifts appear in the FTIR spectra that suggest an interaction. However, a new absorption band appears at 1639 cm-1 for the FTIR spectra containing the probucol mole fraction of 0.9043 (shown by the arrow in Figure 2.46). Absorption bands near this wavenumber typically represent C=O or C=C stretches (53). This absorption band does not appear in the spectra of probucol, poloxamer 407, or any of the other probucol/poloxamer

407 systems except for that of the 0.9043 mole fraction probucol. Figure 2.47 shows a schematic representation of probucol hydrogen bonding with the polyoxyethylene segment of poloxamer 407. Based on this schematic, probucol would bond to poloxamer through its O-H functional group. Since bonding between probucol and poloxamer 407 is suspected, the appearance of the new absorption band at 1639 cm-1 would correspond with shifting of the C=C stretch of the neighboring aromatic ring as a result of hydrogen bonding between the O-

H of probucol and the ether group of poloxamer 407 (see Figure 2.47).

The molecular orientation of probucol may also contribute to favorable bonding. Due to the site of hydrogen bonding, probucol occupies less space along the poloxamer chain in terms of width, but is larger in depth. For example, in Figure 2.47 probucol spans about three oxygens in width, whereas in Figure

2.17 lidocaine spans about four. As a result, more probucol molecules would be able to arrange themselves along the poloxamer molecule and bond. Figure

2.48 shows that rotation of the probucol molecules along the poloxamer chains would occupy less space, thereby facilitating more hydrogen bonds. Figure 2.48

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OH OH

(H3C)3C C(CH3)3 (H3C)3C C(CH3)3

S S

H3C C CH3 H3C C CH3 S S

(H3C)3C C(CH3)3 (H3C)3C C(CH3)3 O O CH H H 3 H H2 H H 2 H 2 H2 2 C O C C O C O CH C C C O HO CH C O C C O H2 H2 H H2 2 H2 y x x CH3

Figure 2.47 Schematic molecular structures of probucol and poloxamer showing hydrogen bonded interactions.

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a. b.

red = oxygen yellow = sulfur grey = carbon white = carbon

Figure 2.48 Schematic molecular structures of probucol and poloxamer showing hydrogen bonding as functions of probucol molecular orientation. Key: a, rotated probucol perpendicular to poloxamer chain; b, favorable probucol orientation.

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also shows that bonding of a probucol molecule with a polyoxyethylene segment next to a polyoxypropylene segment would not be sterically inhibited by the three methyl groups extending from the aromatic ring of probucol (see Figure 2.48 b).

However, folding of probucol at the bridge connecting the two aromatic rings could prevent and/or interfere with the bonding of additional probucol molecules.

Broman, et al. (96) formed solid dispersions of probucol and polyoxyethylene, which is a component of poloxamer 407, using the melting method. It was proposed that some degree of interaction between polyoxyethylene and probucol may occur in the molten state when pressure is applied. Similarly, Ozeki, et al. (119) proposed hydrogen bonding interactions between polyoxyethylene and the poorly water-soluble drug flubiprofen, which increases with polyoxyethylene concentration. Ozeki (119) also suggested that melting of polyoxyethylene during sample preparation increased the mobility of the polymer facilitating contact and interaction with the drug. These studies support the hypothesis that poloxamer 407 is hydrogen bonding with probucol via the polyoxyethylene segment of poloxamer 407. It is further presumed that this intimate contact, as described by Ozeki (119), is also achieved at the processing temperature, 75 °C, which is used to process probucol and poloxamer 407 with supercritical carbon dioxide.

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2.4.6 Probucol, poloxamer 188, and probucol/poloxamer 188 compositions

2.4.6.1 Differential scanning calorimetry

The DSC thermograms for particles of probucol, poloxamer 188, and probucol/poloxamer 188 systems prepared by RESS processing are shown in

Figure 2.49 and are summarized in Table 2.14. Thermal analyses of particles of probucol/poloxamer 188 systems were similar to those of probucol/poloxamer

407 systems (see Figure 2.49) in that similar types of endotherms appear near the same melting points depending on the probucol/poloxamer 188 composition.

At compositions containing 0.9812 mole fraction probucol (Figure 2.49 c) two endotherms appear at 126 °C and 117 °C, respectively for probucol and its polymorph (Form II). An endotherm for poloxamer 188 does not appear apparently due to the small amount of poloxamer 188 present in the system, which may be below the detection limits of DSC. As the poloxamer 188 concentration is increased, an endotherm occurs near its melting point, 55 °C.

