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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.

Permanent Link: http://ufdc.ufl.edu/UFE0021747/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.
Physical Description: Book
Language: english
Creator: Shekhawat, Dushyant
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Dushyant Shekhawat.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Shah, Dinesh O.
Local: Co-adviser: Chauhan, Anuj.
Electronic Access: INACCESSIBLE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021747:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021747/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.
Physical Description: Book
Language: english
Creator: Shekhawat, Dushyant
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Chemical Engineering -- Dissertations, Academic -- UF
Genre: Chemical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Dushyant Shekhawat.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Shah, Dinesh O.
Local: Co-adviser: Chauhan, Anuj.
Electronic Access: INACCESSIBLE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021747:00001


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1 SURFACTANT SYSTEMS FOR DRUG DELIVERY AND WATER EVAPORATION REDUCTION By DUSHYANT SHEKHAWAT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Dushyant Shekhawat

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3 To my parents and sisters who have been my #1 supporters.

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4 ACKNOWLEDGMENTS I would lik e to express my sincere appreciation to Professor Dinesh O. Shah, chairman of my supervisory committee, for allowing me to be a part of the Center for Surface Science and Engineering (CSSE), and for his kind guidance, motivation and encourag ement during my Ph.D. program. I would also like to thank other s upervisory committee members, Professors Anuj Chauhan, Brij Moudgil and Richard B. Dickins on, for their valuable time and suggestions. Thanks go also to Dr. Timothy E. Morey for his numerous inputs in my research and Dr. Jerome H. Modell and Dr. Donn M. Dennis for their help in Animal Experiments. CSSE, a wonderful place with a number of fu lltime and visiting scholars in different areas of expertise, is the ideal pl ace for a perfect brain massage. I thoroughly enjoyed my learning experience here. I wish to thank all my colleag ues for their help and mentorship: Dr. Rahul Bagwe, Dr. Samir Pandey, Dr. Tapan Jain, Dr Manoj Varshney, Dr. Monica James and Dr. Daniel Carter. PERC is acknowledged for allowi ng me to use its research facilities and acknowledgements are due to Gary Scheiffele and Gill Brubaker for teaching me how to use them. Dr. Amar Shah is gratefully acknowledge d for several informal brainstorming sessions, and for proofreading of the dissertation. Speci al mention goes to Dr. Monica James for her critique of my chapters. My heartfelt appreciation goes to my parent s and my sisters. Wit hout their consistent emotional support, I simply could not have come this far. Finally, I want to thank my friends Kamalesh Somani, Saurav Chandra, Vijay Kris hna, Uday Tipnis, Siddharth Gaitonde, Naveen Margankune, Gunjan Mohan, Toral Zaveri, Ya sh Kapoor, Suresh Ye ruva and Madhavan Esayanur, for making my stay in Gainesville enjoyable.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 TABLE OF CONTENTS.............................................................................................................. ...5 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES.........................................................................................................................9 ABSTRACT...................................................................................................................................11 CHAP TER 1 INTRODUCTION AND LITERATURE REVIEW..............................................................13 1.1 Colloidal Drug Delivery...................................................................................................14 1.1.1 Micelles..................................................................................................................15 1.1.2 Macroemulsions..................................................................................................... 16 1.1.2.1 Emulsion droplet size................................................................................... 17 1.1.2.2 Viscosity of emulsions................................................................................. 18 1.1.2.3 Emulsion stability......................................................................................... 19 1.1.2.4 Coalescence and phase inversion................................................................. 20 1.1.2.5 Demulsification............................................................................................21 1.1.2.6 Surfactant selection for emulsification......................................................... 21 1.1.2.7 Applications of emulsions............................................................................23 1.1.3 Microemulsions...................................................................................................... 24 1.1.3.1 Formation of microemulsions...................................................................... 30 1.1.3.2 Applications of microemulsions................................................................... 31 1.1.4 Nano-emulsions...................................................................................................... 31 1.2 Non-Toxic Biomedical and Ph arm aceutical Microemulsions.......................................... 36 1.2.1 Applications............................................................................................................36 1.2.2 Introduction to Pharmaceutical Microemulsions.................................................... 37 1.2.2.1 Biocompatibility........................................................................................... 38 1.2.2.2 Solubility and stability................................................................................. 39 1.2.2.3 Bioavailability.............................................................................................. 39 1.2.3 Method of Drug Delivery.......................................................................................39 1.2.3.1 Parenteral delivery........................................................................................40 1.2.3.2 Oral delivery................................................................................................. 41 1.2.3.3 Topical delivery............................................................................................42 1.2.4 Pharmaceutical Microemulsions Using Nonionic Surfactants ............................... 42 1.2.5 Pharmaceutical Microemulsions Using Anionic Surfactants ................................. 43 1.2.6 Pharmaceutical Microemulsions Us ing Phospholipid s and Cholesterol................ 45 1.2.7 Pharmaceutical Microemulsions Using Sugar based Surfactants .......................... 46 1.2.8 Pharmaceutical Microemulsi ons for Drug Detoxification ..................................... 47 1.3 Retardation of Water Evaporation.................................................................................... 47

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6 1.3.1 Duplex Films.......................................................................................................... 50 1.3.2 Spreading of Oil on Water Surface........................................................................ 50 2 PREPARATION OF PROPOFOL MICROEMULSIONS.................................................... 61 2.1 Introduction............................................................................................................... ........61 2.2 Methods and Materials.....................................................................................................63 2.2.1 Materials.................................................................................................................63 2.2.2 Synthesis of Propofol Microemulsions.................................................................. 63 2.2.3 Viscosity Measurements.........................................................................................63 2.2.4 Stability Evaluation of Microemulsions.................................................................64 2.2.5 Stability on Dilution of Microemulsions................................................................64 2.2.6 Oxidation Studies...................................................................................................64 2.3 Results...............................................................................................................................64 2.3.1 Microemulsion Synthesi s and Characterization ..................................................... 64 2.4 Discussion.........................................................................................................................67 2.4.1 Microemulsion Technology for Drug Delivery......................................................68 2.4.2 Selection of Surfactants..........................................................................................72 2.4.3 Nonionic Surfactants.............................................................................................. 73 2.4.4 Ionic Surfactants.....................................................................................................73 2.4.5 Propofol Anesthetic Action....................................................................................74 2.5 Conclusions.......................................................................................................................76 3 ANESTHETIC PROPERTIES OF PROPOFOL MICROEM ULSIONS............................... 88 3.1 Introduction............................................................................................................... ........88 3.2 Methods and Materials.....................................................................................................89 3.2.1 Materials.................................................................................................................89 3.2.2 Synthesis of Propofol Microemulsions.................................................................. 90 3.2.3 Rat Experiments..................................................................................................... 90 3.2.3.1 Animal experimental protocol...................................................................... 91 3.2.3.2 Statistical analysis........................................................................................ 92 3.2.4 Dog Experiments.................................................................................................... 92 3.2.4.1 Animal experimental protocol...................................................................... 93 3.2.4.2 Blood sample acquisition and processing....................................................94 3.2.4.3 Measurement of plasma propofol concentration.......................................... 95 3.2.4.4 Statistical and pharmacokinetic analysis...................................................... 96 3.3 Results...............................................................................................................................97 3.3.2 Anesthetic Properties in the Rat............................................................................. 97 3.3.2.1 Modification of nonionic su rfactant concentration ...................................... 97 3.3.2.2 Modification of ionic su rfactant concentration ............................................98 3.3.3 Anesthetic Properties in the Dogs.......................................................................... 99 3.3.3.1 Effects on erythroc ytes and leukocytes ......................................................100 3.3.3.2 Effects on platelets and thrombosis............................................................ 100 3.3.3.3 Propofol pharmacokinetics......................................................................... 101 3.4 Discussion.......................................................................................................................101 3.4.1 Microemulsion Fate Upon Injection..................................................................... 102

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7 3.4.2 Propofol Anesthetic Properties.............................................................................104 3.4.3 Propofol Concentration........................................................................................105 3.4.4 Coagulation...........................................................................................................106 3.5 Conclusions.....................................................................................................................107 4 RETARDATION OF WATER EVAPORAT ION THROUGH DUPLEX FILMS OF SURFACTANT IN OI L....................................................................................................... 116 4.1 Introduction............................................................................................................... ......116 4.2 Methods and Materials...................................................................................................117 4.2.1 Materials...............................................................................................................117 4.2.2 Spreading of Duplex Film....................................................................................117 4.2.3 Evaporation Studies..............................................................................................117 4.2.4 Brewster Angle Microscopy Studies.................................................................... 118 4.3 Results and Discussion................................................................................................... 118 4.4 Conclusion......................................................................................................................121 5 SUMMARY AND RECOMMENDATIO NS FOR FUTURE WORK ................................ 127 5.1 Propofol Microemulsions for Anesthesia....................................................................... 127 5.1.1 Summary...............................................................................................................127 5.1.2 Future Work.......................................................................................................... 130 5.2 Reduction of Water Evaporation.................................................................................... 131 5.2.1 Summary...............................................................................................................131 5.2.2 Future Work.......................................................................................................... 132 LIST OF REFERENCES.............................................................................................................134 BIOGRAPHICAL SKETCH.......................................................................................................148

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8 LIST OF TABLES Table page 1-1 Physical characteristics of various drug delivery system s................................................. 56 1-2 Types of breakdown proce sses occurring in em ulsions.....................................................57 1-3 Factors influencing th e stability of emulsions ................................................................... 58 1-4 Parameters that affect phase inversio n in em ulsion and the effect they have.................... 59 1-5 A summary of HLB range s and their application .............................................................. 59 1-6 Microemulsions vs. Nano-emulsions................................................................................. 60 2-1 List of propofol microemulsion......................................................................................... 86 2-2 Size of propofol microemulsions....................................................................................... 87 2-3 Viscosity of propofol microemulsions at 25 C .................................................................87 3-1 Dose and latency intervals for anesthe tic induction and em ergence in rat following intravenous infusion of a propofol macroe mulsion formulation and several propofol microemulsion formulations with C8 fatty acid salt and differing purified poloxamer 188 (Pluronic F68) concentrations................................................................................... 113 3-2 Dose and latency intervals for anesthe tic induction and em ergence in rat following intravenous infusion of a propofol macroe mulsion formulation and several propofol microemulsion formulations containing with C8, C10, or C12 fatty acid salts and 5% purified poloxamer 188 (Pluronic F68)........................................................................... 113 3-3 Effects of propofol microemulsions (Micro) and m acroemulsions (Macro) on parameters of the Red Bl ood Cell population in Dogs....................................................114 3-4 Effects of propofol microemulsions (M icro) and m acroemulsions (Macro) on the White Blood Cell count and population differential in Dogs.......................................... 114 3-5 Effects of propofol microemulsions (Mic ro) and m acroemulsions (Macro) on Platelet population and indices of Thrombosis in Dogs...............................................................115 3-6 Plasma propofol concentration afte r induction of anesthesia in Dogs ............................. 115

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9 LIST OF FIGURES Figure page 1-1 Various types of colloid al drug delivery system s.............................................................. 52 1-2 Schematic diagram of a surfactant molecule, m icelle, and reverse micelle...................... 52 1-3 Properties of surfactant so lutions showing abrupt change at the solution critical m icelle concentration (cmc)............................................................................................... 53 1-4 Schematic diagram of the adsorption of surfactant m onomers from the bulk to the oil/water interface during emulsion formation.................................................................. 53 1-5 Schematic diagram of an oilin-water (O/W ) microemulsion........................................... 54 1-6 Thermodynamic explanation for behavior of m acroemulsions and microemulsions........ 54 1-7 Difference between monolayer and duplex film................................................................ 55 1-8 Problems in forming uniform duplex film......................................................................... 55 2-1 Propofol................................................................................................................... ...........77 2-2 Schematic diagram of an oilin-water (O/W ) microemulsion........................................... 77 2-3 Binary diagram noting the concentrat ions of purified pluronic 68 and the cosurfactant fatty acids necessary to form pr opofol m icroemulsions in a bulk media of normal saline.................................................................................................................. ....78 2-4 Binary diagram noting the concentrat ions of purified pluronic 127 and the cosurfactant fatty acids necessary to form pr opofol m icroemulsions in a bulk media of normal saline.................................................................................................................. ....79 2-5 Effects of purified pluronic 68 conc e ntration and fatty aci d chain length on nanodroplet diameter for pr opofol microemulsions.......................................................... 80 2-6 Effect of dilution on propofol m icroemulsions formulated using various concentrations of purified pluronic 68 and several fatty acid salts with variable carbon chain length............................................................................................................81 2-7 Gas chromatography of propofol microemulsion.............................................................. 82 2-8 Mass spectroscopy of propofol..........................................................................................83 2-9 The pH of F68 microemulsion........................................................................................... 84 2-10 Conductivity of F68 microemulsion.................................................................................. 84

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10 2-11 Formation of propofol dimer and propof ol dim er quinone in propofol emulsions............ 85 2-12 Dynamic behavior of microemulsions............................................................................... 85 3-1 Anesthetic induction parameters in Rats.......................................................................... 109 3-2 Anesthetic emergence in Rats.......................................................................................... 110 3-3 Time-dependent effects of a propofol m icroemulsion or macroemulsion on Dogs........111 3-4 Time-dependent effects of a propof ol m icroemulsion or macroemulsion on respiratory rate in Dogs....................................................................................................112 4-1 Langmuir film balance..................................................................................................... 122 4-2 Evaporation experiment setup.......................................................................................... 122 4-3 Comparison of water evaporation re duction by duplex film and monolayers................. 123 4-4 Effect of film th ickness on evaporation. .......................................................................... 123 4-5 Effect of concentration of Br ij 93 in hexadecane on evaporation................................... 124 4-6 Brewster Angle Microscopy images of duplex film of hexadecane and Brij-93 ............. 124 4-7 Effect of various polymers on evaporation......................................................................125 4-8 Effect of aging and compression /expansion of film on evaporation. ..............................125 4-9 Effect of films of various commer cial products on water evaporation. ........................... 126

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy SURFACTANT SYSTEMS FOR DRUG DELIVERY AND WATER EVAPORATION REDUCTION By Dushyant Shekhawat December 2007 Chair: Dinesh O. Shah Cochair: Anuj Chauhan Major: Chemical Engineering Surfactant systems are used to enhance the quality of products used in every aspect of life; from food to cosmetics, from pharmaceutics to detergency, and even from oil recovery to chemical mechanical polishing of silicon wafers In this dissertation, we investigate these systems for drug delivery as well as wa ter evaporation reduction applications. Microemulsions are excellent candidates as potential drug de livery systems because of their improved drug solubilization, long shelf-life, and ease of prep aration and administration. In the present study, Propofol (2,6-diisopropylphenol) was selected as a test drug to form water external microemulsion. Propofol is intrave nous general anesthetic drug, having several favorable anesthetic character istics, including rapid emergenc e from unconsciousness without drowsiness. Several oil-in-water microemulsions constituting Propofol (oil), biodegradable nonionic polymers, surfactants and fatty acid salts were formulated. Various properties of these microemulsions, like particle size, stability on d ilution, and pH etc. were measured as a function of time, which shows that these systems are th ermodynamically stable. Anesthetic studies of these microemulsion systems were done using ra ndomized crossover design in rats and dogs. Rats randomly received propofol either as a microemulsion or conventional macroemulsion

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12 (Diprivan) to determine endpoints of anesthet ic induction and recovery. Pharmacodynamic and Pharmacokinetic properties of Propofol microemu lsion were measured by experiments in dogs. In conclusion, Propofol microemulsions caused ge neral anesthesia in rat/dogs similar to that resulting from macroemulsions. The surfactant concentration and type markedly affects the spontaneous destabilization and anesth etic properties of microemulsions. Retardation of water evaporation is important from number of viewpoints. In this work, we proposed a new approach to reduce the evaporatio n of water, i.e. multi molecular (duplex) films of micron or sub-micron thickness of oil and surfactants that spontaneously spreads on the water surface and reduce the water evaporation rate. In vestigation of the duplex film of Brij93 and hexadecane has shown some promising results that it can decrease the evaporation rate of water as much as 80%. Addition of polym er (Polyvinyl alcohol) in the water beneath this duplex film helps in further reduction of the evaporation rate. Effect s of various parameters like concentration of surfactant, different surfactan ts in hexadecane, and thickness of the film deposited and various polymeric additives on wa ter evaporation through these films have been studied.

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13 CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW Surfactant system s are used to enhance the quality of products used in every aspect of life; from food to cosmetics, from oil recovery to detergency, and even from pharmaceutics to chemical mechanical polishing of silicon wafers. He re in this thesis, we present studies on two of such system, namely drug delivery by microemu lsions and reduction of water evaporation by duplex films of surfactant in oil. Microemulsions are excellent candidates as potential drug de livery systems because of their improved drug solubilizati on, long shelf-life, and ease of preparation and administration. Depending on the solubility of drug in water and oil, all three type s of microemulsions (i.e. oilexternal, water-external and mi ddle-phase microemulsion) can be used for drug delivery. In the present study, Propofol (2,6-diis opropylphenol) was sele cted as a test drug to form water external microemulsion. Propofol is intrave nous general anesthetic drug, having several favorable anesthetic character istics, including rapid emergen ce from unconsciousness without drowsiness. Propofol is water in soluble and currently used in a macroemulsion form, which has various side effects. In the present study, several oil-in-water microemulsions constituting Propofol (oil), biodegradable surfactants and pol ymers were formulated. Various properties of these microemulsions, like particle size, stability on dilution, and pH etc. were measured as a function of time, which shows that these syst ems are thermodynamically stable. Anesthetic studies of these microemulsion systems were done using randomized crossove r design in rats and dogs. Retardation of water evaporati on is of interest from a number of viewpoints such as in ophthalmology wherein people suffering from dry ey e syndrome show very high water loss from eyes, nuclear industry wherein radioactive water from spen t fuel may escape into the

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14 atmosphere, antiseptic for burned skin victims, wate r conservation in certain arid and dry parts of world where water is scarce etc. But, despite this need to redu ce water evaporation, few efforts have been directed in finding new ways to decr ease the loss of water by evaporation. One of the approaches, which are currently being used, is to spread a monolayer of long chain alcohols/acids on top of water to decrease th e water evaporation. Over the years various researchers1, 2 have worked on reduction on evaporat ion of water by monolayers, but the maximum reduction in evaporation of water ach ieved by monolayers is only around 50%. Also, problem with monolayers is that they can be easily removed by winds and undergo thermal as well as biological degradation. Another approach is to use a multimolecu lar film of oil as a means of reducing the evaporation of water. But in this case small amount of oil does not spread so the amount of oil used is very high, which is not economical. In this work, we proposed a new approach to reduce the evaporation of water, i. e. multimolecular (duplex) films of micron or submicron thickness of oil and surfactants that spontaneously spreads on the water surface and reduce the water evaporation rate. The following is the detailed review of collo idal drug delivery sy stem and reduction of water evaporation. 1.1 Colloidal Drug Delivery The design and developm ent of new drug delivery systems with the intention of enhancing the efficacy of existing drugs is an ongoing proc ess in pharmaceutical research. It is necessary for a pharmaceutical solution to contain a therap eutic dose of the drug in a volume convenient for administration. Of the many types of drug delive ry systems that have been developed, one in particular, i.e. colloidal drug delivery system ha s great potential for the goal in drug targeting. A few of the most widely examined colloidal drug delivery systems are micelles, microemulsions, macroemulsions, liposomes, and nanoparticles. These colloidal systems are

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15 shown schematically in Figure 11. A comparison of physical prope rties of these colloidal drug delivery systems is given in Table 1-1. 1.1.1 Micelles A surfactant, or sur f aceact ive ag ent is defined as a substance that adsorb s onto surfaces or interfaces of solutions to lower the su rface or interfacial te nsion of the system.3 The magnitude of the lowering of the surface or interfacial tension depends on the surfactant structure, concentration, and the physico-chemical conditions of the solution (e.g. pH, salt concentration, temperature, pressure, etc.).3 Surfactants are typically amphiphatic species, meaning that they are made up of a hydrophobic component, referred to as the tail, and a hydrophilic component, referred to as the hea d group (see Figure 1-2). When placed in solution, surfactant molecules tend to orient in such a way as to minimize the interactions of the hydrophobic tail with water in aq ueous solutions, or to minimize the interactions of the hydrophilic head with oil in or ganic solvents. This leads to adsorption of the surfactant molecules onto surfaces or at in terfaces, and above a certain concen tration, known as the critical micelle concentration or cmc, surfactants form aggregates known as micelles. When placed into aqueous solutions, surfactant molecules will form spherical aggregates at the cmc where the hydrophobic tails are pointed inward and removed fr om interaction with water molecules by the hydrophilic head groups as shown in Figure 1-2. When placed into organic solutions, surfactant molecules will form reverse micelles with the hydrophobic tails pointed outward (see Figure 12). When the critical micellar concentration, or cmc, is reached, many of the physical properties of the surfactant solution in water sh ow an abrupt change as shown in Figure 1-3. Some of these properties include the surface tension, osmotic pre ssure, electrical conductivity, and solubilization. The cmc is a measure of th e free monomer concentr ation in surfactant

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16 solutions at a given temperature, pressure, and composition. Mcbain4 first investigated the unusual behavior of fatty acid salts in dilute aqueous solution at the cmc in the 1910s and 1920s and was followed by Hartley5, 6 in the 1930s. Other evidence fo r surfactant aggr egation into micelles was obtained from vapor pressure measurements and the solubility of organic molecules in water. The formation of colloidal-sized clusters of individual surfacta nt molecules in solution is known as micellization. Normal micelles are optically isotropic and thermodynamically stable liquid solution of water and amphiphile. Micelles have low viscosity, long shelf life, and are very easy preparation method. But, micelles do not tolera te large amount of apolar species because of their very limited capacity to solubilize oil. 1.1.2 Macroemulsions It is a commonly known fact th at oil and water do not m ix. However, emulsifying agents, typically surfactants can be added to a mixture of oil and water to promote the dispersion of one phase in the other in the form of droplets. Over the years, em ulsions have been defined in a variety of ways. Emulsions are thermodynamically unstable, heterogeneous systems, consisting of at least one immiscible liquid intimately disp ersed in another in the form of droplets, whose diameters are generally in the range of 1 100 m.7 There are two main types of emulsions: oilin-water emulsions, in which oil droplets are disperse d in a continuous water phase, or water-inoil emulsions, in which wa ter droplets are dispersed in a continuous oil phase. The most fundamental thermodynamic property of any interface is the interfacial free energy, or interfacial tension. The interfacial fr ee energy is the amount of work necessary to create a given interface. The inte rfacial free energy per unit area is a measure of the interfacial tension between two phases. A high value of inte rfacial tension implies that the two phases are

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17 highly dissimilar in nature. Ther e are many methods available to measure the interfacial tension between two liquids including the Du Noy ri ng method, Wilhelmy plate method, drop-weight or drop-volume method, pendant drop method, spinning drop method, and Sessile drop method.3 Emulsification involves the generation of a large total interfacial area. Considering that the two phases in emulsions are not miscible, in orde r to generate this large interfacial area, the interfacial tension must be lowered signifi cantly according to the following equation: W = A (1-1) where W is the work done on an interface, is the interfacial tension, and A is the change in interfacial area associat ed with the work W. According to Equation (1-1), when a constant amount of work is applied to generate an interface, A will be large if is small, and thus the interface will expand significantly to form smaller emulsion droplets. As previously mentioned, the primary means by which the interfacial tension is lowered is through the addition of emulsifying agents, usua lly surfactants. The surf actant molecule also plays a second role in emulsions which is to stabilize the interface for a time against coalescence with other droplets and concomitant phase sepa ration. A large number of methods have been developed to provide the energy needed to achieve complete em ulsification in a given system.710 1.1.2.1 Emulsion droplet size Em ulsions are classified as either wate r-in-oil (W/O) or oil-in-water (O/W) depending on which phase is continuous and which is dispersed. The dispersed phase in emulsions, whether oil or water, is usually compos ed of spherical droplets within the continuous phase. These droplets may be nearly monodisperse in terms of droplet size; or they may have a wide size distribution depe nding on several factors.7 In most cases, the wider the size

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18 distribution, the less stab le is the emulsion. In ot her words, emulsi ons with a more uniform size distribution tend to remain stab le for longer time while those w ith wide size di stribution will usually undergo Ostwald ri pening, a phenomenon where larger dr oplets grow at the expense of smaller droplets.11 In general, emulsions with a narrow size dist ribution and a small mean droplet size tend to exhibi t a greater emulsion stability, all other things being equal.7 The change in the size distribution wi th time reflects the kinetics of coalescence in emulsions. Depending on the surf actant that is used to stabilize th e emulsion, emulsions can have lifetimes ranging from hours to as long as a few years.7 In general, emulsions exhibiting a higher yield stress tend to show higher em ulsion stability and shelf life.7 Emulsion droplet size is also related to the method of preparation that is employed to generate the emulsion. This is a result of the re lationship between interfac ial area and work that is done on the system, according to Equation (1-1 ). As can be seen in this equation, if the interfacial tension is constant with time, the change in interfacial area is directly proportional to the amount of work that is pu t into the system. Some emulsi on preparation methods provide more energy (work) than others, and thereby lead to smaller drople ts and higher in terfacial area. Micellar stability is another factor that affects the droplet size of emulsions.12 If the micelles are very stable, flux of surfactant monomers to the interface of drop lets will be low, resulting in a higher inte rfacial tension at the droplet surface and a large droplet size will occur as predicted by Equation (1-1). The process of monomer diffusion from the bulk to the oil/water interface is ill ustrated in Figure 1-4. 1.1.2.2 Viscosity of emulsions The viscosity, or resistance to flow, of e mulsions could be considered as one of their most important properties. This is true from both a pr actical and a theoretical viewpoint. In a practical sense, certain cosmetic or even food emulsions are only desirable at a specific viscosity (e.g.