Figure 2.49 d, for probucol mole fraction 0.9456 shows endotherms for probucol

(Form I) and its polymorph (Form II), as well as a small endotherm for poloxamer

188 at 52 °C. The shift in the melting point of poloxamer 188 to a lower melting point is indicative of the presence of probucol serving as an impurity in the system. At 0.9043 mole fraction probucol, the endotherm for poloxamer 188 is larger and also has a melting point of 52 °C (see Figure 2.49 e). At this

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Probucol and Poloxamer 188 a. b. c. d. e.

Figure 2.49 Representative DSC thermograms of probucol/poloxamer 188 compositions prepared by RESS processing. Key: a., poloxamer 188 (purple); b., probucol (black); c., probucol MF=0.9812 (blue); d., probucol MF=0.9456 (red); e., probucol MF=0.9043 (green).

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Mole Melting Point, °C Enthalpy, J g-1 Fraction Probucol Poloxamer Probucol Poloxamer of Form I Form II 188 Form I Form II 188 Probucol 1.0000 127.2±0.00 116.7±0.14 - 14±0.00 63.0±0.00 0.00 0.9952 127.2±0.00 117.3±0.07 - 4.9±6.86 65.9±4.03 0.00 0.9936 126.7±0.00 117.3±0.00 - 1.0±1.34 67.2±1.48 0.00 0.9874 127.0±0.42 117.1±0.42 - 1.1±0.14 70.9±3.32 0.00 0.9812 126.7±0.00 117.4±0.07 - 3.9±2.12 65.2±0.28 0.00 0.9720 126.7±0.00 117.1±0.35 - 3.8±4.95 65.1±2.76 0.00 0.9456 126.7±0.00 116.8±0.00 51.5±0.07 0.7±0.21 63.6±0.71 1.75±0.49 0.9220 125.6±0.78 113.5±1.27 52.4±0.00 1.1±0.64 35.3±0.92 42.1±2.47 0.9043 123.2±1.70 110.4±1.63 52.3±0.00 2.5±0.28 23.0±2.76 57.1±6.51 0.8506 - 101.9±8.63 52.4±0.35 0.00 9.60±4.67 80.9±13.7 0 - - 56.0±0.21 0.00 0.00 115±2.26

Table 2.14 Thermal properties of probucol and poloxamer 188 processed with SCCO2 at 75 C, 7100 psi, and restrictor temperature of 40 C, (n = 2).

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concentration the endotherms for probucol and its polymorph (Form II) are reduced in area. For example, the enthalpies for probucol and its polymorph

(Form II) are approximately 2.5 and 23.0 J g-1, respectively, at probucol mole fraction 0.9043, compared to 4.9 and 65.9 J g-1 at probucol mole fraction 0.9952.

This suggests that probucol forms a solid solution of probucol and poloxamer

188, which coexists in equilibrium with excess crystalline probucol randomly dispersed in the matrix. Therefore, a finite amount of probucol is hydrogen bonding with poloxamer 188. Endotherms for probucol (Form I) and its polymorph (Form II) appear in Figure 2.49, while poloxamer 188 is represented by the endotherm at ~52 °C. At 0.8506 mole fraction probucol (see Table 2.14) there is an endotherm for poloxamer 188 along with a small amount of probucol as indicated by its enthalpy. This indicates that as the poloxamer 188 concentration was increased, i.e., the number of polyoxyethylene segments increase and a solid solution forms.

DSC thermograms of particles of probucol/poloxamer 407 and probucol/poloxamer 188 systems indicate that they do not form solid solutions at the same mole fractions. This difference can be attributed to differences in structures between poloxamer 407 and 188. The approximate lengths of the polyoxyethylene segments of poloxamer 407 and 188 differ (101 vs. 80 units), while the polyoxypropylene chain length of poloxamer 188 is approximately half the length of that of poloxamer 407 (27 vs. 56 units). The total length of the polyoxyethylene and polyoxypropylene chains therefore would be shorter for

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poloxamer 188 (see Figure 2.3). As such, fewer probucol molecules may bond to poloxamer 188. As shown in Figure 2.47 e, an endotherm for excess probucol appears at 0.9456 mole fraction probucol compared with the absence of an endotherm at this same concentration for the probucol/poloxamer 407 system.

Figure 2.50 is a phase diagram of enthalpies for probucol (Forms I and II) and poloxamer 188 as functions of probucol mole fraction for probucol/poloxamer

188 systems. Similar to the phase diagram for probucol/poloxamer 407 systems

(see Figure 2.39), the phase diagram for probucol/poloxamer 188 shows a decrease in enthalpy of Form II as the amounts of probucol decrease, while the enthalpies of poloxamer 407 increase. Based on the data in Table 2.14, there is no region in which one endotherm appears. However, the phase diagram follows the same trends as that for probucol/poloxamer 407 in that the amounts of excess probucol decrease due to increased bonding to poloxamer 188. From the enthalpies shown in Table 2.14, it was determined that 15.2 % probucol (Form II) remains in excess at 0.8506 mole fraction probucol, compared to virtually all the drug in excess at 0.9952 mole fraction. Conversely, at 0.9952 mole fraction probucol no poloxamer was detected compared to 70.3 % poloxamer at 0.8506 mole fraction. Furthermore, linear regression of the enthalpies of probucol predicts the concentration at which a solid solution should form (see Figure 2.51).

According to the linear regression shown in Figure 2.51 (r2 = 0.9467), a solid solution should occur at 0.8073 mole fraction probucol. Therefore, at mole

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140 140 Probucol, Form I Probucol, Form II 120 Poloxamer 188 120 Experimental Total Enthalpy

100 100

80 80

60 60 Enthalpy(J/g)

40 40

20 20

0 0 0.04 0.08 0.80 0.84 0.88 0.92 0.960.00 1.00 Mole Fraction Probucol

Figure 2.50 Phase diagram of probucol/poloxamer 188 compositions (1st DSC heating step, data from Table 2.14).

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80 80

60 60

40 40

20 20 Enthalpy (J/g) of Probucol

0 0 0.000.04 0.08 0.80 0.84 0.88 0.92 0.96 1.00 Mole Fraction Probucol

Figure 2.51 Phase diagram of probucol from probucol/poloxamer 188 compositions showing linear regression (in red), r2 = 0.9467 (data from Table 2.14).

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fractions less than 0.8073, solid solutions of probucol in poloxamer 188 would form. Conversely, at higher mole fractions, solid solutions of probucol in poloxamer 188 would coexists in equilibrium with excess probucol.

2.4.6.2 Scanning electron microscopy

Photomicrographs of particles of probucol/poloxamer 188 systems prepared by RESS processing are shown in Figures 2.52 and 2.53. Similar to probucol/poloxamer 407 systems, particles of probucol and poloxamer 188 at high probucol concentrations, i.e., 0.9936 and 0.9812 mole fractions of probucol

(Figure 2.52), are small and irregular in shape and similar in appearance. The dominant characteristics of these particles tend to resemble those of probucol

(see Figure 2.41). However, as the proportion of poloxamer 188 increases, i.e.,

0.9456 and 0.9043 mole fraction probucol, the sizes of the particles increase, yet they remain irregular in shape much like that of probucol (Figure 2.53). At 0.9043 mole fraction probucol (Figure 2.53 c), the particles have hollow interior cavities much like that of particles of lidocaine/poloxamer 188 systems as shown in

Figure 2.23. Such cavities would result from the rapid removal of supercritical carbon dioxide and/or collapse of the exterior walls of the hollow particles after the carbon dioxide has exited at atmospheric pressure. This is likely to occur at concentrations containing greater amounts of poloxamer because they would be more malleable and prone to changes in physical structure compared to crystalline probucol.

188

a. b.

c. d.

Figure 2.52 Scanning electron photomicrographs of probucol and poloxamer 188 compositions prepared by RESS processing. Key: a. and b., probucol MF=0.9936; c. and d., probucol MF=0.9812.

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a. b.

c. d.

Figure 2.53 Scanning electron photomicrographs of probucol and poloxamer 188 compositions prepared by RESS processing. Key: a. and b., probucol MF=0.9456; c. and d., probucol MF=0.9043.

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2.4.6.3 Release of probucol from particles containing poloxamer 188

The release of probucol from probucol/poloxamer 188 particles prepared by RESS processing is shown in Figure 2.54. The release of probucol is enhanced as a function of the amount of poloxamer 188 in the systems. For example, within the first twenty minutes the release of probucol increases from approximately 17 % at 0.9936 mole fraction probucol to 25 % at 0.9812 mole fraction and to 35 % at mole fractions 0.9456 and 0.9043. However, beyond 20 minutes the rates of release decrease from the 0.9456 and 0.9043 systems and are less than that at 0.9812 mole fraction, i.e., ~90 and 75 % release after 180 minutes, respectively. Similar to that of lidocaine/poloxamer 188 systems, the apparent “loss” of drug at these concentrations can be explained in theory by micellar solubilization of probucol by poloxamer 188 which allows the probucol to evade HPLC detection. At 0.9812 mole fraction probucol, the release of probucol reaches 100 %. Extrapolating the curve beyond 180 minutes would give an apparent percent release of greater than 100 %. At this concentration a solid solution would coexist with crystalline probucol. Similar to the 0.9456 and 0.9043 mole fraction systems, micellar solubilization could occur. Simultaneously, crystalline probucol would dissolve forming a saturated solution from which small particles precipitate. Like the lidocaine/poloxamer 188 systems discussed in section 2.4.2.3, these small particles would scatter light and create an apparent increase in absorption, which would over compensate for the apparent loss of probucol. As a result, the release of probucol beyond 180 minutes at 0.9812

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140 Probucol Probucol from RESS 120 Probucol MF=0.9936 Probucol MF=0.9812 Probucol MF=0.9456 100 Probucol MF=0.9043

40 %Release Probucol of

0 0 20 40 60 80 100 120 140 160 180 200 Time (minutes)

Figure 2.54 Release of probucol from probucol/poloxamer 188 particles prepared by RESS processing as functions of composition. (Total weight of each sample = 20 mg, n = 3).