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19 lotions, milk, salad dressings, etc.). Manipulatio n of emulsion viscosity to achieve the desired product specifications is not a tr ivial matter. From a theoretical perspective, the viscosity measurements can be used to provide insightful information about the st ructure and possibly the stability of an emulsion. The overall emulsion stability is affected by the following factors:7, 13 Viscosity of the external phase Concentration (i.e., volume frac tion) of the internal phase Viscosity of the internal phase Nature of the emulsifiers Surface viscoelasticity of the interfacial film formed at the oil/water interface Droplet size distribution 1.1.2.3 Emulsion stability As m entioned previously, one of the most impo rtant parameters in emulsification processes is emulsion stability. For example, milk is a natura l emulsion of the O/W. If the stability of milk was only a week or two, the milk would have to be shaken vigorously before pouring. However, in this case nature has provided us with a stable em ulsion. Another common example is shampoo, anot her emulsion. It would be inconvenient if the shampoo were not a stable emulsion, since shaking would be necessary. There are also cases where it is necessary to break down unwanted, naturally occurring stable emulsions. Such examples are the W/O type emulsions which build up in oil storage tanks, or the O/W type emulsions that arise in effluent waters. So it becomes necessary to un derstand how to develop an emulsion system to be stable or unstable, depending on the n eeds of the industry. To unders tand emulsion stability, it is important be cognizant that there are five t ypes of breakdown processes which can occur in emulsions. These are listed in Table 1-2, along w ith factors that influence that type of breakdown.14

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20 1.1.2.4 Coalescence and phase inversion When two oil drops approach each o ther, a th in film of the continuous water phase is trapped between the drops. The beha vior of the thin film determin es the degree of stability of the emulsion, and the rate of thinning of the film determines the time required for the two drops to coalesce (i.e., coalescence rate). When the film has thinned to a critical thickness, it ruptures, and the two drops unite or coalesce to form one larger drop.15, 16 The rate of film thinning depends on the surface viscosity of the surfactant film adsorbed at th e oil/water interf ace. The film may drain evenly or unevenly depending on the in terfacial tension gradie nt due to adsorbed surfactant.17 The factors that influence the rate of film thinning between droplets therefore influence the emulsion stability. A summary of a ll of the factors influe ncing coalescence of droplets is given in Table 1-3.18 Phase inversion can occur in emulsions due to a number of factors. For a given emulsifier concentration, the viscosity of an emulsion gr adually increases as the phase volume of the dispersed phase is increased. However, at a certain critical volume fraction c, there is a sudden decrease in viscosity, which corresponds to the point at which the emulsion inverts. c was found to increase with increasing emulsifier concentration.19 The sudden decrease in viscosity is due to the sudden reduction in disperse d phase volume fraction. Often c is in the range of 0.74, so that upon inversion, the dispersed phase volume frac tion reduces from 0.74 to 0.26, thus reducing the viscosity significantly. c should theoretically be in the range of 0.74 for spheres of equal radii to be at the maximum packing,20 but c=0.99 was found for paraffin oil/aqueous surfactant solutions21 and c=0.25 was found for olive oil/water emulsions.22 The phase inversion of emulsions can be brought about by several parameter changes, listed in Table 1-4.

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21 1.1.2.5 Demulsification As discussed previously in this section, it is not always desira ble to have a stable em ulsion. Often an emulsion is present in a system in which it is undesirable. One ex ample is the presence of aqueous emulsion droplets dispersed in crude oil. Crude oil is always associated with water or brine in oil reservoirs and also contains natural emulsifying agents, such as resins and asphaltenes. These emulsifying agents form a thick, viscous interfacial film around the water droplets, resulting in a very stable emulsion. Therefore, demulsification is very important in the crude oil industry. Many physical me thods have been developed for demulsification, depending on the industrial application. A wide variety of chem ical additives for demulsification have been developed in recent years. These additives are all relatively high molecular weight polymers capable of being adsorbed at the O/W interface and displacing the film. The primary advantage of these additives is that they can be added to the system even before emulsion formation, so that they act as inhibitors. In the petroleum industry, demulsifiers have been considered for breaking the common fuel oil emulsions. In this area, chemical demulsif iers that have been investigated include ultrahigh molecular weight polyoxiranes23 and micellar solutions cont aining petroleum sulfonates, electrolytes, and cosurfactants.24 It is evident that there are many methods for demulsification. The nature of the emulsion to be separated is th e key factor in determining which method(s) is best for each particular demulsification problem. 1.1.2.6 Surfactant selection for emulsification Often the selection of surfactants in the prep aration of either O/W or W/O emulsions is made on an empirical basis. However, in 1949, Griffin25 introduced a semi-empirical scale for selecting an appropriate surfactant or blend of surfactants. This scale, termed the hydrophilelipophile balance (HLB), is based on the relative percentage of hydrophilic to hydrophobic

PAGE 22

22 groups in the surfactant molecules and ranges from 1 to 40. An HLB of 1 represen ts a surfactant that is highly oil-soluble and an HLB of 40 represents a highly water-soluble surfactant. Surfactants with a low HLB number normally form W/O emulsions, whereas those with a high HLB number often form O/W emulsions.26 A summary of the HLB range required for various purposes is given in Table 1-5. The calculation of the HLB number for a given surfactant, as developed by Griffin,25 is quite laborious and requires a number of trial and error procedures. Simplification methods were later developed by Griffin25 that applied to certain surfactants. Davies26 developed a method for calculating the HLB values of surfactants directly from their chemical formulas, using empirically determined numbers. The HLB number can also be determined experi mental through several correlations that have been developed. These co rrelations relate the HLB number to such parameters as the cloud point,27 water titration va lue for polyhydric alcohol esters,28 and the heat of hydration of ethoxylated surfactants.29 Another method that may be used to select a su rfactant suitable for forming an emulsion is by using the phase inversion temperature (PIT) method. The phase inversion temperature (PIT) is the temperature at which an emulsion experiences phase inversion, as described in a previous section. The PIT of non-ionic emulsifiers has been shown to be influenced by the surfactant HLB number, so the PIT can be used similarly to the HLB nu mber in selecting an emulsifier.22 The primary distinction is that the PIT is a characteristic property of the emulsion, not of the emulsifying agent.22 Due to this, the PIT includes the effect of additives on the solvent, the effe ct of mixed emulsifiers or mixed oils, etc. In other words, the HLB number is actually a function of al l of these properties, but only the PIT completely analyzes a given emulsion system. The PIT method is useful because the PIT is a measurable property

PAGE 23

23 which is related to the HLB number A summary of effects of PIT a nd droplet stability from different investigations is given below:22 The size of emulsion droplets depends on the temperature and the HLB of emulsifiers The droplets are less stable towa rd coalescence cl ose to the PIT Relatively stable O/W emulsions are obtained wh en the PIT of the sy stem is some 20 to 65C higher than the storage temperature A stable emulsion is obtained by rapi d cooling after formation at the PIT The optimum stability of an emulsion is relatively insens itive to changes of HLB value or PIT of the emulsifier, but instability is very sensitive to the PIT of the system The stability agains t coalescence increases markedly as the molar ma ss of the lipophilic or hydrophilic groups increased When the distribution of hydrophilic chains is broad, the cloud point is lower and PIT is higher than when there is a narrow size distribution. The PIT can be measured by the following methods: 1) direct visual assessment,30 2) conductivity measurement,31-33 3) Differential Thermal Analysis (DTA ) or Differential Scanning Calorimetry (DSC),34 and 4) viscosity measurement.35, 36 Both the HUB and PIT methods for selecting an emulsifier in a system have been widely used and adapted to me et industry needs. 1.1.2.7 Applications of emulsions Em ulsions are desirable for many different a pplications because they provide a system having a large interfacial area. Historically, cosmetic emulsions are the oldest class of manufactured emulsions.7 Emulsions are desirable for cosme tic applications because: 1) they increase the rate and extent of penetration into the skin, 2) they open up the possibility of applying both waterand oil-soluble ingredients simultaneously (e .g. deodorants), and 3) they provide for efficient cleansing. Emulsions are also widely used for pharmaceutical applications in the form of creams or ointments and as drug delivery vehicles. They ar e also ideal for use as polishes (e.g. furniture

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24 polishes, floor waxes, etc.) paints, and agricu ltural sprays. Many foods are manufactured in the form of emulsions including ma yonnaise, salad dressings, milk, and margarine. Another industry where emulsion technology is important is the as phalt industry when the principal requirement is the production of water-repellent surfaces. Emulsions are also used as polymerization vehicles to aid in the production of high polymer ic materials such as plastics, synthetic fibers, and synthetic rubbers. These are just a few of th e many applications of emulsions. 1.1.3 Microemulsions A m icroemulsion is a thermodynamically stab le, isotropic dispersion of oil and water containing domains of nanometer dimensions stabil ized by an interfacial film of surface-active agent(s).37 The term microemulsion originated from Jack H. Schulman and coworkers in 1959,38 although Hoar and Schulman originally desc ribed water-in-oil microemulsions, which they referred to as transparent water-in-oil dispersions, in 1943.39 As implied above, microemulsions may be of the oil-in-water (O /W) (see Figure 1-5)) or the water-in-oil (W/O) type depending on conditions of the system and system components. According to Bancroft,40-42 phase volume ratios are less important in the determination of the microemulsion type that will be formed (i.e., W/O or O/W) than the surfactant characteristics (e.g. HLB). As previously mentioned, the Banc roft rule states that whichever phase the surfactant has a greater affinity for w ill typically be the continuous phase. The creation of a microemulsion entails the generation of a huge in terfacial area, which, according to the following equation,43 requires a significant lowering of the interfacial tension (usually << 1 mN/m):44 W = A (1-1)

PAGE 25

25 where W is the work performed, is the surface or interfacial tens ion at the air/water or oil/water interface and A is the change in surface or interfacial area. This ultr a-low interfacial tension in spontaneously formed microemulsion s is achieved by the incorporati on of surfactant(s) (typically a surfactant + a cosurfactant, especia lly when ionic surfactants are used).45 Figure 1-6 shows the thermodynamic explanation for the behavior of macroand microemulsions. As can be concluded from the graph, there is an optimum ra dius for microemulsion systems where the free energy of dispersion becomes negative, there by making the microemulsion stable and its formation energetically favorable.46 Schulman and others first noticed microemulsion systems in 1943 when they observed that the addition of a medium chain-length alcohol made a coarse macroemulsion that was stabilized by an ionic surfactant become transparent.39 Even then, Hoar and Schulman recognized the important role of a very low interfacial tens ion in causing spontaneous emulsification of the added water in oil.39 They concluded that the role of the alcohol is as a stabilizer against the repulsive electrostatic forces that the ionic surfactant head groups would experience. Schulman and others used a variety of expe rimental techniques (e.g. X-ray diffraction,38 ultra-centrifugation,47 light scattering,38 viscosimetry,48 and nuclear magnetic resonance (NMR)49, 50) to elucidate some of the characteristics of these microemulsion systems following the groundbreaking work of Schulman and Hoar. Th ese studies were instrumental in providing them with information about the structure, size, and interfacial film beha vior of microemulsions. They were able to determine the size of the droplets and they found that the presence of the alcohol within the system led to greater interfacial fluidity. Later, in 1967, Prince38 proposed a theory that the forma tion of microemulsions was due to the negative interfacial tension that results from high su rface pressure of the film. Prince

PAGE 26

26 explained this negative interfaci al tension based on the depression of the interfacial tension between the oil and water phase th at occurs when surfactant is added. The principle behind this theory is described by the series of equations that follow. The surf ace pressure of th e film at the air/water interface, aw, is defined as:38, 45 soaw (1-2) where o is the surface tension of the pur e surface (without surfactant) and s is the surface tension of the surface with surfactant. In the case where oil is the second phase (oil/water system), the surface pressure of the surf actant film at the oil/water interface, ow, can be defined as: s wo o wo ow / / (1-3) where (o/w)o is the interfacial tension of the pure oil/water interface (i.e., in the absence of surfactant) and (o/w)s is the interfacial tension of the oil/water interface in the presence of surfactant film. Rearrangemen t of Equation (1-3) gives: ow o wo s wo / / (1-4) Based on Equation (1-3), for a surfactant film that can generate a very high surface pressure ( ow), the interfacial tension of the surfactant film at the oil/water interface ( o/w)s becomes negative. This is only a transient phenomenon because generation of a negative interfacial tension leads to a negative free energy of formati on of the emulsion, which is an unstable situation. This is illustrated by the following equation: Gform = A T Sconfig (1-5) where A is the increase in interfacial area, Sconfig is the configurationa l entropy of the droplets of the liquid that are formed and T is the absolute temperature.51 The negative interfacial tension

PAGE 27

27 accounts for the spontaneous increase in interfacial area that occurs in the formation of microemulsions. When a transient (unstable) ne gative interfacial tensi on is experienced, the system will seek to stabilize by spontaneously ge nerating new interfacial area, thereby raising the interfacial tension back to accepta ble, stable limits. As previously mentioned, in order to form microemulsions, it is required that the concentrat ion of surfactants be gr eater than that required to reduce the oil/water interfacial tension to zero and to cover the total interfacial area of all dispersed droplets. The transient negative interfacial tension that is generated facilitates the spontaneous break-up of droplets. The nature of the thermodynamic stability of microemulsions has long been studied. The stability can be attributed to the fact that the interfacial tension is low enough that the increase in interfacial energy accompanying di spersion of one phase in the other is outweighed by the free energy decrease that is associated with the entropy of dispersion.46 Furthermore, the free energy decrease that accompanies adsorption of surfactant molecules from a bulk phase favors the existence of a large interfacial area and hence pl ays a major role in stabilizing microemulsions.46 One must also understand the role of cosurf actants in microemulsion formulations. The addition of a short-chain alcohol to a surfactant soluti on to enhance microemulsion formation has long been practiced.52 This cosurfactant (shortchain alcohol) serves to 1) fluidize the interface, 2) decrease interfacial viscosity, 3) destroy the lamellar liquid crys talline structures, 4) provide additional interfacial area, 5) re duce electrical repulsi on between droplets and also between polar head groups of surfactants by acting as charge screeners and decreasing surface charge density, and 6) induce the appropriate curvature changes.52 In 1972, Gerbacia and Rosano53 investigated the forma tion and stabilization of microemulsions, with emphasis on the role of th e cosurfactant (in this case, pentanol). They

PAGE 28

28 suggested that a critical aspect of the mechanism of formation of microemulsions involves the diffusion of the pentanol through the interface. This pr ocess has been found to be a necessary, but not sufficient condition for micr oemulsification. In essence, the alcohol transiently lowers the interfacial tension to zero as it diffuses thr ough the interface, thereby inciting the spontaneous dispersion of one phase into the other. The surf actant then acts to stabil ize the system against coagulation and coalescence. NMR data and calc ulation of free energies of adsorption of the pentanol into the interface have proven that a strong associat ion between the surfactant and cosurfactant is not necessary for microemulsion formation.53 The stability of the phases of surfactant in microemulsions results from the competition between entropic and elastic c ontributions to the free energy.54 The two intrinsic parameters that ultimately affect the structure of the aggregates existing in solution are the mean bending modulus, and the Gaussian bending modulus, The bending energy, Fb, is directly related to the mean bending modulus and the Gaussian bending modulus, as can be seen below: 21 2 0 212 2 1 CCCCCFb (1-6) where C1 and C2 are the local principal curvatur es of the surfactant layer and C0 is the spontaneous curvature. Bellocq55 states that the contribution of the bending energy to the total free energy is a crucial determining factor in the type and characteris tic size of the structure. In Equation (1-6) above, the first te rm represents the amount of en ergy needed to bend a unit area of interface by a unit curvature am ount, and the second term is im portant to the change of the membrane topology and the phase transition.55 The spontaneous curvature, C0, is determined by the nature of the interactions between the su rface-active molecules; i.e., the competition in packing of the polar heads and the hydrocar bon tails of the surfact ant. If the dominant interactions are between the polar heads, then the surfactant orientation will be concave to water

PAGE 29

29 and a water-in-oil microemulsion will be formed, whereas, if the dominant interactions are between the hydrocarbon tails, th e surfactant orientation will be convex to water and an oil-inwater microemulsion will be formed. The addition of cosurfactan t (short-chain alcohol) can have a profound effect on the curvature. Bellocq reported that there is a ve ry efficient lowering of the bending constant, of the surfactant film with the dilution of the system with short-chain alcohols.55 This lowering of was attributed to thinning of the interface, and this attributio n was confirmed with deuterium solid-state NMR. The chain length of the cosurf actant (alcohol) was found to be critical in microemulsion formation. The chain length determines what types of phases (bicontinuous, lamellar, sponge, vesicle, etc.) will be of importance. These phases pl ay an important role in the type, structure and size of the microemulsion that will be formed.56 In simplistic terms, a short-chain cosurfactant acts to prevent the formation of or destroy lame llar liquid crystals. Since there is a significant difference between the surfactant ch ain length and the short-chain alcohol, there is a tail wagging effect due to the fact that the excess hydrocarbon tails have more freedom to disrupt molecular packing through conformational disorder, increased tail motion, a nd penetration and/or buckling of the chain into the monolayer.57 This motion of the hydrocarbon tail prevents or disrupts ordering of the molecules and therein prevents fo rmation of or destroys lamellar liquid crystals and enhances microemulsion formation. It must be noted that cosurfactant addition to microemulsion-forming systems is typically only applicable for ionic surfactant systems. Microemulsions that incorporate non-ionic surfactants may be formed without the need for cosurfactant additi on; especially in the case of non-ionic surfactants of the polyeth ylene oxide adducts (POE). This is because these surfactants

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30 are composed of a homologous series of varying composition and molecular weight,45 which serves the same purpose of enhanc ing the interfacial film fluidity. The important factor in the formation of these types of microemulsions is te mperature, because this class of materials is solubilized in water by means of hydrogen bonding between the water molecules and the POE chain. Hydrogen bonding is a temperature-se nsitive phenomenon, which decreases with increasing temperature. Therefore, the temper ature conditions under which a microemulsion is formed are important to the type of microemu lsion that is formed. Above a characteristic temperature, which is commonly known as the phase inversion temperature (PIT),58 the nonionic surfactant changes its affinity from the wa ter phase to the oil phase. Below the PIT, O/W microemulsions will be formed and above th e PIT, W/O microemulsions will be formed. 1.1.3.1 Formation of microemulsions Now that the some of the basic principles of microemulsions have been discussed, it is easier to understand what conditions must be met for microemulsion formation and why they are required. There are three major factors that must be considered in order to form microemulsions.22 First, given the importance of achieving an ultra-low surface tension at the oil/water interface, surfactants must be carefully chosen so that this may be accomplished. Secondly, there must be a large enough concentratio n of surfactants to stabilize the newly formed interface such that phase separation does not occur. It must be mentioned that th e type, structure and characteristics (e.g. Hydrophilic-Lipophilic Balance (HLB), degree of ioni zation, etc.) of the surfactants may potentially play a major role in determining just how high the surfactant concentration needs to be to stabilize the in terface. The third required condition for forming microemulsions is that the interface must be flui d (flexible) enough to faci litate the spontaneous

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31 formation of micro-droplets with a small radi us of curvature (50 500 ). That is where cosurfactant structure can become very important. 1.1.3.2 Applications of microemulsions Microemulsions have a range of industrial app lications. They are useful in technologies such as enhanced oil recovery,59 pharmaceuticals,60 cosmetics,61, 62 food sciences,63 and detergency.64, 65 Microemulsions have been extensively studied in regards to their use as drug delivery vehicles, and are now gaining attention as possible mediums for use in detoxification of blood to remove free drug from th e blood stream of overdose patients.66 Drugs that have significant hydrophobic functionality have been shown to partition into the corona (area at the interface) and/or interior core of O/W microemulsions.66 It is generally accepted that this is largely due to hydrophobic interactions. Recall th at the formation of microemulsions leads to the generation of a large in terfacial area. It is believed that this large interfacial area facilitates the uptake of relatively large amounts of drug into the microemulsion in a time efficient manner, thereby significantly decreasing the bulk drug concentration. 1.1.4 Nano-emulsions As early as 1943, Dr. T. P. Hoar and J. H. Schulman39 discovered that he could prepare transparent water-in-oil dispersions of nanometer size, which displayed stable thermodynamic characteristics (i.e., the water droplets remained st ably dispersed indefinitely if left unperturbed, with respect to temperature, pressure, or co mpositional conditions). Anomalous systems could also be prepared in which oil droplets are disper sed in water. Later, other scientists, including Dr. Stig Friberg,56 found that they could develop transpar ent nanometer size dispersions of one medium within another continuous medium, whic h were not thermodynamically stable, but were kinetically stable (i.e., given time, there will be a breakdown in the stability of the dispersion that will lead to phase separation). Such systems have come to be referred to as nano-emulsions

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32 (thermodynamically unstable systems). Thus, both Schulmans microe mulsions and nanoemulsions start in the nanometer range, but th e microemulsions are thermodynamically stable and maintain the same size whereas the nano-em ulsions coalescence and display an increase in size with time, which ultimately causes phase separation. To sufficiently comprehend the difference be tween microemulsions and nano-emulsions, there must be clarification of the nomenclat ure of the two. As previously mentioned microemulsions is a misleading title consideri ng that their average diameter ranges from 10 to 100 nm. Light scattering and X-ray analysis have indicated that microemulsions are, in fact, coarse mixtures as opposed to molecular dispersions.44 Nano-emulsions lie within the same size range, but may have diameters that considerably exceed the size of a microemulsion droplet (20500 nm). The emphasis must be placed on the fact that nano-emulsions are in fact emulsions of nano-size, meaning that they are kinetically stable, (thermodynamically unstable) heterogeneous systems in which one immiscible liquid is disper sed as droplets in anot her liquid (as emulsions are defined). Although they are on similar size scales, mi croemulsions and nano-emulsions have markedly different characteristics (Table 16). Nano-emulsions are formally defined as thermodynamically unstable, generally opaque, sub-micron-sized (20 500 nm) systems that are stable against sedimentation and creaming.67 They may have the appearance of microemulsions, but they do not necessarily require as much su rfactant concentration in their preparation.67 As early as 1981, Rosano et al.53 found that certain microemulsion systems, which they termed unstable microemulsions, displayed significantly different characteristics from traditional microemulsions. These systems were dependent upon the order of addition of components (i.e., the mixing protocol) and their formation was contingent upon having a large

PAGE 33

33 enough concentration gradient to allow diffusion of amphipathic materials across the oil/water interface. El-Aasser et al.68 performed studies of a miniemulsification pro cess, which produced O/W miniemulsions (another term for what we refer to as nano-emulsions in this paper) having an average droplet diameter in the size range of 100 400 nm. The miniemulsions that they produced were generated by means of a mixed em ulsifier protocol, which was comprised of a mixture of ionic surfactant and long-chain fatty acid in concentrations of 1-3 % by weight in the oil phase. They stated four distinct and si gnificant differentiati ng aspects between the necessary conditions of preparation for mi niemulsification systems and traditional microemulsion systems, which may also be produced by mixed emulsifier systems: Concentration of the mixed emulsifier syst em: only 1 wt% (with respect to the oil phase) is sufficient for the formation a nd stabilization of miniemulsions, whereas microemulsions typically require 15-30 wt %. Droplet size: their miniemul sion droplets range from 100-400 nm in diameter, as opposed to microemulsions, which range from 10-100 nm in diameter. Chain length of the cosurfactan t (fatty alcohols or acids in this case): miniemulsions require at least a 12-carbon chain length, wher eas microemulsions can be prepared with alcohols of shorter chain lengths. Order of mixing of the ingredients (mix ing protocol): successful production of miniemulsions requires that the ionic surfactant and the fatty alcohol be initially mixed in the water phase for 30 minutes to 1 hour at a temperature above the melting point of the fatty alcohol, prior to the addition of the oi l phase, whereas order of mixing does not affect microemulsion formation. El-Aasser et al.68 performed an array of experiments to help them to better understand the miniemulsification process. They found that the process was a spontaneous phenomenon based on results yielded by both dynamic and static sp inning drop experiments. The oddity in this discovery lay in the fact that the measured interfacial tensions were unexpectedly high, ranging from 5 to 15 dynes/cm. They attributed this fi nding to the formation of emulsion droplets by