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mole fraction would appear greater than 100 %. On the contrary, at probucol mole fraction 0.9936 it appears that there is not enough poloxamer 188 in the system to sufficiently enhance the dissolution rate of probucol.

As for the previous binary systems, a comparison of the dissolution profiles were performed using the “Model Independent Approach Using a

Similarity Factor” as described in the Center for Drug Evaluation and Research

(CDER) Guidance for Industry: Dissolution Testing of Immediate Release Solid

Oral Dosage Forms (114). Using equations 2.1 and 2.2 (see section 2.4.1.3) the difference factors (f1) and similartity factors (f2) were calculated and quantitative comparison of the release curves was obtained. Table 2.15 gives the calculated f1 and f2 values for unprocessed probucol, RESS processed probucol, and various probucol/poloxamer 188 binary mixtures based on their respective release data. The data indicates that only the release profiles for probucol mole fraction 0.9456 and 0.9043 are similar with an f1 value of 6.6 and f2 value of 82.8.

This similarity is likely due to the concentration of poloxamer 188 which because of its higher aqueous solubility enhances the rate of release of the drug from the particles. In addition there is a small difference in probucol concentration between the two and therefore it is less likely to give a significant difference between the release profiles.

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Probucol Mole Fraction f1 f2 1.0000 1.0000 0.9936 0.9812 0.9456 0.9043 (As Received) (RESS processed)

x x 113.1 38.78 x x 169.0 27.72 x x 415.1 19.35 x x 648.0 29.87 x x 645.4 30.46 x x 26.26 52.78 x x 114.2 28.06 x x 167.8 37.15 x x 166.9 37.85 x x 71.60 33.45 x x 117.5 40.54 x x 116.8 41.25 x x 27.91 60.12 x x 27.48 58.91 x x 6.574 82.84

Table 2.15 Calculated difference (f1) and similarity (f2) factors from the comparison of dissolution release profiles of probucol / poloxamer 188 binary mixtures.

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For the most part however, comparison of the release profiles show that

they are not similar or equivalent. Furthermore, they show that the release of probucol is enhanced by the presence of poloxamer 188 and the initial release of drug is dependent on the amount of poloxamer 188 present in the system. In general, the addition of poloxamer 188 enhances the initial rates of solution of probucol, but at higher concentrations of poloxamer 188 the release of probucol is reduced as a portion of it is “lost” due to micellar solubilization.

2.4.6.4 Fourier transform infrared (FTIR) analysis

The FTIR spectra of particles of probucol, poloxamer 188, and various compositions prepared by the RESS method are shown in Figure 2.55. Since poloxamer 407 and 188 consist of the same functional groups, the FTIR spectra were similar as expected. The spectra show that, as the proportion of poloxamer

188 increases, its characteristic absorption bands become more intense, while those of probucol weaken in intensity. For example, the phenolic stretching of probucol at 3543 cm-1 and 3624 cm-1 are less intense and begin to broaden as poloxamer 188 concentration increases. This broadening of the peaks is characteristic of poloxamer 188 in this region. Like the spectra for 0.9043 probucol mole fraction in the probucol/poloxamer 407 system, a new absorption band appears at 1639 cm-1. This new absorption band also appears at probucol mole fractions 0.9456 and 0.9043 (as shown by arrows in Figure 2.55) and also corresponds with a shift of the C=C stretch of the neighboring aromatic ring as a

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Figure 2.55 FTIR spectra of probucol, poloxamer 188, and probucol/poloxamer 188 compositions.

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result of hydrogen bonding between the O-H of probucol and the ether group of poloxamer 188 (see Figure 2.47). Similarly, Ozeki, et. al (119) described the appearance of a new absorption band as the result of hydrogen bonding between the hydroxyl group of the carboxyl group of flurbiprofen and ether group of polyoxyethylene in solid dispersions prepared using the melting method.