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34 diffusion of the oil (in this case, styrene) from drops into the adjacent liquid crystal structure of the mixed emulsifier system. They also stated that the presence of mixed emulsifier liquid crystals, which was confirmed by birefringe nce observations, significantly improved the emulsification process and led to greater emulsion stability. They concluded that the mechanism of formation of miniemulsions wa s by swelling of the mixed emulsi fier liquid crystals by oil (in this case, toluene). This swelling of the liquid crystalline structure led to its break-up or subdivision to form small emulsion droplets, which were stabilized by the adsorption of the mixed emulsifier complex at the oil-water interface.68 Although emulsion stability is generally known to increase with droplet surface charge, the miniemulsions that El-Aasser prepared displayed contradictory behavior; th eir stability increase corresponded to a reduction in su rface charge. These results sugge sted that the steric component of stabilization was the dominating factor.68 The mechanism that has been attributed to nano-emulsion breakdown in a ternary system of brine, oil, and non-ionic su rfactant is a 3-stage process.69 The first and last stages of the droplet growth process were found to be due to Ostwald ripening, whereas the droplet size distribution of the second stage became too broad compared to the expected theoretical distribution (as predicted by the Lifshitz-Slezov-Wagner theory) to be due to Ostwald ripening. Katsumoto et al.69 made this assessment after plotting the cube of the z-average radius, rz, as a function of time, and obtaining a linear relations hip. In addition, thei r group found that the volume of bound water on miniemulsion droplets plays an important ro le in obtaining a homogeneous miniemulsion.70 The storage temperature of th e miniemulsion solution and the molecular weight of the surfactant were determined to significantly affect the systems stability: as storage temperature is decreased, the rate of coalescence increases, and as the molecular

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35 weight of the surfactant is increased, the rate of coalescence decreases. The former finding is due to an increase in surface tensi on because non-ionic surfactants become more water-soluble as temperature is decreased. The latter finding is due to steric effects that become predominant as the surfactant size is increased. Nano-emulsions may be formed by a few diff erent experimental methods: condensation,71 low-energy emulsification methods involving phase inversion,67, 72 or by high energy input during emulsification.73 Forgiarini et al.67 reinforced the concept that was proposed by ElAasser68 concerning the importance of the mixing protocol in the formation of nano-emulsions. They found that they only obtained dispersions of nanometer droplet size when the nanoemulsion was formed via stepwise addition of water to a solution of the surfactant in oil. If other methods of addition were used, th e droplet size ra nged from 6 10 m. Izquierdo et al. 72 analyzed the formation and stabili ty of nano-emulsions that were prepared by the phase inversion te mperature (PIT) method, in which emulsions were formed at a temperature near the PIT and then rapidly cooled to room temperature (25 C) by immersion in an ice bath. They proposed that a change in the interfaci al curvature is crit ical to nano-emulsion formation, and that the presence of lamella r liquid crystals was probably a necessary, but insufficient requirement for preparation of nano-emulsions.67 They concluded that the key factor for nano-emulsion formation should be credited to the kinetics of the emulsification process. Nano-emulsions have possible applica tions as drug delivery vehicles,73, 74 in drug targeting, as reaction media for polymerizati on, in personal care and cosmetics, and in agrochemicals. It is also plau sible that nanoemulsions may be used in most industries where extraction will ultimately be required. This idea is based on the premise that the nanoemulsions

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36 may be designed so that their st ability characteristics will coincide with the requirements of the application. 1.2 Non-Toxic Biomedical and Pharmaceutical Microemulsions Microemulsions that are to be used for biom edical or pharmaceutical applications must satisfy basic criteria in order to be efficient. Such microemulsions must first and foremost be non-toxic and biodegradable. In r ecent years microemulsions have been considered as drug delivery vehicles. There are three common means of drug delivery in which microemulsions are incorporated; 1) topical/transderm al delivery, 2) oral delivery, and 3) intravenous delivery. Microemulsions that will be utilized for topical or transdermal applications must have minimal skin irritation and maximum ability for contro lled drug flux across the skin. In oral drug delivery, the microemulsions must not be harmful to the gastrointestinal tract and there should be adequate drug adsorption. In intravenous drug delivery, the microemulsions should be stealthy (i.e. able to escape detecti on as a foreign body by the bodys defense systems) and bloodcompatible. 1.2.1 Applications There are many widespread applications for pharmaceutical microemulsions. As mentioned above, drug delivery via microemulsions is an application that is constantly gaining more and more attention among researchers.75-77 Microemulsions may also be used in reactivity control to manipulate the rates, paths, and stereochemistry of chemical reactions. Other pharmaceutical and biological uses of microemulsions include their use as models for biological membranes, as vehicles for separation and purific ation of proteins and other biopolymers, and in enzymology. One new area of interest for biologi cal microemulsions is their use for the reversal of drug toxicity in the blood of overdose victims. Each of these applications of pharmaceutical microemulsions is discussed in this review.

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37 1.2.2 Introduction to Pharmaceutical Microemulsions The two main processes that govern the releas e of drugs from compartmentalized systems are transfer of drug from the dispersed phase to the continuou s phase, and diffusion of the drug through a membrane or interface from the continuous phase to the sink solution.78, 79 To properly select a suitable microemulsion system for drug solubilization and delivery, it is necessary to have general understanding of the behavior of th e drug, both in delivery system as well as in vivo. Other parameters such as aqueous and oil solubility of drug, parti tion coefficient, droplet size, membrane permeability data acro ss body tissues encountered during delivery,79 and rate of diffusion in both phases are particularly importa nt. Detailed understanding of the components like surfactant, co-surfactant (if required), a nd oil making up the microemulsion for desired performance characteristics is required. As previously mentioned, the major advantag es of microemulsion drug delivery systems include: Ease of preparation Stability Ability to solubilize different kind of drugs ( oi l soluble, water soluble, interface soluble) Ability to be filtered Ability to encapsulate drugs of different HLB in the same system because the microemulsion can solubilize oil-soluble, water-soluble, and interface-soluble drugs Low viscosity Because of these unique physical properties, micr oemulsions have attracted a great deal of interest in recent years as drug delivery vehicles.78 Specific advantages exist for different type s of microemulsion systems. For example, water-in-oil (w/o) microemulsions offers protecti on of water soluble drug s and sustained release

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38 of water soluble material. Similarly, oil-in-w ater (o/w) microemulsions present increased solubility, sustained release, and bioavailabil ity of oil soluble material. Middle-phase (or bicontinuous) microemulsion syst ems provide a concentrated form ulation of both oil-soluble and water-soluble drugs. When tailoring a specific microemulsion system for drug delivery, the following requirements must be followed fo r pharmaceutical microemulsion formulations: 1. All components of microemulsion system must be biocompatible 2. Sufficient drug solubility in microemulsion system 3. Microemulsion system should have long shelf life 4. Rate of release and bioavailability of drug must meet the need of the patient Few studies have been done on the rate of release of drugs from well-defined micellar structures. 1.2.2.1 Biocompatibility As previously mentioned, the major disadvant age of using microemuls ion formulations as vehicles for drug delivery systems are the higher surfactant concentration (>5% usually) and the possible side effects resulting from the surfactants and co-surfactants required to create these formulations. Most of the microemulsion formulati ons used for technological applications in the oil recovery, nanoparticle and other industries ar e composed of surfactants, co-surfactants, and oil incompatible with human physiology. Short ch ain alcohols other than ethanol are also not biocompatible. The surfactants used for pharmaceutical mi croemulsions should be nonirritating. Phospholipids, particularly lecithin, offer a possi ble nontoxic emulsifier for parenteral use. The cosurfactant should be carefully chosen as well. Most existing short and medium chain alcohols currently used as cosurfactants are useless for pharmaceutical microemulsions because of their toxicity and irritancy. Furthermore, evaporati on of these alcohols can destabilize the system, resulting in decrease of formul ation shelf life. Therefore, th e development of biocompatible

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39 surfactants and cosurfactants for use in pharmaceutical microemulsions is becoming a very important technology to challenge. 1.2.2.2 Solubility and stability A microemulsion is not an inert vehicle. A dding new components such as drugs to system may affect its phase behavior. For instance, solubilized drug molecules may affect the spontaneous curvature of the surf actant in different ways, either by being incorporated into the surfactant film or by changing the polarities of the polar and/or apolar phas es. However, it is important to note that in some oil-in-water microemulsions investigated as vehicles for nanoparticles growth, the solubi lization of certain salts was found to actually increase, not decrease, the stability of the microemulsion. Hence, the effect of drug solubilization on microemulsion stability may or may not be a disadvantage in a particular microemulsion formulation. Drug stability is also an important point. The e ffect, whether beneficial or detrimental, that the microemulsion will have on the drug must be determined. 1.2.2.3 Bioavailability Drug bioavailability is anot her factor that must be considered when choosing a microemulsion. Bioavailability is the fraction or percentage of a dose that reaches the systemic circulation intact when not directly injected in to the circulation. By maximizing bioavailability, one maximizes the dose concentration or blood le vel of the drug. Also, if the drugs being administered are expensive to produce, maximizing bioavailability increases cost effectiveness. 1.2.3 Method of Drug Delivery All three types of microemulsions, oil-in-water, middle-phase, water-in-oil, can be used as drug delivery vehicles. Each type of microemu lsion system can dissolve water-soluble, interfacesoluble, and oil-soluble drugs or related com pounds. The extent to which each type of compound

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40 is solubilized, however, depends on the microemulsions structure and composition. Jayakrishnan et al.80 studied the solubilizati on by w/o microemulsions and found that the amount of drug incorporated into the microemulsion depe nds on the concentration of both the surfactants (Brij35/Arlacel 186) and the cosu rfactants (short chai n alcohols). The limit of solubilization depends on the partition coefficient of the drug in oil and water, and on the relative volume of oil and water in the system. The water-external(o/w ) microemulsion presumably can be diluted by the aqueous phase of the stomach and intestine. The oil external (w/o) microemulsion will probably go through an inversion or destabilization upon dilution w ith stomach and intestinal aqueous phase. Depending on the structure of the su rfactant molecules in the microemulsion, the surfactant may enhance the penetration rate or ra te of transport of drug through the intestinal wall. It has been reported that the rate of penetration of drug is much faster from microemulsion formulations than from other drug delivery vehicles. Pharmaceutical microemulsions can be deliv ered by three routes: parenterally (by injection), orally, and topically (to the skin and eyes). The met hod by which the drug is delivered dictates the constraints that will be encountered in formul ation with regards to solubility, bioavailability, toxicity, a nd site targetability of th e microemulsion/drug system. 1.2.3.1 Parenteral delivery Microemulsions have advantage over conventio nal parenteral (inject able) drug delivery systems for a wide variety of reasons. Pare nteral administration (especially via the intraveneneous route) of drugs with limited solu bility is a major problem in industry because of the extremely low amount of drug actually deli vered to a target site. Recently, a number of pharmaceutically acceptable microemulsions for pa renteral drug delivery have been done. Microemulsion formulations have distinct advantages over macroemulsion systems when delivered parenterally, because fine particle micr oemulsions are cleared more slowly than coarse

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41 particle emulsions and, therefor e, have a longer residence time in the body. Both o/w and w/o microemulsions can be used for parenteral de livery. The type of mi croemulsion employed is usually determined by the intended route of delivery as well as the role that the microemulsion will play. Although the literature contains details of many microe mulsion systems, few of these can be used for parenteral drug de livery because of toxicity of the surfactant, cosurfactant, and/or oil phases. As already discussed, most short-ch ain alcohols other than ethanol are not acceptable for parenteral use. 1.2.3.2 Oral delivery Oral drug delivery is generally preferable to parenteral drug formulations. They are less frightening to children as well as easier to admi nister to them as well as people with difficulty swallowing solid dosage forms. Microemulsion formulations offer several benefits over conventional oral formulations for oral administ ration, including increased absorption, improved clinical potency, and decrease dr ug toxicity. Therefore, microemuls ions have been reported to be ideal for oral delivery of drugs such as st eroids, hormones, diuretics, and antibiotics. Pharmaceutical drugs of peptide and protein origins are highly potent and specific in their physiological functions. However, most are difficult to admini ster orally. With an oral bioavailabilty of most peptides in conventiona l (i.e., non-microemulsion based) formulations of less than 10%, they are usually not therapeutically active by oral administra tion. Because of their low oral bioavailability, most protein drugs are only availabl e as parenteral formulations. However, peptides drugs have an extremely short biological shelf life when administered parenterally. Because of this, multiple injec tions are generally required for parenteral administration of peptide drugs. The peptide drug by far most frequently studie d in relation to oral bioavailability is cyclosporine, an immunosuppressa nt drug commonly delivered oral ly to transplant patients to

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42 help prevent organ rejection. A microemulsion-based formulation of this drug, Neoral, has been introduced to replace Sandimmune a crude oil-in-water emulsi on cyclosporine formulation. Neoral is formulated with a fi ner dispersion, giving it a more rapid and predictable absorption profile and less inter-and intrapatient variab ility. It was found that no penalty was paid by converting stable renal transplant recipient from Sandimmune to Neor al. In fact, doing so resulted in effective, safe, increased drug exposur e, reduced intrapatient variability, potential for reduce chronic rejection and thus greater long-term graft surviv al. Therefore, microemulsion formulations have much potential in increasing the viability of oral delivery of cyclosporin, and further research can lead to sim ilar results for other peptide drugs. 1.2.3.3 Topical delivery Topical delivery of drugs can have advantages over other methods for several reasons, one of which is the avoidance of hepatic first-pa ss metabolism of the drug and related toxicity effects. Another is the direct delivery and targetabili ty of the drug to affected areas of the skin or eyes. Recently, there have been a number of studies in the area of drug penetration into the skin. For example, Gasco et al. studied the transport of azelaic acid, a bioactive substance used for treating a number of skin disorders, from a mi croemulsion system to abnormal skin. Several groups have been investigating the use of microemulsion formula tions as ocular drug carriers. The results of these studies seem promising. It was found that in vitro corneal penetration of indomethacin using microemulsions, for example, was more than 3-fold that of currently available eye drops. Microemulsions were also found to prolong the time of drug release, increasing the duration of time the drug spends in the body. 1.2.4 Pharmaceutical Microemulsions Using Nonionic Surfactants Interest in nonionic surfactants for phar maceutical microemulsion formulations is increasing because of their low irritation and high chemical stability. Traditionally, nonionic

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43 surfactants of the polyoxyethyl ene class have been used in the formulation of these microemulsions.81 Ho et al.82 formed microemulsions using pol yglycerol fatty acid esters as nonionic surfactants and short-chai n alcohols as cosurfactants. From tests such as stability and viscosity measurements, they concluded that an oral insulin de livery system employing microemulsions as carriers is applicable. Jayakrishnan et al.80 reported the solubilization of hydrocortisone by w/o microemulsions containing a mixture of th e non-ionic surfactants brij 35 (Polyoxyethylene 23 lauryl ethe r) and Arlacel 186 (glycero l monooleate-propylene glycol), isopropanol as cosurfactant water and n-alkane. The influence of oil soluble su rfactant (Arlacel 186) concentration, the oil chai n length, and the alcohol concentration on the amount of water solubilized in the w/o micr oemulsion were studied. The use of non-ionic surfactant, such as n-alkyl polyoxyethylene ether (CmEn) (where m is hydrocarbon chain length and n is the num ber of oxyethylene units ), to stabilize a microemulsion is particularly attr active because it is generally po ssible to create a microemulsion without the use of a cosurfactant. This ha s important advantages from a pharmaceutical viewpoint. First, as already discussed, most cosurfactants are not pha rmaceutically acceptable. Second, it is not always possibl e to dilute a microemulsion containing cosurfactant, whereas microemulsions stabilized only by surfact ant appear to be infinely dilutable.83 Third, nonionic surfactants are generally recognized as the least toxic of the surfactants and ar e currently used in a variety of pharmaceutical products. 1.2.5 Pharmaceutical Microemulsions Using Anionic Surfactants Many pharmaceutical microemulsions have been developed recently using anionic surfactants with or w ithout nonionic surfactants. For example, addition of non-ionic surfactants sorbitan monolaurate (Span 20, Arlacel 20) to the anionic surfactant Aerosol OT (AOT) increases the phase boundary of w/o microemulsions of AOT.84 With hexadecane, the oil phase

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44 the maximum solubilization occurs for a 1:1 weight ratio of sorbitan monolaurate to AOT. This microemulsion formulation is suitable for topi cal drug delivery applic ation because irritancy caused by the presence of various medium chain length alcohols cosurfactants is absent. Osborne et al.85 have shown that sorbitan monolaurate acts as a cosurfactant for the AOT microemulsion system and significantly affects its phase diagram. They have also observed that the mixing time for the waxy AOT solid with viscous sorbitan monolaurate is quite long, but this can be overcome by ethanolic AOT solution, as ve ry low concentrations of ethanol are accepted pharmaceutically because of ethanol s low irritancy and toxicity compared to other alcohols. Addition of an oil (isopropyl myristate) to the w/o microemulsion system of AOT/water/medium chain alcohol can result in th e formation of o/w micr oemulsions. Trotta et al.86 produced o/w microemulsion with butanol as cosurfactant, but this formulation has limited applicability because of toxicity of butanol. Garcia-Celema et al.87 investigated the use of microemulsion composed of Tween 80 (polyoxuet hylene-20 sorbitan monool eate) surfactant and isopropyl myristate and isopropyl palmitate fo r topical application of antifungal drugs clotrimazole, ciclpirox olamine, and econazole ni trate. They reported th at antifungal drugs with solubilities ranging from slightly soluble to practically inso luble in both water and oil can be successfully dissolved in a pha rmaceutically acceptable ternar y microemulsion system. Berthod et al. attempted to use an o/w microemulsion sy stem as a mobile phase for rapid screening of illegal drugs in sports using reverse-phase HP LC. The system heptane/water/sodium dodecyl sulphate (SDS)/n-pentanol was used to qua ntify 11 drugs in a reversed-phase liquid chromatography column. Now since microemulsion systems have the ability to assay water and oil soluble drugs at once, they have signi ficant potential as drug delivery vehicles.

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45 1.2.6 Pharmaceutical Microemulsions Us ing Phospholipids and Cholesterol Phospholipids, particularly lecithin ar e very good surfactant for microemulsion formulation because of their very low toxicity.88 But, problem in using lecithin as surfactant for microemulsion is that lecithin is slightly too lipophilic to sponta neously form zero mean curvature lipid layer needed for balanced (middle-phase) microemulsion. Also, it is necessary both to adjust the HLB of the lecithin, as well as to destabilize the lamellar liquid crystalline phase that have a strong tendency to form in th ese systems. HLB adjustment is usually done by adding short chain alcohols that makes the polar solvent less hydrophilic. Aboofazeli88 have reported the phase properties of lecithin/pro panol/water/n-hexadecane systems. Around 2-3% lecithin is the minimum amount required to fo rm a microemulsion at all mixing ratios. The propanol concentration should be in the range 10-15 wt% of th e aqueous solvent, with the concentration decreasing slightly with increase in oil content. They have also reported the phase properties of water/lecithin/a lcohol/isopropyl myristate system where alcohols were n-propanol, isopropanol, n-butanol, s-butanol, t-butanol and n-pentanol. Major factor influencing the phase behavior of lecithin based microemulsions is the nature and mixing ra tio of the cosurfactant used. It is possible to produce a large clear isotropic region ex tending over a large range of oil and water composition, called balanced microemu lsion, by using very high amounts of ethanol (60-80% w/w aqueous phase) as cosurfactant in a lecithin-based system. Other workers have used both o/w and w/o lecithin microemulsions as vehicles to deliver a range of drugs.86, 89-91 Considerable interest has b een shown in the use of micr oemulsion drug delivery systems composed of phospholipids for parenteral delive ry of lipophilic drugs. Microemulsion systems containing phospholipids are rapidly captured after intr aveneous injection by the reticuloendothelial system(RES) organs such as liver and the spleen, a nd by inflammatory cells.

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46 This can be advantageous in treating diseases localized to the RES182 or to macrophages. But, accumulation of drugs in the liver and spleen co uld be hazardous in case of other drugs. Microemulsions also provide unique advantages as opthalmological carrier system, as no impairement of visibility is encountered. Su rfactant-containing multicomponent systems for ocular application have been developed and characterized. Kriwet et al.92 studied the relationship between the colloidal structures of a topical formulation and the drug release in vitro as well as the influence of the microstructure on the st ratum corneum drug permeability. They found that phospholipids are able to interact with the structures of the stra tum corneum if they are applied as a microemulsion, as opposed to liposomal form ulations and other investigated systems. Gasco et al.93 compared the effectof topical administrati on of timolol via microemulsion with aqueous solutions. Microemulsion based delivery with lecithin resulted in better concentration-time profile (3.5 times) than the aqueous timolol solutions. It is quite evident that phospholipids and cholesterol based microemulsions have great potential as drug delivery systems, primarily because of their biocompatibility. Such systems can become significant players in the future of targeted drug delivery applications. 1.2.7 Pharmaceutical Microemulsions Using Sugar based Surfactants Sugar based surfactants microemulsion formulat ion have potential applications because of their low toxicity, biocompatibil ity, and excellent biodegradabili ty. They offer an attractive alternative to more convenient ethylen e oxide (EO)-based nonionic polymers.94 Sugar based surfactants are prepared from natural and renewable resources that allow scientists to change structural combinations which ha ve modulating surfactan t properties. Also, the phase behavior of this class of non-ionic surfactants is much less influenced by temp erature than the phase behavior of typical EO-based nonionic surfactants.95, 96 Bolzinger et al.97, 98 investigated the relationship between microetructure and efficacy of a sucros e ester based microemulsion as a drug delivery

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47 system. Sucrose esters are biodegradable surf actants whose hydrophilic and lipophilic properties can be adjusted by varying fatty acid chain lengths. 1.2.8 Pharmaceutical Microemulsions for Drug Detoxification Drug overdose is a major health care problem all over the world. A number of widely used drugs can cause life-threatening t oxicities and are without antidot es. Microemulsion systems that can sequester drugs may offer a solution to this problem. Intravenous injections of such systems can potentially treat overdoses by adsorbing/absorbing drug molecules, thereby, reducing the free drug concentration in blood and tissues. Varshney et al.99 have studied various pluronic based o/w microemulsion system for reducing the free concentration of the local anesthetic bupivacaine in normal saline. They observe that both the molecula r nature and concentration of the constituents in the microemulsion significantly affect extraction efficiency of microemulsions. Extraction was markedly increased by addition of fatty acid sodi um salts due to greater oil/water interface area, increased coulombic interaction between bupivacaine and fatty acids sodium salt, and greater surface activity. Pluronic F127 based microemuls ions extracted bupivacaine more efficiently than microemulsions synthesized using other Pl uronic surfactants (L44, L62, L64, F77, F87, F88, P104). 1.3 Retardation of Water Evaporation Water, is one of the main n ecessity of life. We all know the whole process of water cycle in which it evaporates from sea/river etc., and then condense in upper atmosphere to form clouds and finally rains. There are several instances wh ere we want to reduce th e evaporation of water such as in ophthalmology wherein people suffering from dry eye syndrome show very high water loss from eyes, nuclear industry wherein radioact ive water from spent fu el may escape into the atmosphere, from fruits and vegetables to increas e their shelf life, water conservation in certain

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48 arid and dry parts of world where water is scarce etc. To emphasize the severity of the problem, around 180 million people worldwide suffers from dry eye including 10 million Americans, and nearly 50% of the world populati on will be living in areas, having water shortage by 2050. So, there is a great need to develop methods to reduce the water loss by evaporation. The idea of spreading a layer of a hydrophobic (oily) substance on top of water is an old one, dating back to Benjamin Franklins experime nts of damping of water waves in 18th century. Based on the same concept, in early 20th century, Hedestrand1 and Rideal2 tried to demonstrate the effect of monolayers on the evaporation rate of water on which they were spread. Early experiment on water evaporati on by various researchers demons trated that a monolayer can decrease the evaporation rate of water. In 1943, Irving Langmuir and Vincent Schaefer100 substantiated Eric Rideals2 pioneer finding that monolayers of long chain alcohols were effective suppressants of the evaporation of water. They measured evaporation rates of water by spreading monolayer, over the surface of water in a film balance, with a flat c ontainer with a permeable bottom supporting a solid desiccant CaCl2. Australian researchers, in particular William Mansfield101 and R.G. Vines, after preliminar y laboratory experimentation, then developed successful methods for applying such monolayers on the surf ace of the large water reservoirs to cont rol evaporation. In 1952, La Mer and his co-workers102-106 continued Langmuirs work in an extensive series of laboratory investiga tions in which the properties to be sought in a monolayer for evaporation control were investigated in detail. The primary aim of his research was to gain knowledge of the molecular mechanism involved in the retardation of evaporation, and understanding of the permeability of films, by st udying the influence of molecular architecture. This was accomplished through study of rate of ev aporation, in terms of the state of compression

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49 of the film, and the temperature dependence, by means of Arrhenius equation. He used the method of Langmuir and Schaefer100 for measuring the evaporation of water through monolayers. This method for measuring the rate of evaporation is satisfactory when properly modified, for investigating the influence of the molecular architecture of the molecules composing the monolayer. It give s a quantitative measure of the resistance to evaporation, uncomplicated by motion of air above the m onolayer or by any other disturbances. Benzene and Petroleum ether (Hexane) have b een used for many years as volatile solvent for the less soluble long chain molecules. These solvents facilitate the rate of spreading. However, it has been shown by Archer and La Mer105, 106 that Langmuirs method of spreading the film, by using benzene as spreading solven t and then compressing to the desired surface pressure, yields results that are difficult to repr oduce. This is because the rate of spreading of various acids, alcohols is st rongly dependent upon the chai n length of the molecule. For example, C22OH spreads 2400 times lower than C14OH. In the case of long chain solutes, it is the solvent molecules of the solution, which supply the driving force for spreading.107 They carry the nonspreading solute molecules along leaving, on evaporation of the solvent, a monolayer of supposedly pure solute molecules. In 1970s it has been demonstrat ed conclusively that a comp ressed, molecularly oriented monolayer will be effective in reducing evapor ation of water. Different properties like, evaporation resistance, temperatur e effects, heat transfer thro ugh monolayers have been studied for various monolayers.108-110 Also, in our laboratory we have shown the importance of molecular packing111, 112 achieved by using mixed surfactant monolayers a nd pKa of fatty acids113 in reduction of evaporation of water.

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50 1.3.1 Duplex Films Over the years various researchers have wo rked on water evaporation by monolayers, but till now, maximum reduction in eva poration using monomolecular film of fatty acids or alcohols was around 50%. Moreover, the problem with monolay ers is that they can be easily removed by winds and undergo thermal as well as biological degradation.114 Under such circumstances multimolecular (duplex) films of oil and surfactants would be ideal. Figure 1-7 shows the difference between monolayer and duplex film. Heymann and co-workers115, 116 in 1940s had used multimolecular films of oil as a means of preventing or reducing the evaporation of wate r from exposed water surfaces in arid climates. Using such multimolecular films of paraffin oil and neutral oil from vertical retort tar, they showed a significant reduction in evaporation of water. They found that films of paraffin oil of 1 to 2 m in thickness to which suitable spreading agents have been added would reduce the evaporation by 50 to 60%. Reductions up to 85% were obtained with 1 m films of certain high boiling fractions of neutral oil of vert ical retort tar. Powell and co-workers117 have calculated rate of evaporation of water through su rface films of long chain oils. However, other than that not much literature is found in this ar ea to the best of our knowledge. 1.3.2 Spreading of Oil on Water Surface A quantity of oil placed upon a water surface will spread out by surface-tension forces if the spreading coefficient (S) is positive. This is the net surface tension available to drive the spreading.3 0 fwS (1.7) Where 0 fis the total tension of the oil film at the spreading origin. In the past, 0 f has always been assumed to equal owo the sum of the oil-air and oil-water tensions. If the

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51 deposited volume is small enough, gravity will not be an important spreading force, and the dominant resistance to spreading will be the viscous drag exerted on the oil film by the underlying water.118, 119 Many oils, including the heavier hydrocarbons, ha ve negative spreadi ng coefficients and will not spread on water.120 They will spread, however, if they contain a sufficient concentration, C of surfactant, which reduces to the extent that S becomes positive. So, to form a duplex film of surfactant in oil S should be positiv e, as shown in below (Figure 1-8).

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52 Figure 1-1. Various types of colloidal drug delivery systems. A) Normal Micelles. B) O/W Emulsion. C) O/W Microemulsion. D) Reve rse Micelles. E) W/O Microemulsion. F) W/O Microemulsion. G) Liposom es. H) Soft Nanoparticles. Figure 1-2. Schematic diagram of a surfact ant molecule, micelle, and reverse micelle. Hydrophobic head Hydrophilic head Micelle Reverse micelle Surfactant molecule ABC DEF GH ABC DEF GH

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53 Figure 1-3. Properties of surfactant solutions sh owing abrupt change at the solution critical micelle concentration (cmc). Figure 1-4. Schematic diagram of the adsorption of surfactant monomers from the bulk to the oil/water interface during emulsion formation.

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54 Figure 1-5. Schematic diagram of an oil-in-water (O/W) microemulsion. Figure 1-6. Thermodynamic explanation for behavi or of macroemulsions and microemulsions. R* Droplet radius G of dispersionMacroemulsions (IFT = 1 mN/m) Microemulsions (R* = 10-100 nm, IFT 10-3 mN/m) + 0 Oil WaterOil-in-Water Microemulsion Surfactant Co-Surfactant Oil Aqueous medium Oil Water Oil Oil Oil WaterOil-in-Water Microemulsion Surfactant Co-Surfactant Oil Aqueous medium Surfactant Co-Surfactant Oil Aqueous medium

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55 Figure 1-7. Difference between monolayer and duplex film. Figure 1-8. Problems in forming uniform duplex film. Water Duplex Film of Surfactant and Oil High reduction in evaporation of water Monolayer of Surfactant 25 Low reduction in evaporation of water Oil + Surfactant Water 0.110 Microns Formation of lenses of oil + surfactant molecules due to low energy CH3 terminal groups of monolayer Ordinary Surfactant Brij-93 ( Polyoxyethylene (2) Oleyl Ether) + Hexadecane Water Oil + Brij-93 Oil + Surfactant Water

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56 Table 1-1. Physical characteristics of various drug delivery systems Delivery System Advantages Disadvantages Micelles Low Viscosity Small droplet size Easy Preparation Long shelf-life Low solubilization Potential toxicity of surfactant Microemulsions High solubility of drug Small droplet size Easy preparation Long shelf-life Large amount of surfactant Drug solubility influenced by environmental conditions Potential toxicity of surfactant Emulsions Small amount of surfactant High solubility of drug into carrier High viscosity Instability Short shelf life Large droplets Vesicles and liposomes Made from lecithin and cholesterol Also present in the body High viscosity Difficult to prepare Often disintegrate once administered Nanoparticles Long storage life In vaccinations Slow degradation in body Limited solubility of drug Difficult to prepare Difficult to control size Polymers which represent constituents are usually not bioacceptable

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57 Table 1-2. Types of breakdown pr ocesses occurring in emulsions Breakdown Type Description No change in droplet size (or size distribution) Buildup of an equilibrium dropl et concentration gradient within the emulsion. This phenomenon results from external force fields, usually gravitation, centrifugal, or electrostatic, acting on the system. "Creaming" is a special case in which the droplets collect in a concen trated layer at the top of an emulsion. No change in basic droplet size, but with buildup of aggregates of droplets in the emulsion This process is called "flocculation" and results from the existence of attractive forces between the droplets. Flocculated droplets in an aggregate coalesce to form larger droplets This process also occurs when creaming or sedimentation results in a close-packed array of droplets and these droplets coalesce. The limiting state is the complete separation of the emulsion into two immiscible bulk liquids. Average droplet size increases due to the two liquids forming the emulsion being not totally immiscible This process does not involve ac tual coalescence of droplets, but rather the transfer of di spersed phase across continuous phase after solubization o ccurs. If the emulsion is polydisperse, larger droplets will form at the expense of smaller droplets due to the difference in chemical potential for different size droplets (Ost wald ripening). In principle, the system will tend to an equilibrium state in which all the droplets have combined and are one large droplet, or a separated phase. Emulsion type inverts from W/O to O/W type This is a questionable "breakdow n" process since essentially another emulsion is formed. Th e inversion process can be brought about by numerous parameter changes, which will be discussed in detail later in this section.

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58 Table 1-3. Factors influenci ng the stability of emulsions Factor Description of Effect Physical nature of the interfacial film For mechanical stability, a surf actant film with strong lateral intermolecular forces and high film el asticity is desired. A mixture of two or more surfactants is prefe rred over a simple surfactant (i.e., lauryl alcohol + sodium lauryl sulfate). Electrical barrier Significant only in O/W type emulsions, because of conductivity in continuous phase. In the case of noni onic emulsifying agents, charge may arise due to adsorption of ions from the aqueous phase. The repulsion or attraction ca n be influenced by ch anging the thickness of the double layer, which is described below. Viscosity of the continuous phase or of emulsions A higher viscosity reduces the diffu sion coefficient of the dispersed droplets, resulting in reduced frequency of collision and lesser coalescence. Viscosity can be increa sed by adding natural or synthetic thickening agents. Viscosity also in creases as the number of droplets increases; so many emulsions are mo re stable in concentrated form than when diluted. Size distribution of dispersed droplets Uniform size distribution is more stable than an emulsion with the same average droplet size but ha ving a wider size distribution. Phase volume ratio As volume of dispersed phase increases, stability decreases Phase inversion can occur if dispersed phase volume is increased enough. Temperature Usually, as temperature in creases, emulsion stability decreases because of increased frequency of collision. Steric barrier Addition of polymer that ad sorbs at interface can influence stability. Polymer chains can prevent coalescence due to bulkiness, but they can also enhance flocculation and decrease stability.

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59 Table 1-4. Parameters that affect phase inve rsion in emulsion and the effect they have Parameter Effect on phase inversion Order of phase addition W O + emulsifier W/O O W + emulsifier O/W Nature of emulsifier Bancroft's Rule Making the emulsifier more oil soluble tends to produce a W/O emulsion and viceversa Phase volume ratio Oil/Water Ratio increased in an O/W emulsion W/O emulsion and vice-versa, as described above in the text Phase in which emulsifying agent is dissolved If surfactant can be dissolved at least partially in either water or oil Bancroft's Rule If surfac tant is dissolved in water O/W emulsion Temperature Depends on the surfactant and its temperature dependence. If emulsion is O/W type with po lyoxyethylenated nonionic surfactant, phase inverts to W/O with increase in temperature due to increased hydrophobicity of the surfactant. Addition of electrolytes Strong elect rolytes (polyvalent Ca) added to O/W (stabilized by ionic surfactant) inversion to a W/O type Because of decrease in dou ble layer thickness around oil droplets, droplets coalesce and become the continuous phase. Table 1-5. A summ ary of HLB ranges and their application HLB Range Application 3 to 6 W/O emulsifier 7 to 9 Wetting agent 8 to 18 O/W emulsifier 13 to 15 Detergent 15 to 18 Solubilizer

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60 Table 1-6. Microemulsi ons vs. Nano-emulsions Characteristic Microemu lsions Nano-emulsions Stability Thermodynamically stable Thermodynamically unstable Droplet size 10 100 nm 20 500 nm Surfactant concentration Usually require 10 30 wt% surfactant Can be formed with 4 8 wt % surfactant Formation Independent of mixing protocol Dependent of mixing protocol

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61 CHAPTER 2 PREPARATION OF PROPOFOL MICROEMULSIONS 2.1 Introduction Much interest had been recentl y generates in the pharmaceutical and biomedical fields for the development of microemulsions as a drug de livery system. Microemulsions are transparent, thermodynamically stable, isotropic, low-viscosity dispersions consisting of micro domains of oil and/or water stabilized by inte rfacial film of surf ace active molecules. Such microemulsions contain droplets with the dimension in the rang e of 10-100 nm and, hence appear transparent like single phase liquid. Microemulsions provide a highly attractive carrier system for the delivery of drugs because of high total interfacial area due to their small droplet size, their thermodynamic stability as well as their spontan eous formation. The usefulness mi croemulsions as a drug carrier system stems from their ability to incorporate hydrophilic or hydrophobic drugs which can be protected from direct contact with the body fluids and tissues and can be delivered slowly over a period of time with the decrease in hyper-sensitivity reactio ns and pain during administration. The active substance used in this study is propofol (2,6-d iisopropylphenol) (Figure 2-1). Propofol is a popular intravenous ge neral anesthetic agen t due to several favorable characteristics of this drug. These properties include an an ti-emetic effect and rapid emergence from unconsciousness with minima l residual drowsiness.121-124 However, a primary drawback of propofol is this drugs extreme lipophilicity that necessitates dispersion in soybean macroemulsions to produce white, opaque form ulations. This solvent requirement potentially causes several adverse drug outcomes including bacterial growth leading to postoperative infection, severe pain in a majo rity of patients with peripheral in travenous injecti on, inclusion of egg products, and others.125, 126 Alternative formulations yielding similar pharmacodynamic characteristics as the conventional formulations, but without the associated liabilities, would be

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62 useful to enhance patient safety and comfort. R ecently, efforts have been made to achieve these goals using other lipid solvents and concentrat ions, cyclodextrin formulations, microemulsions technology, and prodrug techniques that depend on native enzymes such as alkaline phosphatase to metabolize a parent compound (i.e., phos phono-2,6 diisopropylphenol ) to the active drug molecule (i.e., propofol).127-131 To address these drawbacks of conventional pr opofol formulations, we hypothesized that this agent could be associated with biocompatible surfactants to form transparent, colorless, thermodynamically stable, low viscosity, oil-in-wat er microemulsions with droplets having a 10 50 nm diameter. Thus, instead of regarding propofols extreme lipophilicity as a hindrance to be overcome, the lipophilicity of propofol (which exists as an oil at room and physiological temperatures) was leveraged to construct the phy sical core of the microemulsions. Therefore, propofol served dual roles as both the lipid oil core of the microemulsion and the pharmacodynamically active agent to cause anesthesia. In addition, we hypothesized that the spontaneous destabilization of the microemuls ion nanodroplets to release propofol could be selectively altered by modifying the concentration and nature of the associated surfactants with resultant changes in latency to anesthetic induction. That is, we hypothesized that the pharmacodynamic and pharmacokinetic properties of propofol microemulsions could be modified by varying the nature and concentration of surfactants. To examine these hypotheses, here, we first determined the physical limitations of possible propofol mi croemulsions using a variety of propofol and surfactant concentrations of these systems. Second, we measured several parameters of anesthetic induction and emergen ce in animal model receiving the experimental and conventional formulations of propofol usi ng a randomized, crossover design in rats and dogs (Chapter 3).

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63 2.2 Methods and Materials 2.2.1 Materials Propofol was obtained from Albe marle Corporation (Baton Rouge, LA). Purified Pluronic, nonionic coblock polymers consisting of pol yethylene and polypr opylene monomers, was purchased from the BASF Corporation (Florham Park, NJ). Tween, Brij, Mrij, Polyethylene Glycol (PEG), Sodium salts of fatty acid (C8, C10, and C12) were supplied by Sigma Chemical Co. (St. Louis, MO). 2.2.2 Synthesis of Propofol Microemulsions Microemulsions were prepared by combining pr opofol (0-100 mg/ml) with surfactant and co-surfactant in normal saline (0.90 mg/ml NaCl) bulk media. Most of the data in this study correspond to pluronic microemulsions which cons ist of purified pluronic 68 (0-70 mg/ml), and a fatty acid salt (0-12.5 mg/ml) in normal saline (0.90 mg/ml NaCl) bulk media. Water was ultrapurified using a water purifica tion system (Nanopure, Barnst ead/Thermolyne, Dubuque, IA) to provide a minimal electrical resistance of 18.2 M Following agitation with a magnetic stirrer, these components combined to form clear, colorless microemulsions with adjustment of pH to 7.40 using either HCl or NaOH. All experiment al formulations were stored under a nitrogen headspace. To characterize the dimension of the i ndividual droplets, the effective droplet size of the nanoparticles was measured by the dynamic light scattering method using a submicron particle sizer analyzer (90Plus, Brookhaven Instruments Co rporation, Holtsville, NY) as previously described.132 2.2.3 Viscosity Measurements A Paar Physica US-200 rheometer with cone a nd plate geometry was used to measure the viscosity of the microemulsions. Viscos ity measurements were performed at 25 C temperature, and the sample temperature was controlled within 1 C using water as the h eat transfer fluid. In

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64 all experiments, a cone of the radius 4.3cm with cone angle 0.5 degree was used. The shear viscosity ( ) of the microemulsions of different co mpositions was measures as a function of shear rate ( ) at 25 C. 2.2.4 Stability Evaluation of Microemulsions The stability test of propofol microemusions were carried ou t at room temperature over a period of two and half years. Th e changes in the droplet size di stribution, visc osity, pH and percentage of oil separation were determined and th e results were used to evaluate the stability of these systems. 2.2.5 Stability on Dilution of Microemulsions The stability on dilution test of propofol microemusions were car ried out at room temperature by adding 100 l of saline solution to 10 ml micr oemulsion in every five minutes. The endpoint was the loss of transparency or in other words onset of turbidity. 2.2.6 Oxidation Studies Oxidation of propofol in microemulsion as well as macroemulsions were done by LC/MS analyses using a Perkin-Elmer PE 200 LC system and an API 4000 triple quadrupole mass spectrometer equipped with an APCI source (App lied Biosystems, Foster City, CA). Samples (25 l injection volume) were separated on a C18 column (Xterra TM RP18, ODS 5 m 250 mm 4.6 mm i.d., Waters) with a guard cartridge (4 mm 3 mm i.d., Phenomenex, Torrance, CA) prior to the C18 column. 2.3 Results 2.3.1 Microemulsion Synthesis and Characterization Microemulsions of propofol were formulated using varying concentrations of propofol, purified pluronic, and fatty acid salt in a normal saline bulk media in order to construct a phase diagram of these systems (Table 2-1). Depending on the concentrations of surfactants, propofol

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65 concentrations of 12-15 mg/ml or less produ ced microemulsions. In contrast, propofol concentrations exceeding 20 mg/ml with these c oncentrations of surfactants formed opaque, white macroemulsions similar to conventional pr opofol formulations. For purposes of this study, the final concentration of propofol in the microe mulsions was selected to be 1% (10 mg/ml), a concentration equal to that in the conven tional commercially available macroemulsion formulation (Figure 2-2). Subsequently, we more closely investigated th e interaction of the concentration and types of surfactants and co-surfactants necessary to form microemulsions (or macroemulsions) while holding the concentration of propof ol constant at 10 mg/ml (Figur e 2-3, 2.4). At the greatest concentration of purified plur onic 68 (70 mg/ml), no fatty acid co-surfactant was required to create microemulsions of propofol as shown in th e binary diagram (Figure 2-3). However, as the concentration of purified pluroni c 68 decreased, the concentrati on of the fatty acid surfactant necessary to form a microemulsion increased. In addition, the nature of the fatty acid significantly affected its concentration of fatty acid salt necessary to create a microemulsion. Thus, the concentration of fatty acid salt required to formulate propofol as the oil core of a microemulsion was markedly diminished by lengt hening the carbon chain of the co-surfactant fatty acid from 8 to 10 or 12 carbon atoms at any concentration of purified pluronic 68 and 127. Minimal-to-no differences were noted for microe mulsions constructed wi th C10 or C12 with respect to formation of a micr oemulsion or a macroemulsion. The measured particle sizes for various formul ations of the microemulsions varied from 11.9 to 47.7 nm (Figure 2-5). In general, as the concentration of purified pluronic 68 was increased, the diameter of the particles also in creased irrespective of the carbon length of the

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66 fatty acid cosurfactant. No change was noted in the dimension of the microemulsion droplets containing after two and half years of storage at room temperature (Table 2-2). We also investigated the relative stability of these propofol microemulsions to dilution with normal saline. That is, we determined the volume of the saline diluent necessary to break the microemulsion into a macroemulsion (Figure 26). The volume of diluent required to cause these changes increased w ith lengthening of the carbon chain in the fatty acid salt. This increase in the diluent volume was most noticeable for the C12 propofol microemu lsions that required approximately a six-fold dilution to break the microemulsion. Propofol in air oxidize to its dimers and tr imers, which are yellow in color. And thus becomes yellow over a period of time. The exact mechanism of oxidation to its dimers and trimers is still debated by the scientists. But ever yone agrees to the fact that this yellow color development is because of oxidation. To remove the problem of oxidati on, we have done two things. First, we kept all the microemulsion samples under nitrogen so that without the oxygen required for oxidation, the oxidati on time period is increased. Figure 2-7 represents the gas chromatography data of propofol microemulsion sy stem purged with nitrogen. As we see from the data, nitrogen purged from the system does not remove any propofol from the microemulsion system. Second, we added chelating agent EDTA (Ethylenedinitrilotetraacetic acid) to which chelates various ions present in the system and t hus reduces the oxidation process. Figure 2-8 (a) represent the mass spectroscopy of propofol in microemulsions as compared to pure propofol (Figure 2-8(b)). As we see from MS/GC results the concentration of pr opofol dimmer (quinine) is much lower in microemulsion system than macroemulsion formulation.

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67 The viscosity values of the microemulsions did not change with time upon (Table 2-3). But the effect of concentration of fatty acid salt on pH as well as conductivity of the micoemulsion was significant (Figure 2-9, 2-10). As sodium salt concentr ation is increased, the pH, conductivity of the microemulsions also increased due to incr ease in the ioni c surfactant concentration. This data of pH conductivity is before adding HCL and NaOH to change the pH of the microemulsion system to 7.4 and tell us the amount of acid/base re quired to change the pH of the system. 2.4 Discussion Propofol is an organic liquid that exists as oil at room temperature with minimal aqueous solubility. This insolubility is both a fundamental property of propofol and a significant problem for purposes of drug delivery. To address this issu e, propofol was formulated in a castor bean oil macroemulsion in the early 1980s, but this so lvent was subsequently abandoned due to its propensity to cause anaphylaxis.133, 134 Instead, a soy bean oil macroe mulsion was selected as an alternative emulsification agent to deliver propofol.135 Currently, these white, opaque macroemulsions (Diprivan, Baxter PPI Propofol ) contain: propofol (10 mg/ml), soybean oil (100 mg/ml), glycerol (22.5 mg/ml), egg lecith in (12 mg/ml), and a preservative (0.05 mg/ml disodium edetate or 0.25 mg/ml sodium meta bisulfite). The requirement that propofol be dissolved in 10% soybean oil with associated surfactant has caused a number of liabilities including support of bact erial growth, addition of egg products that is objectionable to vegans, the necessity of preservatives, and modul ation of the inflammatory pathways.126, 136 The need for a suitable alternative formulati on is evidenced by past and ongoing research into other types of propofol delivery systems. Several investigators ha ve reported favorable results using formulations composed of medium and long-chain triglycer ide macroemulsions to deliver propofol at varying concentrations of 10-60 mg/ml.128, 137, 138 In addition, propofol has

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68 been complexed with sulfobutylether 7-cyclodextrin molecules to form clear, colorless formulations that are stable at a wide range of temperatures (450C), a clear benefit compared to macroemulsion technology, and that have pharmac okinetic properties simila r to the conventional drug delivery system.129, 139 Finally, pro-drug methods have been exploited to develop phosphono-2,6-diisopropylphenol, an ag ent that is metabolized by na tive alkaline phosphatase to generate the active agent, propofol, and two byproducts, formaldehyde and phosphate.140 In this report, we demonstrate that microemulsion met hods represent another su itable technology to deliver propofol intravenously w ith anesthetic parameters simila r to the commercially available macroemulsions. Microemulsions systems are useful for pare ntal administration of drugs. In this study various microemulsion formulations given in Table 2-1 were employed to investigate the physiochemical stability over a period of two and ha lf years. Various concen trations of surfactant solutions (1% to 20%) in 0.9% saline we re employed to solubilize propofol. Some microemulsion developed yellow color in time du e to oxidation of propofol to its dimers and trimers. But as seen in Figure 2-8 the amount of one of the dimer (quinone) is much lower in microemulsion than in macroemulsion formulation. This suggest us that microemulsion system being smaller dynamic system keep the propofol dr oplets separated from the ions present in the system longer and thus reduc ing the oxidation process. 2.4.1 Microemulsion Technology for Drug Delivery Microemulsions have been previously used to deliver several drugs by the oral and transcutaneous routes. Currently, an oral microemulsion of cyclospor ine is available for prevention and treatment of transp lant rejection of solid organs.141 In addition, a number of other drugs have been formulated in microemulsions for oral (e.g., paclitaxel, heparin) and transdermal (e.g., apomorphine, estrogen) delivery.142-145 Fewer drugs have been delivered using an