2.4.7 Probucol, poloxamer 403, and probucol/poloxamer 403 compositions

It was not possible to prepare particles of probucol and poloxamer 403 by the RESS method due to differences in their melting points and solubilities in supercritical carbon dioxide. Poloxamer 403 has a melting point of approximately

35 °C and therefore has to be processed using relatively mild conditions such as an extraction temperature of 35 °C, pressure of 7100 psi, and a restrictor temperature of ~20 °C. This compares to conditions for probucol/poloxamer 407 and 188 systems, which were an extraction temperature of 75 °C, pressure of

7100 psi, and a restrictor temperature of ~40 °C. Since the melting points of poloxamer 407 and 188 are higher, i.e., ~56 °C, and closer to the melting point of probucol (~127 °C), higher processing conditions were used to facilitate dissolution of the two components in supercritical fluid carbon dioxide. These conditions also maintained fluidity of the probucol in the restrictor during expansion, which prevents probucol from precipitating and clogging the restrictor, while preventing melting of the poloxamer. On the other hand, the difference in melting point between probucol and poloxamer 403 is such that processing

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conditions could not be found for simultaneously processing the two components without compromising one or the other. The low restrictor temperature needed for poloxamer 403 would result in probucol precipitating before exiting the system and thereby clogging the restrictor. Furthermore, although probucol is readily soluble in supercritical carbon dioxide at high temperatures, its solubility is limited

(0.21 ± 0.04 % w/w) under the conditions necessary to process poloxamer 403, i.e., 35 °C, 7100 psi, and restrictor temperature of ~20 °C. For comparison, the solubility of probucol at 75 °C, 7100 psi, and restrictor temperature of ~40 °C is

2.11 ± 0.31 % w/w. Consequently, compositions of probucol and poloxamer 403 could not be formed.

2.4.8 Summary: Comparison of probucol/poloxamer 407 and

probucol/poloxamer 188 particles prepared by supercritical fluid

processing

Differential scanning calorimetry of particles of probucol/poloxamer 407 systems indicated the formation of a solid solution at 0.8994 mole fraction probucol as determined by extrapolation of the enthalpies of probucol as functions of mole fraction. This was apparent by progressive decreases in the enthalpies of endotherms for probucol and corresponding increases in the enthalpies of poloxamer 407. At probucol/poloxamer 407 concentrations above

0.8994 mole fraction probucol, solid solutions of probucol/poloxamer 407 coexist in equilibrium with excess crystalline probucol. Probucol/poloxamer 188 systems

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also formed solid solutions coexisting with excess probucol at about 0.8073 mole fraction probucol rather than at 0.8994 mole fraction as indicated for the probucol/poloxamer 407 systems.

The differences in mole fraction for the formation of solid solutions in the probucol/poloxamer 407 and probucol/poloxamer 188 systems can be attributed to differences in their polyoxypropylene chain lengths. The closer proximity of the polyoxyethylene chains of poloxamer 188 due to its shorter polyoxypropylene chain length may sterically inhibit bonding with probucol molecules. In addition, the shorter overall chain length of poloxamer 188, i.e., the polyoxyethylene and polyoxypropylene chains together, has fewer sites for bonding probucol. As a result, less probucol hydrogen bonds to poloxamer 188 and instead remains in excess where it crystallizes as indicated by an endotherm. In the lidocaine/poloxamer systems shown in Figure 2.33, solid solutions were found for each of the poloxamers for which a linear relationship could be established. This relationship shows the dependency of solid solution formation on polyoxyethylene chain length. Unfortunately, probucol and poloxamer 403 could not be simultaneously processed using the RESS method, therefore a similar comparison could not be made with only two values (see Figure 2.56). However, since both lidocaine and probucol appear to hydrogen bond with poloxamer via the same poloxamer moiety it can be speculated that a similar linear dependency exists. Furthermore, these two points lie below those of lidocaine/poloxamer systems perhaps due to differences in lidocaine and probucol molecular weight

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1.00

Poloxamer 0.95 Poloxamer 407 Poloxamer 188 403

0.90 Poloxamer 407

MoleFraction Lidocaine 0.85

● Lidocaine/PoloxamerLidocaine / Poloxamer Systems Systems Poloxamer 188 ▲ Probucol/PoloxamerProbucol / Poloxamer Systems Systems 0.80 0 50 100 150 200 Total Polyoxyethylene Chain Length

Figure 2.56 Relationship between lidocaine (r2 = 0.9934) and probucol (r2 = 1.0000) mole fraction at zero enthalpy and total polyoxyethylene chain length of poloxamers 407, 188, and 403.

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and configuration. In this context, probucol is a larger molecule compared to lidocaine (MW ~ 516 vs. MW ~ 234 respectively) and its three dimensional configuration may sterically inhibit bonding with a poloxamer. Therefore, in order to form a solid solution, increasing amounts of poloxamer would be needed to provide sufficient bonding sites to accommodate excess probucol.