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69 intravenous route, but include tr ans-retinoic acid, flurbiprofen.146 Unlike other oil-in-water microemulsions wherein the active drug is dissolv ed in an oil excipient, in the present study propofol acted in two different, complementary ro les in the present investigation. That is, the need for an excipients oil for propofol dispersal wa s obviated by the fact that propofol is itself an oil at room and physiological temperatures. Th erefore, propofol could serve not only as the active pharmacodynamic agent, but also could exist as the physical platform for the preparation of these microemulsions. By doing so, the need fo r additional excipients oils (e.g., soybean or castor bean oil) is eliminated along with the potential for these excipients to nourish bacteria. Although the we hypothesize that propofol microemuls ions will not support bacterial growth to the same extent as macroemulsions, additional in vestigations are needed to test this thesis. The kinetics of general anesthesia with th e propofol microemulsion should be favorable compared to the commercially available propofol macroemulsion. Whereas the time for general anesthesia (defined by the protocol as loss of le g withdrawal to a pinch) should be higher in the case of microemulsion as they have extra surfact ant at the interface, because of which it takes longer time for the drug to come out in blood. This short delay is favorable when compared to pro-drug technologies that rely on metabolism, a phenomenon that may vary significantly within any patient population.147 The differences in induction tim es between experimental groups reported herein may be caused by differential rele ase of propofol from the individual droplets into the blood. That is the different propofol nanoparticles have markedly different stabilities against dilution by blood based on the emulsifier structure and c oncentration selected for the formulation. In general, a microemulsion is th ermodynamically stable at equilibrium. One can destabilize a microemulsion by si gnificantly changing pressure, temperature, or chemical compositions. The last variable can be changed simply by diluting a microemulsion with saline

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70 or blood. It is well recognized that the forma tion of microemulsions requires an ultra low interfacial tension (e.g., ~10-3 mNewton/m) at the oil/water in terface. Upon dilution with saline, the interfacial tension will increase substantiall y as the emulsifier molecules (i.e., both pluronic 68 and fatty acid salt molecules) desorb from the droplet surface. This event will markedly increase the interfacial tension at the dr oplet surface and ultim ately destabilize the microemulsion, with release of the active pharmaceutical core (i.e., propofol). However, each emulsifier film around the microemulsion droplet has inherent molecular packing and, hence, stability. The extent of dilution required to de stabilize a microemulsion represents its inherent stability. Thus, a microemulsion requiring greate r dilution for destabilization indicates that its emulsifier film has a greater stability. Figur e 2-7 suggests that th e C12 fatty acid salt microemulsions are the most stab le as they require the greatest dilution to become a turbid macroemulsion. It is likely; however, that microe mulsion destabilization is affected by more than just dilution in vivo as the surfact ant can go to various other places in vivo dilution than in vitro. Further understanding and selec tively modifying these destab ilization rates by use adapting surfactant type and concen trations alludes to the possibility of controlling release times of active pharmaceuticals from nearly immediate (e.g., propof ol) to longer times (e.g., chemotherapeutic or antifungal agents). Since propofol is water insoluble and causes severe pain on inject ion, it has been formulated as a 1% solution in a fat emulsi on containing 10% soybean oil consisting of long chain triglycerides (LCT). However, about half of patients still experien ce moderate to severe pain on injection.148, 149 Thus, several methods have been devised to prevent this pain, for example injection into a larger vein, use of various drugs (aspirin, fentanyl, alfentanil, metoclopramide, nitroglycerin prilocaine, pethidine),150-154 and co-administration with either

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71 lidocaine or nafamostat mesilate (a kallikrein inhibitor),149, 155 of which the most effective and common are use of a larger vein and mixing with lidocaine. Regarding one of the mechanisms of injecti on pain, recent studies demonstrated generation of the bradykinin caused by propofol and its inhi bitory effect of lidocaine and nafamostat mesilate, so that these two drugs are considered to decrease the pain by reducing the plasma bradykinin levels.149, 156 Bradykinin is produced by contact between the lipid solvent for propofol and the plasma kallikrein-kinin system, and results in modification of the injected vein, such that the propofol in the aqueous phase has easy access to the free nerv e endings of the vessel, causing aggravation of the pain.156 The reason why propofol causes pain on injection was studied by Ohmizo150 using nafamostat mesilate, a kallikrein inhibitor, as follows: the lipid solvent (long chain triglycerides (LCT)) for propofol activates the plasma ka llikrein-kinin system during injection, generating bradykinin th at causes hyperpermeability of the vessel and thus dilates the injected local vein. As a result there is increased contact be tween the aqueous phase propofol and the free nerve endings of the vessel, resu lting in aggravation of propofol-induced pain.156 When bradykinin generation is reduced by the pharmacological effect of nafamostat mesilate, the propofol-induced pain is decrease d. Alternatively, the decrease in pain when using long chain triglycerides/ medium chain trig lycerides (LCT/MCT) propofol is c onsidered to be attributed to the lipid solvent that decreases the propof ol concentration in the aqueous phase.137 Association between the aqueous phase propofol concentration and injection pa in has also been reported in some studies.137, 157 Thus, it is possible that a reduction in either bradykinin generation or the aqueous phase propofol concentrat ion decreases the pain on inject ion with propofol. In addition, it has recently been reported that use of 10% long and medium chain triglycerides (LCT/MCT) mixed at a 1:1 ratio in the carrier emulsion, as an alte rnative to LCT, reduces pain without any

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72 changes in the pharmacokinetics and pharmacodynamics. It was suggested that this can be attributed to a decreased concentr ation of the aqueous phase propofol.127, 158 According to the above reports, reduction of bra dykinin generation or propofol con centration in the aqueous phase may be the main reasons for reduced pain on in jection with propofol em ulsified in LCT/MCT. Although a decrease in the aque ous phase propofol in this em ulsion has been demonstrated, 150 the effect on bradykinin genera tion has not been examined. Apart from the pain, it has been demonstrated that complement (C3a) is activated by LCT.159 Although the clinical implication of this complement activation is unclear, it may be important, because it has been associated with pulmonary edema.159 One benefit of slightly delayed propofol releas e due to these differential stabilities may be reduced pain on injection. That is, we hypothesize that the concentration of aqueous propofol in peripheral veins will be sufficiently low so that minimal-to-no pain will be experienced during induction. If the relaxation rate is sufficiently long to ensure na noparticle integrity from the time of injection in a peripheral vein to the time they enter the central circulation (e.g., 10-15 s), then no pain should be caused during injection. This hypothesis is based on th e observations of Seki and colleagues160 who noted that patients with propofol administered through a central venous catheter do not experience pain on injection whereas those patie nts injected via a peripheral intravenous catheter have a 53-76% incidence of pain. 2.4.2 Selection of Surfactants The nonionic and ionic surfactants used to formulate propofol microemulsions were carefully selected not only fo r their ability to combine w ith the drug to form stable microemulsions, but also because both surfactants have been previously injected intravenously into humans without reported adverse incidents. Justification for choosin g these surfactants is presented subsequently.

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73 2.4.3 Nonionic Surfactants The selection of purified plur onic 68, an ethylene oxide propy lene oxide block copolymer, was justified based on number characteristics re lated to chemical and medical requirements. First, this surfactant a llowed rapid and reproducible preparation of propofol microemulsions that possessed good stability over time. In general, mi croemulsions are thermodynamically stable and hence exhibit infinite shelf life. These formula tions are destabilized upon dilution with blood or plasma or saline beyond its tole rance limit. Second, purified pl uronic 68 has been previously administered intravenously in large doses to hu mans without any apparent adverse effect. For example, this surfactant was infused to patients su ffering chest crisis due to sickle cell disease in an attempt to reduce the duration and severity of the crisis.161 Although not efficacious to treat chest crisis at large doses of (e.g., 88g), no appare nt untoward effects were noted in the subjects. Third, this surfactant is primarily (>95%) excret ed by kidneys and does not stimulate or inhibit human cytochrome P450 systems.162 In fact, others have hailed this coblock surfactant as a model surfactant for pharmacological use.162 2.4.4 Ionic Surfactants Sodium caprylate was chosen as the best ioni c surfactant for the following reasons. First, the surfactant allows reliable synthesis of propofol microemulsi ons with particle diameters of <50 nm range. Second, sodium caprylate is al ready present in many blood products (e.g., human albumin) currently in clinically use to stabilize proteins.163, 164 Third, the smaller length of the carbon backbone reduces the risks of cellular injury associated with fatty acids with longer carbon chains such as sodium oleate that is us ed to cause lung injury as a model of acute respiratory distress syndrome.165 These data suggest that sodium caprylate is the best choice currently available for additional investigations using microemulsions of propofol, although the

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74 information from use of C10 and C12 allowed us to understand how su rfactant selection can affect the pharmacokinetic and pharmacodynami c properties of propofol microemulsions. 2.4.5 Propofol Anesthetic Action Propofol is a 2,6-diisopropylphenol with se dative-hypnotic properties used for general anesthesia or as a sedative during surgeries and intense care.166 Because of its slight solubility in water, the drug is formulated as an emulsion for clinical use. It is highly lipophilic and distributes extensively in the body. Due to the shor t duration of its anesthe tic effects propofol is also utilized for anesthesia maintenance in asso ciation with narcotics, benzodiazepin derivatives and inhalatory agents. Its mechanism of action is uncertain, but it is postulated that its primary effect may be potentiation (enhancement) of the GABA-A receptor, possibly by slowing the channel closing time.167 Gamma-aminobutyric acid (usually abbrevia ted to GABA) is the major inhibitory neurotransmitter found in the in the vertebrate central nervous system (CNS), whose action is produced by its selective interaction with at least two classes of GA BA receptors, namely GABAA and GABAB receptors..167 While GABAB receptors are members of the Gproteinlinked receptor superfamily and are coupled with K+ and Ca2+ channels, GABAA receptors are ligand-gated ion channels coupled to an integral chloride channel.168 Propofol induces central depression by means of a potent agonistic effect on the GABAergic transmission, mediated by binding to the GABAA receptor, and by reducing the metabolic activity in the brain. GABAA receptors are composed of a number of phylogenetically related subunits ( 1-6, 1-4, 1-3, 1-3), that coassemble to form a pentameric structure which contains a central Clchannel.169 Indeed, a number of distinct classes of drugs (benzodiazepines and benzodiazepinelike compounds, beta-carbolines, steroids, barbiturates, alc ohols, picrotoxin,

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75 tertbutylbicyclophosphorothionate (T BPS)) exert their effects by interacting w ith specific modulatory sites on this receptor.168, 169 One of the major outcomes of the recent research in the field of general anesthetics is the observation that a large number of chemically unrelated anesthetics possess the common ability to in fluence the function of GABAA receptors.170 There is evidence that propofol also exerts a presynaptic action, leading to an accumulation of gaminobutyric acid (GABA) in the sy naptic cleft and contributing to its hyperpolarizing action in postsynaptic receptors, which are coupled to chlorine channels.170 Recent research has also suggested the endocannabinoid sy stem may contribute significantly to propofol's anesthetic action and to its unique properties. During induction, propofol decreases the systolic and diastolic blood pressure by approximately 20-30 percent with minimal change in heart rate; apnea is also common. The cardiovascular and respiratory e ffects of propofol, however, shoul d not cause major concern in otherwise healthy patients. By virtue of its pharmacokinetic pr ofile, the drug lends itself to continuous infusion for maintenance of anesthesia. When used as the main anesthetic agent, it produces satisfactory anesth esia with rapid recovery and without major adverse effects in healthy individuals. In continuous infusion propofol can be used as an alternative to inhalation anesthetics.171 Propofol permeates the cell membrane at very hi gh rates and has a very high affinity for tissues so that its apparent volume of dist ribution, depending on the conditions and kind of tissue, can exceed several folds as compared to the aqueous space. Since it interacts intensely with membranes and proteins, the drug also exerts many metabolic effects, including impairment of energy metabolism. Studies of interactions of propofol with tissues, esp. those ones occurring

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76 at the interface between membrane s and proteins are likely to reveal new details about the general mode of action of the drug as well as about the mechanism of its anesthetic action. Propofol is highly protei n bound in vivo and is metabolised by c onjugation in the liv er. Its rate of clearance exceeds hepatic blood flow, suggesting an extrahepatic site of elimination as well. The elimination half-life of propof ol has been estimated to be between 2 hours. Propofol is extensively metabolized by the liver prior to its elimination by the kidney. However, its duration of clinical effect is much shorter because propof ol is rapidly distribute d into peripheral tissues, and its effects therefore wear off considerably within ev en a half hour of inje ction. This, together with its rapid effect (within minutes of injecti on) and the moderate amnesia it induces makes it an ideal drug for IV sedation. 2.5 Conclusions Here, we report the successful preparati on, characterization, and use of propofol microemulsions for anesthesia in rat simila r to that caused by propofol macroemulsions. Formulation of propofol microemulsions was ach ieved although no excipient oil (e.g., soybean or castor bean oil) was used because propofol exists as an oil and can serve as its own core of an oil-in-water microemulsion. Thus, propofol was us ed both to solubilize it self and as an active pharmaceutical. The differential diluent volumes in vitro based on changing the concentration and type of surfactant alludes to the possibility of selectively modifying the pharmacokinetic and pharmacodynamic properties of propofol, a nd potentially other lipophilic drugs.

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77 Figure 2-1. Propofol. Figure 2-2. Schematic diagram of an oil-in-water (O/W) microemulsion.

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78 0 0.2 0.4 0.6 0.8 1 1.2 203040506070 Pluronic F68 (mg/ml)Fatty Acid Concentration (mg/ml) C8 co-surfactant C10 co-surfactant C12 co-surfactant Figure 2-3. Binary diagram noting the concen trations of purified pluronic 68 and the cosurfactant fatty acids necessary to form propofol microemulsions in a bulk media of normal saline. The concentration of propofol was constant at 10 mg/ml. For a given fatty acid cosurfactant, the region above the line is a microemulsion whereas the area below the line represents a macroemulsion formulation.

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79 0 0.1 0.2 0.3 0.4 0.5 0.6 203040506070 Pluronic F127 (mg/ml)Fatty Acid Concentration (mg/ml) C8 co-surfactant C10 co-surfactant C12 co-surfactant Figure 2-4. Binary diagram noting the concen trations of purified pluronic 127 and the cosurfactant fatty acids necessary to form propofol microemulsions in a bulk media of normal saline. The concentration of propofol was constant at 10 mg/ml. For a given fatty acid cosurfactant, the region above the line is a microemulsion whereas the area below the line represents a macroemulsion formulation.

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80 0 10 20 30 40 50 60 203040506070 Pluronic F68 (mg/ml)Particle Size (nm) C8 co-surfactant C10 co-surfactant C12 co-surfactant Figure 2-5. Effects of purified pluronic 68 concentration a nd fatty acid chain length on nanodroplet diameter for propofol microemulsions. In these systems, the concentration of propofol was held consta nt at 1% (10 mg/ml) and the minimum necessary amount of fatty acid salt was a dded to formulate propofol microemulsions.

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81 0 100 200 300 400 500 600 700 800 203040506070 Pluronic F68 (mg/ml)Dilution with Saline Solution (%) C8 co-surfactant C10 co-surfactant C12 co-surfactant Figure 2-6. Effect of diluti on on propofol microemulsions formulated using various concentrations of purified pluronic 68 and se veral fatty acid salts with variable carbon chain length. Shown are the relative volumes of 0.9% NaCl dilu ents that caused the microemulsions to break into macroemulsions (i.e., a transition from a transparent to a turbid state). The concen tration of propofol in the undiluted microemulsions was constant at 1% (10 mg/ml).

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82 Figure 2-7. Gas chromatography of propofol microemulsion. Effect of Purging on Propofol Content of Microemulsion Sample4% F68 + Sodium Caprilate (Microemulsion) Avg. Concentration ( g/mL) 1. No Purging 66.5 2. N2 Purging 65.6

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83 Sample Avg. Concentration of Quinone ( g/mL) 4% F68 + Sodium Caprilate 0 Deprivan (Macroemulsion) 1.5 Figure 2-8. Mass spectroscopy of propofol. From Literature (Pure Propofol) Our Propofol (Yellow)

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84 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 203040506070 Pluronic F68 (mg/ml)pH C8 co-surfactant C10 co-surfactant C12 co-surfactant Figure 2-9. The pH of F68 microemulsion. 5 10 15 20 25 30 203040506070 Pluronic F68 (mg/ml)Conductivity (mS) C8 co-surfactant C10 co-surfactant C12 co-surfactant Figure 2-10. Conductivity of F68 microemulsion.

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85 Figure 2-11. Formation of propofol dimer and propofol dimer quinone in propofol emulsions. Figure 2-12. Dynamic behavior of microemulsi ons. A) Collision with merging/breakdown. B) Fragmentation/coagulation. B A B A

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86 Table 2-1. List of propofol microemulsion S. No. Surfactant Co-surfactant 1 Pluronic 68 Sodium Octanoate, Sodium Decanoate Sodium Dodecanoate 2 Pluronic 77 Sodium Octanoate, Sodium Decanoate Sodium Dodecanoate 3 Pluronic 88 Sodium Octanoate, Sodium Decanoate Sodium Dodecanoate 4 Pluronic 108 Sodium Octanoate, Sodium Decanoate Sodium Dodecanoate 5 Pluronic 127 Sodium Octanoate, Sodium Decanoate Sodium Dodecanoate 6 Tween 20 Polyethylene Glycol 20 0, Polyethylene Glycol 400, Polyethylene Glycol 600 7 Tween 40 Polyethylene Glycol 20 0, Polyethylene Glycol 400, Polyethylene Glycol 600 8 Tween 60 Polyethylene Glycol 20 0, Polyethylene Glycol 400, Polyethylene Glycol 600 9 Tween 80 Polyethylene Glycol 20 0, Polyethylene Glycol 400, Polyethylene Glycol 600 10 Brij Series Tween 20, Tween 40, Tween 60, Tween 80 11 Span Series Tween 20, Tween 40, Tween 60, Tween 80 12 Polyoxy Fatty acid Esters Polyethylen e Glycols 200, Polyethylene Glycols 400 13 Tween 60 No cosurfactant required 14 Tween 80 No cosurfactant required 15 Polyethylene Glycol 400 No cosurfactant required 16 Polyethylene Glycol 600 No cosurfactant required

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87 Table 2-2. Size of propofol microemulsions Microemulsion Drop let Size (nm) Droplet Size (nm) (2 years old) Pluronic 68(5%)/Sodium Octanoate 27.6 28.4 Pluronic 68(5%)/Sodium Decanoate 25.4 25.6 Pluronic 68(6%)/Sodium Dodecanoate 21.8 25.4 Pluronic 127(4%)/Sodi um Octanoate 34.1 34 Pluronic 127(5%)/Sodium Decanoate 29.5 30.6 Pluronic 127(6%)/Sodium Dodecanoate 24.5 25 Table 2-3. Viscosity of propofol microemulsions at 25 C Microemulsion Viscosity (cps) 10 (1/S) Viscosity (cps) 100 (1/S) Viscosity (cps) 1000 (1/S) Pluronic 68(5%)/Sodium Octanoate 1.25 1.19 1.15 Pluronic 68(5%)/Sodium Decanoate 1.25 1.19 1.14 Pluronic 68(5%)/Sodium Dodecanoate 1.27 1.19 1.15 Pluronic 127(5%)/Sodium Octanoate 1.42 1.39 1.36 Pluronic 127(5%)/Sodium Decanoate 1.42 1.38 1.37 Pluronic 127(5%)/Sodium Dodecanoate 1.42 1.39 1.37

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88 CHAPTER 3 ANESTHETIC PROPERTIES OF PROPOFOL MICROEMULSIONS 3.1 Introduction Propofols is a unique compound compared to th e other intravenous anesthetics. Its lone ionizable functional group, th e hydroxyl, has a pKa of 11, which renders it unsuitable for forming salts in solution. The remaining portion of the molecule, the benzene ring and isopropyl side groups, are highly lipophilic. The result is a molecule with a poor water miscibility (146 mg/L).172 So, on an average human being with 4L of blood, assuming the blood solubility is the same as water can solubilize 0. 584 gm of free propofol in blood a ssuming propofol solubility in blood is the same as water. But in human syst em, even though the dose of propofol is 200 mg/L to 400 mg/L for induction and maintenance of anesthesia.173 Free concentrati on of propofol in blood is 25 mg/L,173 because propofol being hydrophobic compound partition or attaches to various lipophilic compounds as well as other bios urfaces in the blood. Thus, the aqueous phase of blood never reaches to its sa turation limit by propofol. The con centration of propofol in blood can be considered as the transient phenome non coupled with its qui ck partitioning into hydrophobic components of the blood (e.g. red bl ood cells, membranes, hydrophobic proteins). Also, propofol being hydrophobic is highly prot ein bound in vivo173. High lipophilicity (logP 4.16)174 of propofol means that good miscibility can only be achieved in lipophilic substances or organic solvents. Because vehicles for clinical delivery of anesthetics should be devoid of sedative and anesthetic properties, as well as toxi c side effects, nearly all small molecular weight organic solvents into which propofol is freely miscible are not useful. However, propofol can also be combined with biocompatible surfactants to form transparent, colorless, and thermodynamically stab le, oil-in-water microemulsions of nanometer diameter without the need for excipient oils such as soybean oil as already discussed in previous

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89 chapter. In these microemulsions, propofol serves the physical role as the core of the particles since this drug exists as oil at room temperatur e, and as the active pharmaceutical agent to cause general anesthesia. To examine anesthetic properties, we measured several parameters of anesthetic induction and emergence in rats receiving the experiment al and conventional formulations of propofol using a randomized, crossover design. Previously, we have shown that these microemulsions can be formed using various non-ioni c and ionic surfactants and have physical properties similar to those of a propofol macroemulsion, the conventiona l formulation. In addition, the surfactant type and concentration used to formulate these propofol microemulsions should affect the pharmacokinetic properties in animals. However, the limited blood volume (e.g., ~50 ml) of the rat compared to that volume (e.g., ~30 ml) necessary to measure plasma propofol concentrations repeatedly precluded determining pharmacokinetic properties. Therefore, a large animal model (i.e., dog) study was conducted in using a randomized, crossover design in orde r to compare the pharmac okinetics of a propofol microemulsion with those of a macroemulsion. In addition, several pharmacodynamic effects were noted including anesthetic induction and emergence ti mes, possible alteration of erythrocytes, leukocyte, and plat elet cell populations, and potentia l changes in the coagulation indices. 3.2 Methods and Materials 3.2.1 Materials Propofol was obtained from Albemarle Corpor ation (Baton Rouge, LA). Purified pluronic F68, a nonionic coblock polymer consisting of polyethylene a nd polypropylene monomers, was purchased from the BASF Corporation (Florham Park NJ). Sodium salts of fatty acid (C8, C10, and C12) were supplied by Sigma Ch emical Co. (St. Louis, MO).

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90 3.2.2 Synthesis of Propofol Microemulsions Microemulsions for rat studies were prepared by combining propofol (0-100 mg/ml) with purified Pluronic F68 (0-70 mg/ml), and a fatty ac id salt (0-12.5 mg/ml) in normal saline (0.90 mg/ml NaCl) bulk media. Water was ultra-purifie d using a water purification system (Nanopure, Barnstead/Thermolyne, Dubuque, IA) to provide a minimal electrical resistance of 18.2 M Following agitation with a magnetic stirrer, these components combined to form clear, colorless microemulsions with adjustment of pH to 7.40 using either HCl or NaOH. All experimental formulations were stored under a nitrogen head space. To characterize the dimension of the individual droplets, th e effective droplet size of the nanoparticles was measured by the dynamic light scattering method using a submicron pa rticle sizer analyzer (90Plus, Brookhaven Instruments Corporation, Holtsvill e, NY) as previously described.132 Microemulsion for dog studies were prepared by combining propofol (10 mg/ml) with purified Pluronic F68 (50 mg/ml), and a fatty acid salt (2.1 mg/ml) in a normal saline (0.90 mg/ml NaCl) bulk media. Water was ultra-purifie d using a water purification system (Nanopure, Barnstead/Thermolyne, Dubuque, IA) to provide a minimal electrical resistance of 18.2 M Following agitation with a magnetic stirrer, these components combined to form clear, colorless microemulsions. We then adjusted the pH to 7. 40 using either HCl or NaOH. The experimental formulation was kept under a nitrogen head to delay oxidation of propofol. Before administration, the propofol microemulsion was filtered through a 0.45 m sterile filter, but the macroemulsion ((Diprivan, AstraZeneca Pharmaceu ticals, Wilmington, DE) was not filtered. 3.2.3 Rat Experiments All experimental protocols i nvolving the use of animals were reviewed and approved by the University of Florida Institutional Animal Care and Use Committee. Sprague-Dawley rats (350-500 g; either sex) were purchased from Charles Rive rs Laboratories (Wilmington, MA).

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91 Before delivery of animals to th e investigators by the vendor, ra ts underwent catheterization of the left femoral vein with subcut aneous tunneling to an exit site between the scapulae. Thus, rats were supplied by the vendor with pre-implanted, heparinbonded, femoral vein catheters and did not require additional instrumentation at the time of the experiments that might require sedation or anesthesia that could confound interpretation of results. Rats were caged singly in order to avoid damage to the catheter by cage mates. They were allowed unlimited access to food and water with a 12:12 hour light:dark cycle. On the day of experimentation, each rat was weighed. Thereafter, the central venous lin e of each rat was easily accessed in conscious, unrestrained rats. All catheters were aspirated until blood was obs erved and then flushed with 0.5 ml of normal saline. Subsequently, rats entered the animal experimental protocol. 3.2.3.1 Animal experimental protocol Rats were randomized to receiv e either 1) an experimental propofol microemulsion (n=6 per microemulsion) after filtration through a 200 nm pore filter in orde r to ensure sterility or 2) a conventional macroemulsion of propofol in a soybean-based solvent (Diprivan, AstraZeneca Pharmaceuticals, Wilmington, DE). In both cases, the formulation was infused at a rate of 10 mg/kg/min propofol via a micr oprocessor-controlled syringe pump (sp2000i, World Precision Instruments, Sarasota, FL) in order to avoid va rying rates of infusion associated with manual injection that could po tentially confound anesth etic induction time parame ters. The endpoints of anesthetic induction were total drug dose, time to loss of explor atory behavior (i.e., stunned), time to loss of righting reflex, time to loss of lash reflex to gentle stroking (i.e., canthal reflex), and time to loss of reflexive withdrawal of th e leg following a great toe pinch every 10 s by a metal clamp. In these experiments, the meta l clamp was rubber shod to avoid tissue damage during the experiment. Following loss of withdrawal, the drug infusion was discontinued. Endpoints of anesthetic recovery we re return of the withdrawal res ponse to a toe pinch, recovery

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92 of spontaneous eye blinking, recovery of a sust ained head lift, and recovery of the righting reflex. Following the first anesthetic, each rat was recovered for at least 14 days before enrollment in the experimental limb opposite the original assignment. Thus, each rat was anesthetized with both experimental and convent ional propofol formulations with random initial assignment and with a 14 day interval before crossover between the fo rmulations. Following a 14 day period of observation after the participa tion in the crossover limb, the experiment was ended. 3.2.3.2 Statistical analysis Measurements are reported as meanstandard de viation. Statistical analysis was performed with SigmaStat 3.1 (Systat Software, Inc., Point Ri chmond, CA). Prior to parametric testing, the assumption of normality was validated using the Kolmogorov-Smirnov test with Lilliefors correction. For normally distributed data, one-w ay repeated measures analysis of variance (factor: propofol formulation) was used to test for overall statistical significance followed by Bonferonni post-hoc pairwise testing, when appr opriate, with tw o-tailed protection to analyze multiple comparisons between different formul ations of propofol. For nonparametric data, Friedman repeated measures analysis of va riance on ranks was used followed by Bonferonni testing with two-tailed protection when appropriate. P<0.05 was considered to be statistically significant. 3.2.4 Dog Experiments The Institutional Animal Care and Use Comm ittee of Calvert Laborat ories (Olyphant, PA) approved the study protocol prior to investigatio n. Treatment of animals was in accordance with the conditions specified in the Guide for the Ca re and Use of Laborator y Animals. Ten purposebred and experimentally naive beagles (Mar shall Bioresources, North Rose, NY) aged 7-8 months and weighing 7.0-8.9 kg were identified by ear tattoo. All animals had access to certified

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93 canine diet (Teklad, Harlan, I ndianapolis, IN) up to 400 g/day. Tap water was available ad libitum to each animal via an automatic watering device. Study animals were acclimated to their housing for a minimum of five days prior to en tering the experimental protocol. After being fasted for 16-20 hours, dogs were weighed and prepared for the experiment. Peripheral intravenous catheters were inserted in the right and left le g veins for intravenous administration of propofol formulations a nd the collection of whole bl ood samples, respectively. 3.2.4.1 Animal experimental protocol On the day of experimentations, dogs (n=10) were randomized to receive either the propofol microemulsion (n=5) or macroemulsion (n =5) intravenously at a rate of 10 mg/kg/min via a microprocessor-controlle d syringe pump in order to obviate potential confounding influences caused by variable administration rate s from manual injecti on. During the infusion, the endpoint of anesthetic induction was the ti me necessary to cause the loss of reflexive withdrawal of the leg following a toe pinch every 15 sec with a rubber-shod clamp. After loss of withdrawal of the leg, the infu sion was discontinued. The total drug dose to cause induction was the mass of propofol required to cause loss of leg withdrawal. Thereaf ter, the subject was observed for recovery of the withdrawal to a to e pinch. Also, the inve stigators measured the heart rate (Pagewriter, Hewlett Packard Compa ny, Palo Alto, CA), indirect peripheral blood pressure (9301V, Cardell Veterina ry), and respiratory rate at th e following time points: pre-dose, immediately postinduction at one minute, and every five minutes thereafter until dogs emerged from anesthesia. The electrocardiogram was examined for arr hythmias. In addition to these parameters, animals were observed for apnea or other signs of respiratory distress. Body temperature was measured with a thermometer and was maintained using a heating pad placed under the animal.

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94 Following this anesthetic, dogs were allowed to recover and then return ed to the kennel. The dogs were allowed to recover and were then returned to the kennel. After the first set of experiments, dogs were recovered for at least 7 days before being crossed over to receive the formulation in the opposite limb of the study. Thus, dogs previously enrolled to receive microemulsion (or macroemu lsion) were switched to receive the opposite formulation. Following anesthetic induction in th e opposite limb of the i nvestigation, dogs were recovered for a least 7 days before the investigation was terminated. 3.2.4.2 Blood sample acquisition and processing During these experiments, blood samples were taken for two purposes. First, samples were secured before induction and at times 1, 5, 10, 15, 20, 25, and 30 min after induction (or until the dog emerged from anesthesia) to measure the propofol concentration in order to study pharmacokinetics of these propofol formulati ons. The blood samples were acquired from a second intravenous cannula placed in vein on a leg contralateral to that used for propofol injection. These blood samples (1-2 ml) for assessment of propofol concentration were stored in non-additive blood tubes and centrifuged at 3,000 re volutions/min. The resultant supernatant was aspirated and stored at -20 C until thawed for measurement of propof ol concentrations as described subsequently (see Measurement of Plasma Propofol Concen tration). Second, blood samples were acquired to determine the eff ects of the formulations on the hemogram, prothrombin time, activated partial thromboplasti n time, and fibrinogen concentrations. These blood samples (4 ml) were collected pre-indu ction, immediately postinduction when concentration of propofol and excipients would be expected to peak in the plasma, and during recovery of anesthesia. In all cases, the maximum amount of bl ood collected was limited to 1% of the body weight over a period of two weeks.

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95 3.2.4.3 Measurement of plasma propofol concentration Plasma propofol concentrations were meas ured using a method previously reported by Dennis and coauthors.175, 176 Briefly, the mobile phase was passed through a 0.22 m nylon filter (Millipore, Bedford, MA) and degassed by sonica tion. Stock solutions (10 mg/ml) of propofol and thymol, the internal standard, were prepared in methanol and st ored at 4 C. Vials were filled with nitrogen after opening to prevent drug oxidati on. Standard solutions of propofol (2, 20, and 200 g/ml) were made by further dilution of the stock solution with meth anol. Quality control samples (10, 100 and 1000 ng/ml) were prepared by adding 50 l of appropriate propofol standard solution to 10 ml of blank plasma. These samples were vortexed and kept frozen in 1 ml aliquots at -20 C until analysis. For calibration curves, blank plasma sa mples (1 ml) were spiked with 25 l of the appropriately diluted standard solutions to final propofol concentrations of 5, 10, 50,100, 500, 1000 and 2000 ng/ml. Control samples containing no added propofol were also prepared. SPE cartridges were activated with 1 ml of methanol and washed with 1 ml of water. Plasma samples (1 ml) were diluted with 1 ml of phos phate-buffered saline after adding 20 l of thymol (50 g/ml) as an internal standard, acidified with 20 l of phosphoric acid, vortexed, and loaded onto activated cartridges. The cartridges were washed three times (1 ml of water followed by 1 ml of 1% KHCO3 in acetonitrile:water (1:9), followed by 1 ml of acetonitrile:water (2:3)) and dried in a low vacuum for 30 sec. Solutes were eluted wi th 1 ml of methanol without vacuum. Vacuum was used briefly to remove remaining solven t in the cartridge. Solu tes were vortexed and transferred to autosampler vials for analysis LC/MS/MS analyses were performed using a Perkin-Elmer PE 200 LC system and an API 4000 triple quadrupole mass spectrometer equipped with an APCI source (Applied Biosystems, Foster City, CA). Samples (25 l injection volume) were separated on a C18 column (Xterra TM RP18, ODS 5 m 250 mm 4.6 mm i.d., Waters)

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96 with a guard cartridge (4 mm 3 mm i.d., Phenomenex, Torrance, CA) prior to the C18 column. The isocratic mobile phase consisted of meth anol and 0.05% aqueous ammonium hydroxide solution (98:2) at a flow rate of 1 ml/min. The deprotonated precursor molecular ions were selected and fragmented by nitrogen gas collisio n in the Q2 region with a collision energy of 45 eV. The resulting mass spectra were acquired in full scan mode from m/z 100. The most abundant product ion at 161 for propof ol and the ion at 133 for thymol were selected for multiple reaction monitoring (MRM) quantitation. Calibrati on curves were construc ted by plotting the ion abundance peak area ratios (propof ol/IS) as a function of plasma propofol concentration. These data were then fitted with an unweighted least sq uares regression analysis to the equation: y = ax + b, where a defines the slope and b the intercept of the ordinate. The prop ofol concentrations of unknown samples were calculated using the results of the regression analyses. 3.2.4.4 Statistical and pharmacokinetic analysis Measurements are reported as meanstandard de viation. Statistical analysis was performed with SigmaStat 3.1 (Systat Software, Inc., Point Ri chmond, CA). Prior to parametric testing, the assumption of normality was validated using the Kolmogorov-Smirnov test with Lilliefors correction. For normally distributed data with two groups, the paired t-test was used. For normally distributed data with mo re than two groups, one-way re peated measures analysis of variance (factor: propofol formulation) was used to test for overall statistical significance followed by Bonferonni post-hoc pairwise testin g, when appropriate, to analyze multiple comparisons between different formulations of propofol. For nonparametric data, Friedman repeated measures analysis of variance on ranks was used followed by Bonferonni testing when appropriate. P<0.05 was considered to be statistically significant. Pharmacokinetic analysis of each dogs blood concentration versus time profile was performed using the data analysis program Wi nNonLin (Scientific Consulting, Inc., USA)

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97 according to Knibbe and colleagues.128 The following biand triexponential equations were used in order to describe the blood concentration-time profiles af ter injection to determine the best fitted relationship: t teCeCtC 2 12 1)( (3-1) t t teCeCeCtC 3 2 13 2 1)( (3-2) where C(t) is the blood concentration of pr opofol at time t, C1, C2 and C3 are the coefficients and 1, 2, 3 the exponents of the equation. Sta tistical analysis of pharmacokinetic data was performed using analysis of variance (ANOVA) with a statistical software package (Epistat v3.0, T.L. Gustaf son, Wound Rock, TX, USA). 3.3 Results 3.3.2 Anesthetic Properties in the Rat Two separate series of experiments were perf ormed to determine the anesthetic properties of propofol microemulsions in rats. In the firs t set, the concentration of the nonionic surfactant purified Pluronic F68 was primarily changed and compared to the macroemulsion control to cause anesthesia in rats. In the second set, the carbon chain length of the fatty acid was increased (C8, C10, C12) to determine if this modifica tion affected anesthetic induction and emergence times compared to the macroemulsion control. 3.3.2.1 Modification of nonionic surfactant concentration In the first series of experiments, rats were administered propofol microemulsions with different concentrations of the nonionic surfac tant purified Pluronic F68 (3%, 5%, and 7%) or they received the conventional macr oemulsion. Twenty-three rats were enrolled in this protocol, but five rats completed only one limb of this pr otocol due to retraction of the catheter under the skin during the recovery interval from the first limb of the study. Data from these five rats (n=3

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98 in microemulsion groups, n=2 macroemulsi on group) were removed from the study. The remaining rats (n=18) completed the crossover study and were anes thetized with these microemulsions (n=6 per group) and with th e macroemulsion control (n=18). All rats experienced rapid general anesth esia without apnea and recovere d for at least 14 days without apparent injury. Following the 14 day observation period after crossover, the experiment was terminated. The summary data from these inducti on experiments are presented in Figure 3-1 and Table 3-1. All microemulsions required greater do ses for anesthetic induction as defined by loss of leg withdrawal to a toe pinch compared to the required dos e of macroemulsion (P=0.005, <0.001, and <0.001 compared to 3, 5, and 7% purified Pluronic F68 microemulsions, respectively). In addition, the time to onset of anesthesia caused by the 5% purified Pluronic F68 microemulsions was significantly longer than that caused by the macroemulsion for stunning (P<0.001), loss of the righting reflex (P<0.001) and loss of the lash reflex (P=0.01). For emergence parameters, the 3% microemulsion caused more rapid emergence than the macroemulsion control as assessed by return of righting (P= 0.03), but not for return of leg withdrawal (P=0.71), sustained h eadlift (p=0.10), or lash reflex (P=0.40). Finally, rats emerged more rapidly following anesthesia as assessed by return of leg reflex (P=0.01) and return of the lash reflex (P=0.01) with the 7% microemulsion compared to the 3% microemulsion. 3.3.2.2 Modification of ionic surfactant concentration In a separate series of experiments, rats were administered microemulsions with a constant concentration of purified Pluronic F68 (5%), but with ionic surfactants (i.e., fatty acid salts) of increasing carbon chain length (C8, C10, and C12). Twenty-one ra ts entered this protocol, but three rats did not complete the crossover experiments due to cat heter retraction under the skin (n=2 for microemulsion group, n=1 for macr oemulsion group) during the 14 day recovery interval between crossover. The information from these three rats was removed from the data set.

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99 The remaining rats in this protocol were infused with the th ree different propofol microemulsions (n=6 per group) and with the m acroemulsion (n=18). As with the first set of animal experiments all rats experienced general anesthesia without apnea and recovered for at least 14 days without apparent injury. After 14 days following the crossover limb, the experiment was ended. The summary data for these experiment s are presented in Table 3-2. (Note: the rats listed for the C8 fatty acid salt experimental pr opofol microemulsion were the same as those noted for purified poloxamer 5% in the Table 31 and are presented again in Table 3-2). The dose of propofol necessary to cause anesthesia wa s greater for microemulsions compared to the macroemulsion. In addition, the length of time to induction was longer for all the microemulsions compared to the macroemulsion for all induction parameters measured. Also, the microemulsion formulated with C10 fatty acid sa lt required a larger dose to cause anesthesia than for the microemulsions made with C8 (P <0.001) or C12 (P=0.01) fatty acid salts. For emergence, rats receiving the C 12 fatty acid salt microemulsion required a longer time to emerge as assessed by return of leg withdrawal (P=0 .008), righting reflex (P=0.002), and sustained headlift (P=0.008) compared to the macroemulsi on. Similarly, these rats receiving the C12 microemulsion took longer to emerge than th e animals receiving the C8 microemulsion as measured by return of the righting reflex (P =0.004) and a sustained headlift (P=0.004). 3.3.3 Anesthetic Properties in the Dogs All dogs (n=10) experienced gene ral anesthesia with subsequent recovery when injected with either formulation of propofol. The mean do se of microemulsion or macroemulsion to cause loss of leg withdrawal was 10.3.2 and 9.7 1.6 mg/kg, respectivel y (P=0.39). Although anesthetic induction caused marked changes in the heart rate (P <0.001), blood pressure (P<0.001), and respiratory rate (P<0.001) over time, these changes were similar for animals assigned to receive either the microemulsion or macroemulsion (Figure 3-3, Figure 3.4). There

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100 were no significant differences w ith respect to group assignment in the heart rate (P=0.62), blood pressure (P=0.81), or respiratory rate (P=0. 60) for dogs administered the microemulsion or macroemulsion. Apnea or respiratory distress was not observed. The duration of anesthesia caused by these doses of anesthetic were 1046 275 and 1094 sec for the microemulsion and macroemulsion, respectively (P=0.70). 3.3.3.1 Effects on erythrocytes and leukocytes Data stratified by group assignment (micro emulsion, macroemulsion) and time (preinduction, post-induction, recovery period) for parameters of th e erythrocyte (Red Blood Cells) population are presented in Table 33. No differences were observed between formulations with respect to indices of erythrocyt e populations as noted by the P va lues in Table 3-3. However, significant reductions in some indices were observe d with respect to time. That is, there were decrements in the red blood cell count (P <0.001), hemoglobin concentration (P<0.001), hematocrit (P<0.001), absolute reticulocyte co unt (P=0.002), and relative reticulocyte count (P=0.030) over time for both groups. Similar results were observed after measuring the concentr ation of white blood cells and subtypes (i.e., neutrophils, lymphocytes, eosinophils mast cells, basophils) as tabulated in Table 3-4. That is, although no differen ces were noted between formula tion types of propofol as noted by the P values in Table 3-4, significant reductio ns in the total white blood cell concentrations and the concentrations of white cell sub-popul ations were noted in both groups. However, neither formulation assignment nor time significantly affected the fraction (i.e., differential count) of different white blood cell types (data not shown). 3.3.3.2 Effects on platelets and thrombosis Neither the microemulsion nor macroemulsion of propofol affected platelet concentration, fibrinogen concentration, prothrombin time, or activ ated partial thromboplastin time (Table 3-5).

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101 In one dog that received the propofol macroemulsion, the platelet concentration was measured to be 8,000 platelets/ l in the recovery period although th e concentrations in the same dog determined at pre-induction and postinduction were 240,000 and 233,000 platelets/ l, respectively. Following microscopic examination of the specimen, no clumping of platelets was noted. Therefore, this outlying data point was re tained and accounts for the increased standard deviation for this gr oup (111,000 platelets/ l) compared to the deviations for other groups (39,000-50,000 platelets/ l). 3.3.3.3 Propofol pharmacokinetics In order to understand the pharmacokinetic prof iles of these formulations of drugs, the plasma concentration of propofol was measured at various times after ad ministration of propofol as noted in Table 3-6. After a bol us of propofol at 0 min, a larg e peak propofol concentration was measured followed by rapid decrements in the con centrations at 1, 5, and 10 min. At 15 and 20 min, increases in the propofol concentrations were observe d for both themacroemulsion and microemulsions groups. Notwithstanding these ti me-related changes, no significant differences in plasma propofol concentrations were noted at 1, 5, 10, 15, or 20 min between the dogs receiving the microemulsion or macroemulsion formulations. 3.4 Discussion In rat study, kinetics of gene ral anesthesia with the propofol microemulsion were favorable compared to the commercially available propofol macroemulsion. Whereas the time for general anesthesia (defined by the protocol as loss of leg withdrawal to a pinc h) during macroemulsion injection was 122-126 s, the latency periods du ring microemulsion treatment were 166-203 s. This short delay is favorable when compared to pro-drug technologies that rely on metabolism, a phenomenon that may vary significan tly within any patient population.147 The differences in induction times between experimental groups re ported herein may be caused by differential

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102 release of propofol from the individual droplets into the blood. That is, the different propofol nanoparticles have markedly different stabil ities against dilution by blood based on the emulsifier structure and concentration selected fo r the formulation. In general, a microemulsion is thermodynamically stable at equilibrium. One can destabilize a microemulsion by significantly changing pressure, temperature, or chemical co mpositions. The last variable can be changed simply by diluting a microemulsion with saline or blood. It is well recogni zed that the formation of microemulsions requires an ultra low interfacial tension (e.g., ~10-3 mNewton/m) at the oil/water interface.31 Upon dilution w ith saline, the interfacial tens ion will increase substantially as the emulsifier molecules (i.e., both poloxamer 188 and fatty acid salt molecules) desorb from the droplet surface. This event will markedly increase the interfacial tension at the droplet surface and ultimately destabilize the microemulsi on, with release of the active pharmaceutical In dog study, we investigat ed the dose of propofol, mi croor macroemulsions, administered to dogs to induce anesthesia, along with possible effects on blood, the first tissue encountered by the formulations. The measured va riables of anesthesia, vital signs, indices of blood cell populations or thrombosis, and plasma concentrations of propof ol did not significantly differ between the groups of animals given either the propofol microemulsion or macroemulsion. 3.4.1 Microemulsion Fate Upon Injection In a macroemulsion or microemulsion, propofol is highly concentrated in the emulsified oil droplets (defined as the di scontinuous phase), with only sma ll quantities in the aqueous phase (i.e., continuous phase), the latt er of which constitut es the largest volume of the microemulsion or macroemulsion (1% propofol formulation). Upon administration of a propofol-containing emulsion, propofol diffuses across the droplet in terface and passes into the bloodstream. Major factors that govern this proce ss for propofol or any lipophilic drug are the drug concentration gradient, the partition coefficient, the drug diffusivity in both pha ses, and the interfacial area of

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103 the drug-containing oil droplets. Therefore, similar to any drug releasing particle, microemulsions and macroemulsions slow the availability of free drug as compared with drugs administered in solutions in whic h they are molecularly dissolved. The drug diffusivity from dispersed phase (oil phase) to continuous phase (water phase) is different in macro and microemulsions. In micr oemulsions, propofol molecules take longer time to come out of the microemulsions because of the extra barrier of the very low interfacial tension surfactant film. These microemulsions grow in size when they get diluted in the blood, as more and more surfactant leave the oil/w ater interface for other places in the blood and vein. And after a certain dilution these microemulsion convert to macroemulsion. So, this extra barrier for drug to come out of the microemulsion to the blood slows the availability of free drug in microemulsion than in macroemulsion. Eventu ally the bioavailability of propofol in blood reaches to its pharmacological limits at which we s ee the start of the onset of anesthesia, but it would take longer time in the case of microemulsion than in macroemulsion. The total interfacial surface area is a highly important factor in the rate of drug release from a propofol containing droplet. This in tu rn is dependent on the size and number of oil droplets resulting from the injection. In a propofol macroemulsions made from identical emulsifiers, if they contain uniform droplets (monodisperse) of 1.0 m in diameter, the total oil water surface area, or dropletaqueo us phase interface, would be 0.66 m2/ml. However, if the particle size were reduced to 0.1 m, it would have a total oil water surface area of 27.6 m2/ml, nearly 42 times greater. The latter allows for a more rapid rate of rel ease of propofol to the blood, or in other case faster anesthesia. Thus, a microemulsion has greater total interfacial area but higher resistance to diffusion across interface. Since experimens showed greater induction

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104 time, it implies that the interfacial resistance must be a dominating factor as compared to total interfacial area. 3.4.2 Propofol Anesthetic Properties In dog studies, all animals experienced rapi d anesthesia as assessed by loss of leg withdrawal with mean propofol doses of appr oximately 10 mg/kg for both the microemulsion and macroemulsion. This dose of propofol is gr eater than that previously reported in other studies investigating anesthetic induction in unpremedicated dogs using propofol formulated in a macroemulsion. In those reports, the mean dose of propofol noted ranged from 3.8-6.9 mg/kg with a meanstandard error of the mean calculated from these six studies of 5.8.5 mg/kg.177-179 Because the mean dose observed by the present in vestigators was greater for the macroemulsion (in addition to the microemulsion), this incr ease was likely due to differences in the study protocol including a higher rate of propofol infusion (10 mg/kg/min) and use of younger animals (i.e., 8 month old dogs) in the present report. Al though we can not identify any studies detailing age-related effects on propofol dos e in dogs, previous investiga tions conducted in rats and humans demonstrate that greater doses are re quired in younger, compared to older, subjects.180, 181 For example, Schnider and colleagues observed th at the mean plasma propofol concentrations at which 50% of the human participants experi enced anesthetic induction were significantly greater (1.88-fold increase) for 25 year old (2.35 g/ml) compared to 75 year old (1.25 g/ml) subjects.181 Likewise, we observed a si milar proportional increase in propofol macroemulsion dose (9.7 mg/kg / 5.8 mg/kg or 1.67-fold grea ter) for younger dogs compared to the mixed age dog populations from the previously reported dog studies.179 In addition, the duration of anesthetic was remarkably similar to that prev iously reported for dogs. That is, whereas we observed a mean duration of anesthesia of 18. 2 min whereas Watkins an d colleagues reported a value of 18 min. Therefore, the pharmacodynamic effects of propofol administered in either

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105 formulation are consistent with previously reported values in dogs and are similar for the microemulsion and macroemulsion preparations. On e limitation of this type of investigation is that any conclusions are limited to the dose studie d. Extrapolation to other doses and sustained infusion of propofol require additional work and ev idence. In addition, any extrapolation of these data to potential human use should be done cauti ously because of possible species differences and the need for safety investigations (e.g., dose escalation studies). 3.4.3 Propofol Concentration The plasma propofol concentrations reported in the present study demonstrated a high peak concentration (25 M) followed by rapid decline. Similar findings for the propofol macroemulsion were noted by Zoran et al.182, who observed peak concentrations of 2.3 g/mL (12.9 M) and 3.29 g/mL (18.5 M) in mixed breed dogs and Greyhounds, respectively. Similarly, Cockshott et al.183 noted peak concentrations of approximately 4 g/mL (22.4 M) in specimens obtained from dogs after injecti on of a 7 mg/kg propofol bolus. The lesser concentrations reported for previous investigations may have been due to the fact that the dogs enrolled in their previous studies received le ss propofol (5 mg/kg) over a longer time period than did dogs in our investigation. The observat ion of secondary peaks in plasma propofol concentrations at 15 min in our study was une xpected. We had previously hypothesized the existence of a single,large peak concentration of propofol imme diately after bolus injection followed by steady decrements. Two possible reasons may account for these secondary peaks in propofol concentrations. First, this finding ma y be an artifact of the protocol. We stopped sampling blood for propofol concentrations when dogs emerged from anesthesia. Therefore, dogs with the least plasma propofol concentrati ons were excluded from the summary data at 15 and 20 min. This event would tend to cause th e mean propofol concentr ations to increase. Second, this phenomenon has been observed by others studying the pharmacokinetics of