Consequently, the mole fraction at which a solid solution of probucol in poloxamer forms would be less than that of the lidocaine/poloxamer systems.

The existence of hydrogen bonding between probucol and poloxamer was shown by FTIR. As the poloxamer concentration was increased, more probucol bonds with it to form a solid solution. An additional absorption band appears at

1639 cm-1 due to the C=C stretching of the aromatic ring as a result of hydrogen bonding between the O-H group of probucol and the ether oxygen of the polyoxyethylene segment of the poloxamer.

Furthermore, SEM showed small irregular shaped particles of probucol/poloxamer were produced by RESS processing. These particles would have larger apparent surface areas compared to that of RESS processed or unprocessed probucol, which in addition to the formation of solid solutions would contribute to enhanced dissolution rates of probucol.

Release studies also showed that the initial release of probucol is dependent on the amount of poloxamer in the systems. Initial rates of release

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were directly proportional to the amounts of poloxamer in the system. However, the presence of the surface-active poloxamers would result in the formation of micelles and the solubilization of probucol. For example, steady states at ~75 and 90 % release of probucol at 0.9043 and 0.9456 mole fraction probucol, respectively, would indicate the apparent loss of probucol due to micellar solubilization. At higher probucol concentrations (or lower poloxamer concentrations), i.e., 0.9936 and 0.9812 mole fraction probucol, micellar solubilization would also occur along with precipitation of small particles of excess probucol. These particles would scatter light in the flow cell during HPLC detection resulting in apparent increases in absorption, which could over compensate for the loss of drug due to micellar solubilization and cause release profiles for probucol to exceed 100 % at 180 minutes and beyond. Nevertheless, comparison of the release profiles indicate that both RESS processed probucol or probucol/poloxamers (407 and 188) binary mixtures are potential methods of enhancing the rate of release of probucol.

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CHAPTER 3

GENERAL SUMMARY

Supercritical fluid processing is a relatively new technology being utilized in the pharmaceutical industry for the purposes of recrystallization and small particle formation. In the present work supercritical fluid processing was used to form small particles consisting of solid solutions of binary mixtures of a drug and an appropriate water-soluble excipient for drug delivery with the goal of enhancing drug dissolution rates. In this context, solid solutions are classified as solid dispersions in which the drug is molecularly dispersed. The objective was to investigate the formation of solid solutions consisting of the relatively insoluble model drugs lidocaine or probucol and a block copolymer surfactant, a poloxamer. Such solid solutions would be expected to enhance dissolution rates since particle size is reduced to its absolute minimum, i.e., the drug molecule.

Through enhancement of drug dissolution rates, solid solutions prepared by supercritical fluid processing have the potential to improve the bioavailability of a relatively insoluble drug.

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Using the method of rapid expansion of supercritical solutions (RESS), small particles consisting of a relatively insoluble drug (lidocaine or probucol) and a water-soluble component (a poloxamer) were formed. The physicochemical properties of these particulate systems were characterized to determine the mechanism(s) by which solid solutions form and their role in enhancing the dissolution rates of such relatively insoluble drugs.

Lidocaine and probucol are nonpolar drugs that are soluble in supercritical carbon dioxide (36.2 ± 0.45 % w/w and 2.11 ± 0.31 % w/w, respectively, at 75 °C and 7100 psi), and have relatively low melting points of 68 °C and 125 °C, respectively. To form a solid solution, a water-soluble component that is at least slightly soluble in supercritical carbon dioxide is necessary to form binary mixtures with relatively insoluble drugs. Poloxamers are suitable candidates because they are block copolymers consisting of a nonpolar polyoxypropylene chain and two polar polyoxyethylene chains. The polyoxypropylene chain facilitates solubility of the poloxamer in the nonpolar supercritical carbon dioxide, while the polyoxyethylene component allows the poloxamer to dissolve in aqueous dissolution media. In addition, it can be expected that poloxamer dissolved in the dissolution medium will enhance wetting of the particles thereby contributing to increasing the rate of release of the drug from the solid solution.

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RESS processing of the relatively insoluble drugs lidocaine or probucol with the water-soluble poloxamers 407, 188, or 403 produced solid solutions and solid solutions in equilibrium with excess drug depending on the relative compositions of the systems. The final step of RESS processing is an instantaneous expansion of the supercritical solutions to atmospheric pressure during which carbon dioxide is lost and the two components are coevaporated.

Therefore, it is likely that the molecular arrangement of the drug and surfactant in solution in the supercritical fluid can be retained in the particles during this process. In this context, both lidocaine and probucol are relatively small molecules with molecular weights of approximately 234 and 516 g mol-1. By contrast, poloxamers are relatively large molecules compared to lidocaine and probucol, with molecular weights ranging from 5,750 g mol-1 for poloxamer 403 to

12,500 g mol-1 for poloxamer 407. These differences in molecular sizes therefore could allow molecules lidocaine or probucol to disperse within the interstitial spaces of the poloxamer where intermolecular hydrogen bonding would occur.