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106 propofol in both dogs182 and humans.183, 184 For example, Zoran182 described a secondary peak concentration of propofol in five of eight mixed breed dogs and eight of ten Greyhounds. Similar to the present investigation, they noted that this peak occurred at the time that the dogs emerged sufficiently to achieve a sternal position (i.e., righting reflex). Th e exact cause of this secondary peak is unknown, but previously suggested reason s include changes in ca rdiac output associated with movement after anesthesia, influx of propofol from peripheral tissues with loss of venodilation caused by propofol, use of venous (vis-a`-vis arterial) sampling, and changes in blood components due to splenic contraction in dogs associated with the phlebotomy necessary for acquiring multiple blood specimens.182 3.4.4 Coagulation One possible drawback of using intravenous microemulsions to delivery propofol is potential interference by the components of the fo rmulations with the coa gulation system. In the present study, we used purified Pluronic F68 as a nonionic surfactant to reduce the interfacial tension between propofol oil droplets and the aqueous environment in order to construct microemulsions. Purified Pluronic F68 is a polymer consisting of poly(ethylene oxide)/poly(propylene oxide) copolymers ((C 2H4O)70(C3H6O)35(C2H4O)70) and has been thought to possibly modify platel et adhesion. This and related polymers have been used for coating biological implants constructed from va rious materials and have been shown to reduce platelet adherence, an effect primarily dependent on the num ber of propylene oxide (C3H6O) residues vis--vis ethylene oxide (C2H4O) residu es (Note: the number and proportion of the residues are varied by th e manufacturers to construct unique polymers).185, 186 Previously, Morey and colleagues studied human blood using th romboelastography and demonstrated that microemulsions similar to those used in the curre nt investigation reduced platelet function (i.e., thromboelastograph maximal amplitude, elastic m odulus) in a concentration-dependent manner,

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107 but did not decrease the overall platelet population or detectably interfere with clotting factor function.187 Furthermore, this effect of the microe mulsions was highly correlated (r2=0.94) to that caused by the concentration of the co-block polymer component of the microemulsion, but was not caused by the oil or fatty acid components. In contrast to these in vitro investigations, minimal-to-no effects have been observed during in vivo investigations. For example, Orringer and colleagues hypothesized that high-dose (i.e ., 100 mg/kg for 1 hour followed by 30 mg/kg per hour for 47 hours), intravenous infusion use of purif ied Pluronic F68 as an investigational drug to treat acute vaso-occlusive cr isis of human patients with sickle cell disease would reduce the clotting associated with sickled erythrocytes, but observed only mild effects in a pediatric subgroup also receiving hydroxyurea.161 Likewise, no changes were noted in dogs due to administration of formulations in the platelet count, fibrinogen concentration, prothrombin time, or activated partial thro mboplastin time in the present study. Although useful as screening tools, these parameters of coagulation are not sensitive to detect subtle changes in clotting or platelet functions. Therefore, this limita tion of the present study should be further addressed with investigations specifically dedicated to obser vations of possible cha nges in clotting using sensitive tests (e.g., thromboelastography, platelet contractile fo rce, clot elastic modulus). 3.5 Conclusions Here, we report the successful use of propofol microemulsions to cause anesthesia in rat similar to that caused by propofol macroemulsions. The differential diluent volumes in vitro and destabilization times in vivo based on changing th e concentration and type of surfactant alludes to the possibility of selectively modifying the pharmacokinetic and pharmacodynamic properties of propofol, and potentially other lipophilic drugs. Future work will be aimed towards exploring these pharmacokinetic /pharmacodynamic differences for pro pofol and other lipophilic agents

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108 and to determining if these propofol microemu lsions reduce the liabilities (e.g., stinging on injection, support of b acterial growth) associat ed with soybean oil-based macroemulsions. Here, we have shown that propofol formulated as a microemulsion can be used successfully to anesthetize dogs in a way similar to a comm ercially available propofol macroemulsion. Furthermore, no detectable a dverse effect was observed on indices of the hemogram or parameters of clotting.

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109 Figure 3-1. Anesthetic induction parameters in Rats. A) Propofol dose. B) Stunned. Propofol Dose Propofol Dose (mg/kg) 0 10 20 30 40 *Oils:Propofol 10 mg/ml Soybean Oil 100 mg/mlSurfactants:Glycerol 22.5 mg/ml Egg Lechithin 12.5 mg/ml No fatty acid Disodium edetate 0.05 mg/ml Oils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 30 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Exta Oil Surfactants:Pluronic F68 50 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 70 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialFormulationDiprivanP<0.05: *, vs. Diprivan vs. F68 (30 mg/ml)n=6 n=6 n=6 n=4ME1ME2ME3 Propofol Dose Propofol Dose (mg/kg) 0 10 20 30 40 *Oils:Propofol 10 mg/ml Soybean Oil 100 mg/mlSurfactants:Glycerol 22.5 mg/ml Egg Lechithin 12.5 mg/ml No fatty acid Disodium edetate 0.05 mg/ml Oils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 30 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Exta Oil Surfactants:Pluronic F68 50 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 70 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialFormulationDiprivanP<0.05: *, vs. Diprivan vs. F68 (30 mg/ml)n=6 n=6 n=6 n=4ME1ME2ME3 ME1ME2ME3 Induction: Stunned Formulation Latency (s) 0 60 120 180 240 Latency (min) 0 1 2 3 4Oils:Propofol 10 mg/ml Soybean Oil 100 mg/mlSurfactants:Glycerol 22.5 mg/ml Egg Lechithin 12.5 mg/ml No fatty acid Disodium edetate 0.05 mg/ml Oils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 30 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Exta Oil Surfactants:Pluronic F68 50 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 70 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialn=6 n=6n=4 n=6Diprivan*P<0.05: *, vs. Diprivan vs. F68 (30 mg/ml)*ME1ME2ME3 Induction: Stunned Formulation Latency (s) 0 60 120 180 240 Latency (min) 0 1 2 3 4Oils:Propofol 10 mg/ml Soybean Oil 100 mg/mlSurfactants:Glycerol 22.5 mg/ml Egg Lechithin 12.5 mg/ml No fatty acid Disodium edetate 0.05 mg/ml Oils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 30 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Exta Oil Surfactants:Pluronic F68 50 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 70 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialn=6 n=6n=4 n=6Diprivan*P<0.05: *, vs. Diprivan vs. F68 (30 mg/ml)*ME1ME2ME3 ME1ME2ME3 A B

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110 Figure 3-2. Anesthetic emergence in Rats. Emergence: Return of Lash Reflex Formulation Latency (s) 0 300 600 900 1200 Latency (min) 0 5 10 15 20Oils:Propofol 10 mg/ml Soybean Oil 100 mg/mlSurfactants:Glycerol 22.5 mg/ml Egg Lechithin 12.5 mg/ml No fatty acid Disodium edetate 0.05 mg/ml Oils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 30 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Exta Oil Surfactants:Pluronic F68 50 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 70 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialDiprivanOverall P value: 0.11 n=6 n=6 n=6 n=4ME1ME2ME3 Emergence: Return of Lash Reflex Formulation Latency (s) 0 300 600 900 1200 Latency (min) 0 5 10 15 20Oils:Propofol 10 mg/ml Soybean Oil 100 mg/mlSurfactants:Glycerol 22.5 mg/ml Egg Lechithin 12.5 mg/ml No fatty acid Disodium edetate 0.05 mg/ml Oils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 30 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Exta Oil Surfactants:Pluronic F68 50 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialOils:Propofol 10 mg/ml No Extra Oil Surfactants:Pluronic F68 70 mg/ml No Egg Products Octanoic (C8) fatty acid No antimicrobialDiprivanOverall P value: 0.11 n=6 n=6 n=6 n=4ME1ME2ME3

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111 Figure 3-3. Time-dependent eff ects of a propofol microemulsion or macroemulsion on Dogs. A) Heart rate. B) Mean ar terial blood pressure. A) Heart rate. B) Blood Pressure.

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112 Figure 3-4. Time-dependent effects of a propofol microemulsion or macroemulsion on respiratory rate in Dogs. Data is ex pressed as meanstandard deviation.

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113 Table 3-1. Dose and latency intervals for anesth etic induction and emergence in rat following intravenous infusion of a propofol macroe mulsion formulation and several propofol microemulsion formulations with C8 fatty acid salt and differing purified poloxamer 188 (Pluronic F68) concentrations Prameter Propofol Formulation Macroemulsion Microemulsion 3% 5% 7% N 18 6 6 6 Anesthetic Induction Dose (mg/kg) 21.0 4.9 26.6 4.0 30.4 4.5 30.2 4.8 Stunned (s) 38 8 48 8 69 13 64 12 Loss of Righting Reflex (s) 55 15 68 11 91 12 78 15 Loss of Lash Reflex (s) 87 19 93 17 129 24 106 16 Loss of Leg Withdrawal (s) 126 29 160 23 182 26 181 28 Anesthetic Induction Return of Leg Withdrawal (s) 375 147 368 77 405 193 274 105 Return of Lash Reflex (s) 306 170 347 84 362 195 230 107 Return of Righting Reflex (s) 681 125 460 64 629 126 581 155 Return of Sustained Headlift (s) 720 105 495 83 644 133 596 143 Data expressed as meanstandard devi ation of N number of experiments. Table 3-2. Dose and latency intervals for anesth etic induction and emergence in rat following intravenous infusion of a propofol macroe mulsion formulation and several propofol microemulsion formulations containing with C8, C10, or C12 fatty acid salts and 5% purified poloxamer 188 (Pluronic F68) Prameter Propofol Formulation Macroemulsion Microemulsion C8 C10 C12 N 18 6 6 6 Anesthetic Induction Dose (mg/kg) 20.2 4.0 30.4 4.5 41.9 3.4 33.9 1.6 Stunned (s) 41 9 69 13 69 10 83 12 Loss of Righting Reflex (s) 55 8 91 12 110 13 100 13 Loss of Lash Reflex (s) 90 11 129 24 156 11 142 33 Loss of Leg Withdrawal (s) 122 24 182 26 251 20 203 10 Anesthetic Induction Return of Leg Withdrawal (s) 270 140 405 193 318 63 516 176 Return of Lash Reflex (s) 358 193 362 195 334 57 528 240 Return of Righting Reflex (s) 645 104 629 126 712 192 889 175 Return of Sustained Headlift (s) 667 116 664 133 725 197 850 119 Data expressed as meanstandard devi ation of N number of experiments.

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114Table 3-3. Effects of propofol microemulsi ons (Micro) and macroemulsions (Macro) on parameters of the Red Blood Cell population in Dogs Prameter Pre-Induction Post-I nduction Recove ry Recovery Micro Macro Micro Macro Micro Macro RBC (106/ l) 6.04 0.45 6.13 0.43 5.99 0.41 6.07 0.34 5.47 0.71 5.40 0.50 0.41 Hemoglobin (103/ l) 14.1 0.80 14.3 0.80 14.0 0.70 14.4 0.50 12.7 1.40 12.6 1.00 0.66 Hematocrit (%) 42.0 2.30 42.7 2.70 41.9 2.00 42.4 1.50 37.9 4.00 37.5 2.80 0.78 MCV (fl) 69.6 2.40 69.7 2.30 70.0 2.50 69.9 2.20 69.6 2.30 69.5 2.20 0.84 MCH (pg) 23.4 0.70 23.3 0.70 23.4 0.80 23.7 0.70 23.3 0.70 23.3 0.80 0.45 MCHC (g/dl) 33.7 0.50 33.5 0.60 33.5 0.40 33.9 0.40 33.5 0.50 33.6 0.40 0.48 Reticulocytes (103/ l) 67.3 18.7 73.9 33.8 67.4 17.5 64.3 20.3 53.7 11.5 50.8 12.4 0.97 Reticulocytes (%) 1.12 0.30 1.21 0.50 1.15 0.28 1.06 0.36 1.01 0.22 0.96 0.30 0.88 Table 3-4. Effects of propofol microemuls ions (Micro) and macroemulsions (Macro) on the White Blood Cell count and population differential in Dogs Prameter Pre-Induction Post-I nduction Recove ry Recovery Micro Macro Micro Macro Micro Macro WBC (103/ l) 8.55 1.04 9.48 2.20 7.24 1.50 8.81 2.23 7.72 1.30 8.15 2.79 0.14 Neutrophils (103/ l) 5.03 0.69 5.93 1.41 4.15 1.09 5.36 1.35 4.49 0.96 5.03 1.94 0.11 Lymphocytes (103/ l) 2.60 0.71 2.61 0.68 2.37 0.64 2.37 0.72 2.35 0.65 2.24 0.62 0.82 Monocytes (103/ l) 0.58 0.17 0.59 0.30 0.44 0.13 0.55 0.29 0.57 0.16 0.57 0.29 0.58 Eosinophils (103/ l) 0.19 0.13 0.21 0.21 0.16 0.12 0.17 0.17 0.17 0.11 0.19 0.18 0.47 Basophils (103/ l) 0.06 0.03 0.08 0.07 0.05 0.02 0.05 0.05 0.06 0.03 0.05 0.03 0.59

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115 Table 3-5. Effects of propofol microemulsi ons (Micro) and macroemulsions (Macro) on Platelet population and indices of Thrombosis in Dogs Prameter Pre-Induction Post-I nduction Recove ry Recovery Micro Macro Micro Macro Micro Macro PlAtelets (103/ l) 338 43 330 48 319 39 322 50 331 42 299 111 0.55 Fibrinogen (mg/dl) 202 30 219 80 202 23 227 87 204 30 206 81 0.52 PT (s) 8.7 0.4 8.7 0.3 9.0 0.4 9.0 0.4 8.9 0.4 8.9 0.5 0.89 aPTT (s) 10.1 0.6 10.2 0.4 10.3 0.5 10.3 0.4 10.2 0.6 10.1 0.6 0.76 Table 3-6. Plasma propofol concentration after induction of anesthesia in Dogs Plasma propofol concentration ( Time (min) Microemulsion n Macroemulsion n -1 <0.05 10 <0.05 10 1 25.2 8.7 10 31.7 14.1 10 5 2.9 1.2 10 3.7 1.9 10 10 1.9 0.6 10 2.1 0.7 10 15 2.3 1.3 9 1.6 0.5 9 20 1.9 1.4 4 2.4 1.1 6

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116 CHAPTER 4 RETARDATION OF WATER EVAPORATION THROUGH DUPLEX FILMS OF SURF ACTANT IN OIL 4.1 Introduction Early experiment on retardation of water ev aporation can be traced back in 1924, where Hedestrand1 tried to demonstrate the effect of monol ayers on the evaporation rate of water on which they were spread. In 1943, Langmuir and Vincent Schaefer100 measured evaporation rates of water by suspending, over the surface of water in a film balance (Figure 4-1), a flat container with a permeable bottom supporting a solid dessican t (calcium chloride). Also, in our laboratory we have shown the importance of molecular packing achieved by using mixed surfactant monolayers and pKa of fatty acids in reduction of evaporation of water. The maximum reduction in evaporation using monomolecular film of surfactants was around 50%. However, the problem with monolayers is that they can be easily re moved by winds and also they undergo thermal and biodegradation easily.114 Under such circumstances multimol ecular films of oil and surfactants could be ideal. Heymann and co-workers115, 116 in the 1940s had used multimolecular films of oil as a means of preventing or reducing th e evaporation of water from e xposed water surfaces in arid climates. Using such multimolecular films of paraffin oil and neutral oil from vertical retort tar, they showed a significant reducti on in evaporation of water. However, other than that not much literature is found in this area to the best of our knowledge. In our research, we found one such surfactant, Brij-93, after scr eening various surfactants which can mix with hexadecane and uniformly spread on water surf ace and effectively reduce the ev aporation. The effect of temperature, various polymeric additives, volume of the duplex film deposited and concentration of surfactant in the mixture was studied. The st ructure of the film at the interface was studied using Brewster Angle Microscopy.

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117 4.2 Methods and Materials 4.2.1 Materials The surfactant Brij-93 (HLB 4.9) or Polyoxyeth ylene (2) Oleyl Ether was supplied by ICI, Speciality Chemicals, (Wilmington, DE). Stearic acid, Stearyl alcohol, Poly Vinyl alcohol, Bovine Serine Albumin was purchased from Sigma Aldrich. Hexadecane and anhydrous Calcium Chloride was from Fisher Scientific, (Fair Lawn, NJ). Artificial tear solutions were purchased from local Walmart pharmacy. Water used in all the experiment was de-ionized water, unless specifically mentioned. All experiments were carried out at 23 1 C unless otherwise stated. 4.2.2 Spreading of Duplex Film All the experiments were carried on Langmuir film balance (Figure 4-2), having trough of dimensions lxbxh=25x12x1.25 cm3. The trough was f illed with 385ml of water each time and the evaporation measurements were carried out on pure water surface first, before spreading of the duplex film. The duplex film was then depos ited on top of water by putting some drops of solution (oil+surfactant) using microsyringe on various areas of the trough. Depending on the volume deposited, the average thickness of the film ranged from 4 to 10 micron in thickness. For the monolayer studies: 1% solution of compound is made in 1:1:3 mixtures of chloroform, methanol and hexane. 4.2.3 Evaporation Studies A known amount of anhydrous calcium chloride wa s measured and placed in a petri dish. The petri dish was then covered with nylon cl oth, inverted, and placed 3 mm above the surface of a water or water covered with film. The rate of evaporation was measured by the increase in weight of the desiccant, anhydrous CaCl2. The change in desiccant weight was measured every 20 min over a period of 1 hour. To reduce the contribu tion of air, i.e. moisture of air absorbed by

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118 CaCl2, the Langmuir trough is covered with aluminum foil and increase in weight of the desiccant (CaCl2) was measured every 20 min over a period of 1 hour. This is then subtracted from earlier measurement, to ge t the actual value of water ev aporated from the surface. 4.2.4 Brewster Angle Microscopy Studies The Brewster angle microscope used wa s a BAMl from Nanofilm Technologie Gmbh (Gottingen, Germany). A He-Ne laser (p-polarized ) was used as the light source. The angle of incidence was initially set to 53' and then adjusted to minimize the reflected intensity of the clean water surface prior to spreading of each film. All photos presente d were taken with a rotatable analyzer set at 90' placed in between the refl ected signal and the CCD camera. A frame-grabbing program (Videopix, Sun Microsystems) was used to generate photos from videotape and convert them to a computer-readable format. 4.3 Results and Discussion Evaporation experiments on these films are done using Langmuir film balance. We have studied the effect of following factors on the rate of evaporati on for duplex film of Brij 93 and hexadecane. Comparison with Monolayers Thickness of the Film Deposited Concentration of Surfactant Various Polymeric Additives Stability of the Film Structure of this film usi ng Brewster Angle Microscopy Figure 4-3 shows the comparison of duplex film of Brij 93 and Hexadecane with monolayers of Stearic acid and St earyl alcohol. Stearic acid monolayer alone does not have significant effect in reduction in evaporation rate. However, when replaced by monolayer of Stearic reduction in evaporation by more than 40%. Duplex film of Brij93 in Hexadecane shows

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119 very high reduction of evaporation, this is due to high resistance provided by interfacial layer of surfactant at water/hexadecane interface as well as hexadecane/air interface. Figure 4-4 shows the effect of addition of duplex film of Brij-93 and hexadecane on retardation of evaporation. It can be seen that increase in addition of the surfactant plus oil mixture results in retardation of evaporation up to 85%. Not much significant evaporation reduction was observed above addition of 400 microliters or 16-micron thick film. Because the film is not uniform after 16 micron thickness, as the surfactant and oil combination form emulsion droplet rather than continuous film. The diffusion of water across hexadecane oil layer can be described in terms of the equation: dx d mcJ (4-1) Where, J is net flux/area, m is mobility of water in hexadecane, c is concentration and d/dx, the gradient of chemical potential, is the dr iving force. This equation is a general form of the Nernst-Planck equation. The chemical potential of water () can be expanded in terms of water vapor pressure (p). dx dp p c RTmJ (4-2) The diffusion coefficient (D) is defined by RTm, where R is ga s constant and T is absolute temperature. For a dilute solution, such as water in hexadecane, the ratio c/p is constant and is termed a solubility coefficient (). Then: dx dp DJ (4-3) Integrating across th e hexadecane layer: p D J (4-4)

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120 Where, is the thickness of the layer. If water is in equilibrium across the interfaces on either side of the hexadecane layer, p is the difference in water vapour pressure between the media on either side of the hexadecane layer. As, we see from equation 4-4 water flux is inversely proportional to the thickness of the film, because of which we will see less and less differential decrease in water evap oration rates as we increase the thickness of the film (Figure 44). Figure 4-5 shows that increase in concentrati on of Brij-93 in hexadecane the retardation of evaporation increases first (till Brij93 conc. 0.025%) after which it remain constant. This can be explained as due to incomplete surface coverage of the oil film on wa ter surface at surfactant concentration lower than 0.025%, further addition of surfactant result in formation of oil in water emulsion. This can be further confirmed using our Brewster Angle Micr oscopy results wherein one finds clear pattern initially ending up into circular emulsion droplet (Figure 4-6 (c)). Figure 4-7 shows the effect of different polym ers on retardation of water evaporation. 100 microliters of 1 wt. % polymer were first de posited on water trough followed by addition of 100 microliters of duplex film of Brij-93 and hexade cane. Addition of 100 micr oliters of duplex film on top of a Polyvinyl alcohol (PVA, MW-10,000) film in water reta rds evaporation by more than 80 % much more than the duplex film of hexadecan e + Brij-93 alone (60%). This is due to tight packing of polyvinyl alcohol at th e interface between surfactant and oil layer. We further studied the effect of polymers such as Bovine Seru m Albumin, Hydroxyethyl cellulose, Hydroxypropyl cellulose, Carboxymethyl cellulose, but none of th em show retardation of evaporation. Which further underscore the fact that Polyvinyl alc ohol being very surface active polymer crowd the water/hexadecane much more than with the surf actant alone, and thus reducing the effective evaporation of water.

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121 This duplex film is very elastic as seen in Figure 4-8 shows the effect compression/expansion and aging of duplex film on water evaporati on. It can be seen deduced that this duplex film (Brij93 + Hexadecane) is ve ry effective in reducing the evaporation of water after one day. These duplex films are much is mu ch more stable than other films, as well as compression/expansion does not reduce it s evaporation reduction significantly. Figure 4-8 shows how commercially available tear solutions reduces water evaporation compared to duplex film. As we see from the gr aph they do not reduce an y water evaporation as compared to duplex film. This proves to the possibil ity that if we can able to find this type of duplex films in sub-micron size range we would be able to apply them to solve the problem of Dry Eye. 4.4 Conclusion Brij 93 in Hexadecane spreads uniformly on th e water surface forming a Duplex film. Duplex film reduces water ev aporation much more in comp arison to long chain acids and alcohols. Rate of evaporation of water can be suppressed to as much as 80%, by increasing film thickness. Duplex film is elas tic i.e. continuous expansion or compression of the film in a manner akin to the blinking of the eye does not decrease the evaporati on reduction efficiency. Addition of surface active polymer PVA further decreases the evaporation rate of water, whereas other polymers do not reduce evaporation rate. This oil/surfacta nt combination can form an emulsion with water which can be used to re duce evaporation of water. Brewster Angle Microscopy pictures shows that surface structure of the film changes by varying surfactant concentration, volume etc.

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122 Figure 4-1. Langmuir film balance. Figure 4-2. Evaporatio n experiment setup. Langmuir Trough Plexiglas Plate Desiccant, CaCl2 Multilayer-films Top View Cross Compression Barrier Monolayer { } 3mm

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123 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0102030405060 Time (Min)Water Evaporation (gm/cm2)Stearic Acid Stearyl Alcohol Pure Water Brij 93 (0.2%) + Hexadecane Desiccant CaCl2 Volume Deposited 100microliters A rea 250 cm2, RH 60% Figure 4-3. Comparison of wa ter evaporation reduction by d uplex film and monolayers. 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0102030405060Time (Min)Water Evaporation (gm/cm2) Pure Water 4 Micron 8 Micron 12 Micron 16 Micron Brij93 (0.2%)+ Hexadecane Desiccant CaCl2 RH 60% Figure 4-4. Effect of film thickness on evaporation.

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124 0 10 20 30 40 50 60 70 00.20.40.60.81 Conc. of Brij 93 in Hexadecane, %W/W% Reduction in Evaporation Desiccant CaCl2 Thickness of Film 4 Micron RH 60%, Time 1 hrFigure 4-5. Effect of co ncentration of Brij 93 in hexadecane on evaporation. Figure 4-6. Brewster Angle Microscopy images of duplex film of hexadecane and Brij-93. A) 0.2 wt.% Brij-93 in hexadecane. B) 0.5 wt .% Brij-93 in hexadecane. C) 0.8 wt.% Brij-93 in hexadecane. 0.2 mm AB C 0.2 mm AB C

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125 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 02 04 06 0 Time (Min)Water Evaporation (gm/cm2) Pure Water Brij 93 + Hexadecane Brij 93 + Hexadecane + PVA Brij 93 + Hexadecane + BSAPolymer (PVA, BSA) -1% Solution in Water (0.1ml) Film Thickness -4 Micron Desiccant CaCl2, RH 60% PVA (MW)--10000g/mol Brij 93 (0.2%) in Hexadecane Figure 4-7. Effect of vari ous polymers on evaporation. 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 02 04 06 0 Time (Min)Water Evaporation(gm/cm2) Pure Water Hexadecane + Brij 93 Hexadecane + Brij 93 (After One Day) Hexadecane + Brij 93 (After one hour of compression/expansion @ 10 times per minute) Desiccant CaCl2 Conc. of Brij 93 in Hexadecane = 0.2% Thickness of Film 4 Micron RH 55% Figure 4-8. Effect of aging and compression/expansion of film on evaporation.

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126 0 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0102030405060 Time (Min)Water Evaporation(gm/cm2) Pure Water Refresh Tears Tears Naturale Visine Moisture Eyes Thera Tears Refresh Endura Brij 93 + Hexadecane Pure WaterDesiccant CaCl2 Volume Deposited 100 Micoliters A rea 250 cm2 RH 55%Brij 93(0.2%) in Hexadecane Figure 4-9. Effect of films of various commercial products on water evaporation.

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127 CHAPTER 5 SUMMARY AND RECOMMENDATIONS FOR FUTURE WORK 5.1 Propofol Microemulsions for Anesthesia 5.1.1 Summary Microemulsions offer a potential alternative carrier system for drug delivery because of their high solubilization capacity, transparency, thermodynamic stability, ease of preparation, and high diffusion and absorption rates. A number of factors must be considered when using microemulsions as drug delivery vehicle. First, th e appropriate type of oil and surfactant must be used in order to dissolve and protect the drug. The surfactant can be nonionic, or anionic (in combination of non-ionic). The type of drug diss olved and conditions of the target site will dictate the type of surfactant used to carry the drug. Second, the concentrati on of surfactant largely influences the carrier system, because this surfactant concen tration will ultimately determine drug delivery effectivenes s. Last, the conditions of the ta rget site are critical to the effectiveness of a surfactant delivery system. Para meters such as temperature, pH will influence the solubility of a drug inside the microemulsion system. Although o/w microemulsions are promising so lvent systems for drug delivery, compared to other drug delivery methods, their potential has not been reached. There are several barriers that need to be overcome before this technology can be used in practice. The main challenge is the control of drug diffusion and partitioning between the dispersed and continuous phases present. Another factor influenci ng the use of microemulsions is the biological tolerance of the constituents of microemulsions, particularly th e surfactants and cosurfact ants, because they can cause disruptions in biological membranes. A high surfactant concentrat ion in the body over a long period of time may disturb so me bodily processes. Also, co surfactants like medium chain length alcohols are skin or eye irritants, so th eir use in topical drug delivery systems is limited.

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128 Nonionic surfactants such as ethoxylated alkyl ch ain ethers and sobitan easters, as well as nonionic block copolymers are gene rally less toxic than ionic surfactants. Also, many nonionic surfactants have an advantage over charged surfac tants, as they can form microemulsions even without cosurfactants. The need for a suitable alternative formulati on is evidenced by past and ongoing research into other types of propofol delivery systems. In this thesis, we have demonstrated that microemulsion methods represent another suitable technology to deliver propofol intravenously with anesthetic parameters similar to the comm ercially available macroemulsions. Fewer drugs have been delivered using an intravenous rout e. Unlike other oil-in-water microemulsions wherein the active drug is dissolved in an oil exci pient, in the present st udy propofol acted in two different, complementary roles in the present investig ation. That is, the need for an excipients oil for propofol dispersal was obviated by the fact that propofol is itself an oil at room and physiological temperatures. Therefore, pr opofol could serve not only as the active pharmacodynamic agent, but also could exist as the physical platform for the preparation of these microemulsions. By doing so, the need for additional excipients oils (e.g., soybean or castor bean oil) is eliminated along with the potential for these excipients to nourish bacteria. The kinetics of general anesthesia with the propofol micr oemulsion was favorable compared to the commercially available propofol macroemulsion. Whereas the time for general anesthesia (defined by the protocol as loss of leg withdraw al to a pinch) during macroemulsion injection was 122-126 s, the latency periods during microe mulsion treatment were 166-203 s. This short delay is favorable when compared to prodrug technologies that rely on metabolism, a phenomenon that may vary significan tly within any patient population.147 The differences in induction times between experimental groups reporte d herein is caused by differential release of

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129 propofol from the individual drople ts into the blood. That is, the different propofol nanoparticles have markedly different stabilities against dilu tion by blood based on the emulsifier structure and concentration selected for the formulation. In general, a microemulsion is thermodynamically stable at equilibrium. But, one can destabilize a microemulsion by significantly changing pressure, temperature, or chemical compositions The last variable can be changed simply by diluting a microemulsion with saline or blood. It is well recognized that the formation of microemulsions requires an ultra low interfacial tension (e.g., ~10-3 mNewton/m) at the oil/water interface. Upon dilution with sali ne, the interfacial tension will increase substantially as the emulsifier molecules (i.e., both pl uronic 68 and fatty acid salt molecules) desorb from the droplet surface. This event will markedly increase the interfacial tensi on at the droplet surface and ultimately destabilize the microemulsion, with release of the active pharmaceutical core (i.e., propofol). However, each emulsifier film around the microemulsion droplet has inherent molecular packing and, hence, stability. The extent of dilution re quired to destabilize a microemulsion represents its inhe rent stability. Thus, a microemu lsion requiring greater dilution for destabilization indicates that its emulsifier film has a greater stability. C12 fatty acid salt microemulsions are the most stab le as they require the greatest dilution to become a turbid macroemulsion. It is likely; however, that microe mulsion destabilization is affected by more than just dilution in vivo as the surfact ant can go to various other places in vivo dilution than in vitro. Further understanding and selec tively modifying these destab ilization rates by use adapting surfactant type and concen trations alludes to the possibility of controlling release times of active pharmaceuticals from nearly immediate (e.g., propof ol) to longer times (e.g., chemotherapeutic or antifungal agents).

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130 One benefit of slightly delayed propofol releas e due to these differential stabilities may be reduced pain on injection. That is, we hypothesize that the concentration of aqueous propofol in peripheral veins will be sufficiently low so that minimal-to-no pain will be experienced during induction. If the relaxation rate is sufficiently long to ensure na noparticle integrity from the time of injection in a peripheral vein to the time they enter the central circulation (e.g., 10-15 s), then no pain should be caused during injection. In dog study, we investigat ed the dose of propofol, mi croor macroemulsions, administered to dogs to induce anesthesia, along with possible effects on blood, the first tissue encountered by the formulations. The measured va riables of anesthesia, vital signs, indices of blood cell populations or thrombosis, and plasma concentrations of propof ol did not significantly differ between the groups of animals given either the propofol microemulsion or macroemulsion. In these studies, all animals experienced rapid anesthesia as a ssessed by loss of leg withdrawal with mean propofol doses of approximately 10 mg/kg for both the microemulsion and macroemulsion. This dose of propofol is greater than that previously re ported in other studies investigating anesthetic induc tion in unpremedicated dogs usi ng propofol formulated in a macroemulsion. In the end, the use of microemulsions as drug delivery vehicle is an exciting and attractive area of research, offering not only chal lenges to be overcome but also many potential extraordinary benefits. 5.1.2 Future Work 1. Development of non-ionic microemulsion systems: As already discussed ionic surfactants (even anionic) have very low accep tability limits in body as higher irritancy as well as cell response mechanisms. Whereas non-ionic systems can be used in high concentration without thes e side effects. So, non-ionic systems particularly Tween/PEG microemulsion system should be pursued for higher animal models for their efficacy for drug delivery. 2. Franz diffusion cell experiments for skin ir ritation: In Franz cell model, penetration studies are conducted to measure the rate a nd extent of penetration and absorption of

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131 drug as well as surfactants into the skin (a nd various cell surfaces). From such studies, the optimal surfactant, co-sur factant combination can be se lected and issues regarding penetration can be identified and resolved be fore costly clinical studies are embarked upon. 3. Pharmacokinetics and pharmacodynamics model for propofol system: Even though after 25 years of use of propofol we still dont know the exact m echanism of action of propofol. We need to develop a model for how the drug and surfactants included in the formulation affect the various system s in body. An analytical model of pharmacokinetics and pharmacodynamics is th erefore required to minimize the animal experiments for efficacy studies. 4. Pharmacodynamics and pharmacokinetics in higher animal system like Pig: Once optimum system is defined in vitro, pharmacokinetics as well as pharmacodynamics studies needs to be done in bigger animal model like pigs. These studies will help us determine what various drug/surfactant doe s to the body as well as what body does to them. 5. Extension of this work on the water insol uble drugs: As new water insoluble drugs are being discovered, these microemulsion system s can be developed as a fingerprint of delivery system, based on solubility, stability, partition coefficient and various other process parameters. Few of the drugs for example can be rapamycin, geldanamycin, and various other water insoluble protein drugs. 6. Effect of various excipients on the perf ormance of microemulsions: As we know pharmaceutical formulations consist of various excipients like antimicrobial agents, fillers, disintegrants, lubricants, glidants a nd binders. They are required for stability of drug and formulation, to stop bacterial gr owth, to ensure easy disintegration, disaggregation, and dissolution etc. So, it is necessary to study the effect over various excipients as mentioned above on the pharmaceutical performance of microemulsions made in a system containing all required excipients both in vivo and in vitro. 5.2 Reduction of Water Evaporation 5.2.1 Summary Reduction of water evaporation directly touches many aspects of our lives. Knowledge, understanding of various methods to reduce wate r evaporation has revol utionary potential. A multimolecular (duplex) film of oil and surfactants is one of the approaches to reduce evaporation of water, can be a potential solution to this problem. Mixture of Brij-93 and hexadecane forms a duplex film, which uniformly spreads on surface of water and retards evapor ation of water. Increase in deposited volume of Brij-93 and

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132 hexadecane reduces the evaporation rate and th e maximum retardation in evaporation achieved was 80%. Corresponding changes in surface structur e of duplex film due to increase in Brij-93 concentration were seen using Brewster Angle Microscope. Addition of polymer (Polyvinyl alcohol) was found to improve the retardation effici ency of the duplex film as compared to other polymeric additives. 5.2.2 Future Work 1. To find different kind of systems: Determin e the different kind of surfactant and oil systems, which can spread on the water surf ace and form a duplex film. The work will include try using natural biode gradable long chain hydrophobic oils with different kind of superspreading surfactants. These surfactants decrease the interfaci al tension at the oil/water interface to very low value a nd helps in spreading the duplex film. 2. Determine different kind of mixed surfact ant monolayers, which reduce the rate of evaporation of water. In this case we want very compact interface so that through which water permeation rate is very slow, so we w ill try to form a very compact monolayer of surfactant. Especially mixed monolayers of Stear ic acid, Oleic acid, Caster oil, Olive oil etc., with combination w ith long chain alcohols. 3. Effect of these films on perm eation of gases: To study the rate of oxygen, carbon dioxide and other gases through these films, as c ontinuous flow of oxyge n/carbon dioxide is necessary for aquatic life and on the eye surface where oxygen is needed by top epithelium cell of the eye surface. So a conti nuous flow of oxygen, which is required by the epithelium cells, and carbon dioxide, whic h is excreted by the cells, is required for maintaining the normal vision. 4. Different kind of efficiency experiments on different systems of duplex film and mixed surfactant monolayers found in the first part, like surface structure behavior, film balance behavior, surface rheological properties to ch aracterize these kinds of films. Various effects like lubrication efficacy, viscosity, and interaction with contact lenses of this film needs to be investigated. Following that, be st surfactant and oil systems need to be identified which can directly spread on the surface and reduce water evaporation. These systems can be used directly to reduce eva poration of water or if incorporated in emulsion, it can be used in artificial tear so lutions to reduce evapor ation of water from tear film. 5. Identify the optimum systems weather monolayer or duplex film which reduces evaporation in vitro studies (film studies). De termine different properties of these films, and characterize these films. Determine m echanism by which these films spread and reduce the evaporation of water.

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133 6. Incorporate the best duplex film/monolay er system as nanoemulsion in aqueous formulations and test its efficacy in e fficiently reducing the evaporation rate.

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134 LIST OF REFERENCES 1. Hedestrand, G., On the influence of thin surface films on the evaporation of water. Journal of Physical Chemistry 1924, 28, 1245-1252. 2. Rideal, E. K., The influence of thin surface films on the evaporation of water. Journal of Physical Chemistry 1925, 29, 1585-1588. 3. Rosen, M. J., Surfactants and Interfacial Phenomena. 3rd ed.; John Wiley & Sons: Hoboken, New Jersey, 2004. 4. McBain, J. W.; Salmon, C. S., Colloidal electrolytes. Soap solutions and their constitution. J. Am. Chem. Soc. 1920, 42, 426. 5. Hartley, G. S.; Collie, B.; Samis, C. S., Transport numbers of paraffin-chain salts in aqueous solution. I. Measurement of tr ansport numbers of cetylpyridinium and cetyltrimethylammonium bromides and their interpretation in terms of micelle formation, with some data also for cetanesulfonic acid. Trans. Faraday Soc. 1936, 32, 795-815. 6. Hartley, G. S., Aqueous Solutions of Paraffin-Chain Salts. Paris, 1936. 7. Becher, P., Emulsions: Theory and Practice. 2nd ed.; Reinhold Publishing Corporation: New York, 1965. 8. Cosgrove, T.; Phipps, J. S.; Richardson, R. M., Neutron reflection from a liquid/liquid interface. Colloids Surf. 1992, 62, (3), 199-206. 9. Torza, S.; Cox, R. G.; Mason, S. G., Electrohydrodynamic deformation and burst of liquid drops. Philos. Trans. Royal Soc. London Series A 1971, 269, (1198), 295. 10. Gormally, J.; Gettings, W. J.; Wyn-Jones, E., In Molecular Interactions, Ratajczak, H.; Orville-Thomas, W. J., Eds. Wile y: New York, 1980; Vol. 2, p 143. 11. Kabalnov, A. S.; Pertzov, A. V.; Shchuki n, E. D., Ostwald ripening in emulsions .1. Direct observations of Ostw ald ripening in emulsions. J. Colloid Interface Sci. 1987, 118, (2), 590-597. 12. Oh, S. G.; Jobalia, M.; Shah, D. O., The e ffect of micellar lifetime on the droplet size in emulsions. J. Colloid Interface Sci. 1993, 156, (2), 511-514. 13. Sutheim, G. M., Introduction to Emulsions. Chemical Publishing Co., Inc.: Brooklyn, N. Y., 1947. 14. Tadros, T.; Vincent, B., Emulsion Stability. In Encyclopedia of Emulsion Technology: Basic Theory, Becher, P., Ed. Marcel Dekker, Inc.: New York and Basel, 1983; Vol. 1, p 129. 15. Menon, V. B.; Wasan, D. T., Encyclopedia of Emulsion Technology. Marcel Dekker, Inc: New York, 1983; Vol. 2.

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135 16. Barreleiro, P. C. A.; Olofsson, G.; Brown, W.; Edwards, K.; Bonassi, N. M.; Feitosa, E., Interaction of octaethylene glycol n-dodecyl monoether with dioctadecyldimethylammonium bromide and chloride vesicles. Langmuir 2002, 18, (4), 1024-1029. 17. Wu, X. Y.; Pelton, R. H.; Tam, K. C. ; Woods, D. R.; Hamielec, A. E., Poly(NIsopropylacrylamide) .1. Interact ions with sodium dodecyl-sulfate measured by conductivity. J. Polym. Sci., Part A: Polym. Chem. 1993, 31, (4), 957-962. 18. Liem, A. J. S.; Woods, D. R., Review of Coalescence Phenomena. 1974; Vol. 70. 19. Ross, S., Emulsion control. J. Soc. Cosmetic Chem. 1955, 6, 184-192. 20. Ostwald, W., Emulsions. Wilmersdorf. Z. Chem. Ind. Kolloide 1910, 8, 103-109. 21. Pickering, S. U., Emulsions. J. Chem. Soc. Faraday Trans. 1907, 91, 2001-2021. 22. Shinoda, K.; Nakagawa, T., Colloidal Surfactants: Some Physicochemical Properties. Academic Press: New York, 1963. 23. Mange, F. E.; Buriks, R. S.; Fauke, A. R. Demulsification with ultrahigh-molecularweight polyoxiranes. 3617571, 19711102, 1971. 24. Webb, T. O. Use of micellar solution as a hydrocarbon-water emulsion \"breaker\". 3554289, 19710112, 1971. 25. Griffin, W. C., Calculation of HLB values of non-ionic Surfactants. J. Soc. Cosmetic Chem. 1954, 5, 249-355. 26. Davies, J. T.; Rideal, E. K., Interfacial Phenomena. 2nd ed.; Academic Press: New York and London, 1963. 27. Greenwald, H. L.; Brown, G. L.; Finema n, M. N., Determination of the hydrophilehipophile character of surface active agen ts and oils by a water titration. Anal. Chem. 1956, 28, (11), 1693-1697. 28. Racz, I.; Orban, E., Calorimetric dete rmination of hydrophile-lipophile balance of surface-active substances. J. Colloid Sci. 1965, 20, (2), 99. 29. Esumi, K.; Miyazaki, M.; Arai, T.; Koid e, Y., Mixed micellar properties of cationic gemini surfactants and a nonionic surfactant. Colloids Surf., A 1998, 135, (1-3), 117-122. 30. Bhatnagar, S. S., Studies in emulsions. I. A new method for determining the inversion of phases. J. Chem. Soc., Trans. 1920, 117, 542-552. 31. Aoki, K.; Hori, J.; Sakurai, K., Interaction between surface active ag ents and proteins .3. Precipitation curve of the system sodium dodecy l sulfate-egg albumin at various pHs and the determination of the concentration of pr otein by the titration using surfactant. Bull. Chem. Soc. Jpn. 1956, 29, (7), 758-761.

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136 32. Shimamoto, T., Studies on the emulsion I. Phase inversion by ho mogenizer processing. Jpn. J Pharmacol. 1962, 82, 1237-1240. 33. Attwood, D.; Florence, A. T., In Surfactant Systems, Chapman & Hall: London, 1983. 34. Matsumot, S.; Sherman, P., A DTA technique for identifying phase inversion temperature of O/W emulsions. J. Colloid Interface Sci. 1970, 33, (2), 294. 35. Lin, T. J.; Lambrechts, J. C., Effect of initial surfactant location on emulsion phase inversion. J. Soc. Cosmetic Chem. 1969, 20, (3), 185-198. 36. Sunderla, V.; Enever, R. P., Influence of formulation variables on phase inversion temperatures of emulsions as determined by a programmed viscometric technique. J. Pharm. Pharmacol. 1972, 24, (10), 804. 37. Lindman, B.; Friberg, S. E., MicroemulsionsA Historical Overview. In Handbook of Microemulsion Science and Technology, Kumar, P.; Mittal, K., Eds. Marcel Dekker, Inc: New York, 1999; p 1. 38. Schulman, J. H.; Stoeckenius, W.; Prince, L. M., Mechanism of formation and structure of micro emulsions by electron microscopy. J. Phys. Chem. 1959, 63, (10), 1677-1680. 39. Hoar, T. P.; Schulman, J. H., Transparent water-in-oil dispersions: the oleopathic hydromicelle. Nature (London, U. K.) 1943, 152, 102-103. 40. Bancroft, W. D., The theory of emulsification, V. J. Phys. Chem. 1913, 17, 501. 41. Bancroft, W. D., The theory of emulsification, VI. J. Phys. Chem. 1915, 19, 275. 42. Ruckenstein, E., Thermodynamic in sights on macroemulsion stability. Adv. Colloid Interface Sci. 1999, 79, (1), 59-76. 43. Walstra, P., Formation of Emulsions. In Encyclopedia of Emulsion Technology, Becher, P., Ed. Dekker: New York, 1983; Vol. 1, pp 57-128. 44. Kegel, W. K.; Overbeek, T. G.; Lekkerkerker, H. N. W., Thermodynamics of Microemulsions I. In Handbook of Microemulsion Science and Technology, Kumar, P.; Mittal, K., Eds. Marcel Dekker, Inc: New York, 1999; pp 13-44. 45. Myers, D., Surfaces, Interfaces, and Colloid s: Principles & Applications. 2nd ed.; John Wiley & Sons: New York, 1999; p 253-292. 46. Ruckenstein, E.; Chi, J. C ., Stability of microemulsions. J. Chem. Soc. Faraday Trans. II 1975, 71, 1690-1707. 47. Bowcott, J. E.; Schulman, J. H., Emulsions Control of droplet si ze and phase continuity in transparent oil-water dispersions stabilized with soap and alcohol. Zeitschrift Fur Elektrochemie 1955, 59, (4), 283-290.

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137 48. Cooke, C. E.; Schulman, J. H., Surface Chemistry. Munksgaard: Copenhagen, Denmark, 1965; p 231-235. 49. Sears, D. F.; Schulman, J. H., Influence of water structures on surface pressure surface potential + area of soap monolayers of lithium sodium potassium + calcium. J. Phys. Chem. 1964, 68, (12), 3529. 50. Zlochowe, I.; Schulman, J. H., A study of molecular interactions and mobility at liquid/liquid interfaces by NMR spectroscopy. J. Colloid Interface Sci. 1967, 24, (1), 115. 51. Somasundaran, P.; Lee, L. T., Polymer-surf actant interactions in flotation of quartz. Sep. Sci. Technol. 1981, 16, (10), 1475-1490. 52. Shah, D. O., Microemulsions and their Technological Applications. In Surfactants: Principles and Applications (Short Course on Surfactants), Gainesville, 2004; p Chapter 8. 53. Gerbacia, W.; Rosano, H. L., Microemulsions Formation and stabilization. J. Colloid Interface Sci. 1973, 44, (2), 242-248. 54. Gelbart, W. M.; Ben-Shaul, A.; Roux, D., Micelles, Membranes, Microemulsions and Monolayers. Springer-Verlag: New York, 1994. 55. Bellocq, A. M., Handbook of Microemulsion Science and Technology. Marcel Dekker, Inc.: New York, 1999; p 139-184. 56. Shinoda, K.; Friberg, S., Emulsions and Solubilization. Wiley: New York, 1986. 57. Shiao, S. Y.; Chhabra, V.; Patist, A.; Free, M. L.; Huibers, P. D. T.; Gregory, A.; Patel, S.; Shah, D. O., Chain length compatibility eff ects in mixed surfactant systems for technological applications. Adv. Colloid Interface Sci. 1998, 74, 1-29. 58. Brooks, J. T.; Cates, M. E., The role of added polymer in dilute lamellar surfactant phases. J. Chem. Phys. 1993, 99, (7), 5467-5480. 59. Pillai, V.; Kanicky, J. R.; Shah, D. O., A pplications of Microemulsions in Enhanced Oil Recovery. In Handbook of Microemulsion Science and Technology, Kumar, P.; Mittal, K. L., Eds. Marcel Dekker Inc.: New York, 1999; pp 743-754. 60. Malmsten, M., Microemulsions in Pharmaceuticals. In Handbook of Microemulsion Science and Technology, Kumar, P.; Mittal, K. L., Eds. Ma rcel Dekker, Inc: New York, 1999; pp 755-772. 61. Myers, D., Physical Properties of Surfactants Used in Cosmetics. In Surfactants in Cosmetics, Rieger, M. M.; Rhein, L. D., Eds. Marc el Dekker, Inc.: New York, 1985; Vol. 68, pp 29-82.

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148 BIOGRAPHICAL SKETCH Dushyant Shekhawat was born in S ardargarh, Rajasthan, India. He completed his high school from Kendriya Vidyalaya, Kota, Rajasthan. Later on, he jo ined the prestigious Indian Institute of Technology (IIT), Bombay from wher e he graduated with a bachelors degree in chemical engineering in 2002. Here, at IIT, he was greatly inspired by his Professors (Dr. Jayesh Bellare, Dr. K. C. Khilar) and decided to pursue a Ph.D. degree in US universities. Of the several graduate school offers, he decide d to join University of Florida owing to Dr. Dinesh O. Shahs fame in colloid and interface science area. In January 2003, Dushyant joined Professor Shahs group and completed his degree under his supervision in December of 2007.