Small particles consisting of drug and poloxamer varied in physical appearance as functions of composition despite the high mole fractions of drug used. At the higher concentrations of poloxamer, the surface of the particles appeared smooth and the shapes were near spherical, similar to that of poloxamer alone. At higher concentrations of drug however, the particle surfaces were textured, resembling the appearance of particles of the drug alone. In all cases, small invaginations due to partial collapse of the particle appeared on the

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surfaces of the particles due to the expansion and subsequent loss of carbon dioxide during the last step of RESS processing. Photomicrographs of fractured particles did not show separate particles of drug within the poloxamer particles suggesting the formation of homogeneous or near homogeneous particles.

However, darker regions in the walls of these particles may represent regions of excess crystalline drug separate from that of the solid solution.

Probucol is a virtually insoluble compound compared to the relatively insoluble lidocaine, i.e., with an aqueous solubility of less than 5 ng mL-1 versus

0.004 g mL-1 for lidocaine at 25 °C. In general, substances with low aqueous solubilities generally tend to have slow dissolution rates due to their inability to readily enter into solution. However, dissolution studies indicated that the apparent rates of release of lidocaine and probucol from solid solutions consisting of the drug and poloxamer 407, 188 or 403 were significantly enhanced. In the case of systems in which all of the drug is molecularly dispersed in a solid solvent and none is in excess, dissolution of the water- soluble poloxamer would expose molecules of drug whose rate of solution would in turn be expected to be maximal since they would be in the state of ultimate surface area attainable for contact with the dissolution medium. Conversely, in the presence of excess drug the overall rates of solution would not be maximal, yet would be expected to be enhanced compared to the dissolution rates of the drug alone due to the contribution of drug from the solid solution.

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Comparison of the release profiles of lidocaine and poloxamer 407 or 188 binary mixtures indicate that both compositions of lidocaine in equilibrium with excess drug and solid solutions have enhanced rates of release. This could suggest that the formation of a solid solution may not be necessary to enhance the rate of release of drug from a binary mixture of lidocaine and a poloxamer prepared using the RESS method. However, RESS processing of probucol and a poloxamer at certain compositions is not sufficient to enhance the rate of release of probucol compared to probucol processed alone. Therefore, in addition to RESS processing, it can be inferred that a certain degree of interaction, such as the formation of a solid solution, is necessary to enhance the rate of release of such a relatively insoluble drug.

Furthermore, dissolution studies indicated that the apparent rates of release of lidocaine and probucol from particles containing poloxamer 407, 188, or 403 was dependent on the particular poloxamer present. Since poloxamer

403, and particles consisting of drug and poloxamer 403, were semi-solid at ambient conditions, it was not possible to adequately determine the release of drug by the flow-through dissolution method. On the other hand the initial release rates of lidocaine were enhanced from systems containing different amounts of poloxamer 407 or 188. This was likely due to the presence of small particles in the samples collected during the first 10-15 minutes, which would give an apparent increase in absorption due to light scattering. However, above

0.9812 lidocaine mole fraction the total percent release of drug was greater than

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100 %, while below this mole fraction the total percent release was less than 100

%. This anomaly also occurred in systems consisting of probucol and poloxamer

407 or 188. These variables in amounts released can be attributed to the precipitation of small particles and/or possibly the solubilization of drug in micelles of poloxamer. For small particles, light scattered in the flow cell during detection could mimic absorption thereby giving the appearance of an increase in drug release. Furthermore, the formation of micelles in the vicinity of the particle surface would create a hydrophobic interior environment in the micelle favorable to the drug. As such, the hydrophobic drug would be solubilized and release from the particle surface would be enhanced, thereby facilitating the dissolution process. In the case of micellar solubilization, it can be hypothesized that drug within the micelles appears to evade HPLC detection giving the appearance of less than 100 % release. Consequently, micelles can also scatter light in the flow cell, however, the apparent loss of lidocaine due to micelle solubilization could more than compensate for the apparent increase in absorption due to light scattering. Although evasion of drug detection through solubilization within a micelle is theoretically possible, such an occurrence would require confirmation through additional studies beyond the scope of the research. For the purpose of the study it is sufficient to note that at certain compositions of drug and a poloxamer the initial release rates are enhanced significantly compared to the rate of release of the drug alone.

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As indicated in Chapter 2, depending on the relative concentrations of systems, solid solutions, or solid solutions in equilibrium with excess drug may form. In binary systems where excess drug coexists in equilibrium with a solid solution, both components can be individually identified by their characteristic endotherms in a DSC thermogram. This is consistent with the concept of partial solid solubility where portions of drug may be molecularly dispersed in a solid solvent and yet coexist with crystalline drug (36). On the other hand, in solid solutions all of the drug would be molecularly dispersed and would not be detected by DSC.

Phase diagrams of the enthalpies of drug and poloxamer from drug / poloxamer systems as functions of mole fraction of drug indicate that a linear relationship exists between the enthalpies of both the drug and the poloxamer with the mole fraction of drug. Progressive decreases in mole fraction of drug result in corresponding decreases in its enthalpy. From DSC it is apparent that the amounts of excess drug decrease while the proportion of solid solution increases until the excess solid disappears as indicated by zero enthalpy, i.e., where its endotherm disappears. Solid solutions therefore are represented in the phase diagrams as occurring in the region of drug concentrations below that at which zero enthalpy occurs. At zero enthalpy all of the drug is in the form of the solid solution which in turn coexists with solid poloxamer as indicated by the presence of a single endotherm assigned to the poloxamer. No endotherm for the drug appears at this point since it is molecularly dispersed. At higher

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concentrations of poloxamer, solid solutions coexist with excess drug as indicated by the presence of endotherms for poloxamer and for the drug. Linear regression of the enthalpies of the drug as functions of mole fraction of drug to zero enthalpy could therefore be used to predict the mole fraction of drug at which a solid solution should form. This appears to be a novel means of confirming the formation of solid solutions, the mechanism by which they occur in these systems, and a method for predicting the relative concentrations at which they occur.

In addition, the degree of drug-surfactant bonding was found to be a function of the length of the polyoxyethylene chains of a poloxamer. Lidocaine and probucol hydrogen bond with poloxamers 407 and 188 via the ether oxygen of the polyoxyethylene moieties. DSC indicated no apparent differences in thermal properties between lidocaine/poloxamer 407 systems and lidocaine/poloxamer 188 systems despite differences in the lengths of their polyoxypropylene chains. The polyoxypropylene chain length of poloxamer 188 is approximately half the length of that of poloxamer 407. For both systems, the endotherm for lidocaine gradually decreased in area and eventually disappeared indicating that increasing amounts of lidocaine were bonding with the poloxamer to form a solid solution. On the other hand, poloxamer 403 has approximately half the polyoxyethylene chain length of poloxamers 407 and 188. DSC thermograms of lidocaine/poloxamer 403 systems have endotherms for lidocaine and poloxamer 403 at all compositions because fewer polyoxyethylene moieties

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would be available for hydrogen bonding with lidocaine. Therefore, it can be assumed that the polyoxypropylene segment of the three poloxamers does not play as significant a role in the degree of drug-surfactant bonding as does the polyoxyethylene segment.

Bonding between poloxamer and lidocaine or probucol was also evident from FTIR analysis. FTIR spectra of lidocaine/poloxamer 407 and lidocaine/poloxamer 188 systems at lidocaine mole fraction 0.9456 indicate shifts in the amine stretch of lidocaine from 3249 cm-1 to 3257 cm-1. Lidocaine appears to hydrogen bond with poloxamers 407 and 188 via its amine group and the polyoxyethylene ether group of the poloxamer. Although hydrogen bonding with the polyoxypropylene segment is also possible, it is assumed that bonding primarily occurs with the polyoxyethylene segment due to the greater number of sites for bonding and the ability to bond with minimal steric interference. The presence of the additional methyl group of the polyoxypropylene segment may sterically interfere with bonding. On the other hand, probucol has the potential to hydrogen bond to the ether oxygens of poloxamer via the hydroxyl groups of its aromatic rings. FTIR analysis indicates the presence of this interaction through the appearance of an absorbance band near 1639 cm-1 at 0.9043 probucol mole fraction. This band represents changes in the C=C stretching of the aromatic ring as a result of hydrogen bonding between probucol and poloxamer 407 or

188. Since there are many sites on the polyoxyethylene segments for lidocaine

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or probucol to bond and an infinite number of structural orientations, it is likely that in three dimensions many molecules of lidocaine or probucol will be positioned along the polyoxyethylene segments with minimal steric interference.

Based on the work presented herein, supercritical fluid processing by the

RESS method may serve as a general process for preparing drug delivery systems consisting of a relatively insoluble drug and a water-soluble excipient in the form of solid solutions, which can be used for various therapeutic purposes.

Such particles could be compressed into tablets for oral administration or utilized as individual particles. For example, small particles of a local anesthetic could be distributed within a surgical wound to help relieve post surgical pain. Small particles of an appropriate drug and excipient could also be used to deliver drug to the lungs via an aerosol drug delivery device.

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