FORMULATION OF AN ANTI-TUBERCULOSIS DRUG DELIVERY SYSTEM LISA CLAIRE DU TOIT A dissertation submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfillment of the requirements for the degree of Master of Pharmacy Supervisor: Professor Michael Paul Danckwerts Department of Pharmacy and Pharmacology, University of the Witwatersrand, South Africa Co-Supervisor: Professor Viness Pillay Department of Pharmacy and Pharmacology, University of the Witwatersrand, South Africa Johannesburg, 2007 ii DECLARATION I, Lisa Claire du Toit, declare that this dissertation is my own work. It has being submitted for the degree of Master of Pharmacy in the Faculty of Health Sciences in the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University. ?????????????. This ??.. day of February 2007 iii RESEARCH OUTPUTS 1. Research Publications a. Lisa Claire du Toit, Viness Pillay and Michael Paul Danckwerts. Application of Synergism and Variation in Ionic Compatibilities within a Hydrophilic Polymeric Sodium Starch Glycolate-?-Carrageenan Combination: Textural Profiling of the Suspension Behavior. Journal of Bioactive and Compatible Polymers. 2006, 21: 107- 122. b. Lisa Claire du Toit, Viness Pillay and Michael Paul Danckwerts. Tuberculosis Chemotherapy: Current Drug Delivery Approaches. Respiratory Research. 2006, 7: 118. 2. Conference Outputs a. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. Formulation of an Anti-Tuberculosis Drug Delivery System: Microencapsulation of Isoniazid. Young Scientist Competition Abstract Entrant Presented at 26th Annual Conference of the Academy of Pharmaceutical Sciences, 29 September ? 2 October 2005, Port Elizabeth. b. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. A Novel Salting-Out Approach for the Formulation of Cross-linked Enterospheres Incorporating Isoniazid. Poster Presented at 33rd Annual Meeting & Exposition of the Controlled Release Society, 22-26 July 2006, Vienna, Austria. c. Lisa Claire du Toit, Viness Pillay and Michael Paul Danckwerts. Textural Characterization of Synergism in Hydrophilic Polymeric Sodium Starch Glycolate- kappa-Carrageenan Sol and Gel Systems. Poster Presented at 33rd Annual Meeting & Exposition of the Controlled Release Society, 22-26 July 2006, Vienna, Austria. iv d. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. Formulation of an Anti-Tuberculosis Site-Specific Drug Delivery System: A Comparative Study of Two Methods for Enteric-Coating Isoniazid Particles. Poster Presented at 26th Annual Conference of the Academy of Pharmaceutical Sciences, 29 September ? 2 October 2005, Port Elizabeth. e. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. Evaluation of Novelly-Formed Nano- and Entero-spheres for Targeted Delivery of an Anti- Tuberculosis Drug. Podium Presentation at ICPPS Conference 2006, Vanderbijlpark, South Africa. f. Lisa Claire du Toit, Viness Pillay, Michael Paul Danckwerts, Craig Cremen, Ismail Ravat, Jayendran Subramoney and Harshen Vassan. Approaches to Fabricating Anti- TB Nanosystems Embodying a Salted-Out and Cross-linked Architecture. Poster Presented at ICPPS Conference 2006, Vanderbijlpark, South Africa. g. Lisa Claire du Toit, Viness Pillay, Michael Paul Danckwerts, Craig Cremen, Ismail Ravat, Jayendran Subramoney and Harshen Vassan. Preliminary production of enteronanoparticles based on a salted-out and cross-linked architecture. Poster Presented at the American Association of Pharmaceutical Scientists Annual Meeting and Exposition Conference, October 2006, Texas, USA. 3. Additional Research Outputs a. Lisa Claire du Toit, Oluwutoyin Kolawole, Viness Pillay and Michael Paul Danckwerts. The Wits Approach: A Bioactive Research Network (Article), South African Pharmacy Journal, October 2005. b. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. Formulation of an Anti-Tuberculosis Site-Specific Drug Delivery System. Podium Presentation at School v of Therapeutic Sciences Research Afternoons, University of the Witwatersrand, 10-11 August 2005. c. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. Formulation of an Anti-Tuberculosis Site-Specific Drug Delivery System: A Comparative Study of Two Methods for Enteric-Coating Isoniazid Particles. Podium Presentation at Faculty Reseach Day, University of the Witwatersrand, 23 August 2006. d. Lisa Claire du Toit, Michael Paul Danckwerts and Viness Pillay. Formulation of an Anti-Tuberculosis Drug Delivery System. Podium Presentation at the 1st Symposium for Biomaterials Science and Applications, University of the Witwatersrand, 4 September 2006. vi ABSTRACT Tuberculosis (TB) is a leading killer of young adults worldwide and the global scourge of multi-drug resistant tuberculosis is reaching epidemic proportions. A number of novel drug delivery systems incorporating the principle anti-tuberculosis (anti-TB) agents have been fabricated that either target the site of TB infection or reduce the dosing frequency with the aim of improving patient outcomes; however, there is a requisite to manufacture an oral system, which directly addresses issues of unacceptable rifampicin (RIF) bioavailability recently reported in a number of fixed-dose combinations (FDCs). There is an urgent need to segregate the delivery of RIF and isoniazid (INH) upon co-administration, such that INH is not released in the stomach owing to the induction of accelerated hydrolysis of RIF in acidic medium to the poorly absorbed insoluble 3-formyl rifamycin SV in the presence of INH. The fabrication of a polymeric once-daily oral multiparticulate fixed-dose combination of the principal anti-TB drugs, which attains segregated delivery of RIF and INH for improved RIF bioavailability, could be a step in the right direction in addressing issues of treatment failure due to administration of poor quality FDCs and patient non-compliance. Novel approaches were implemented for the fabrication of an oral multiparticulate system for differentiated release of RIF and INH in the gastrointesinal tract. The envisaged system comprised INH-loaded enterosoluble multiparticulate entities for targeted delivery of the INH to the small intestine and reconstitutable multiparticulate entities incorporating the poorly water-soluble RIF and appropriate gel-forming hydrophilic suspending agents, which were required to disintegrate rapidly in tepid water to form a gel network suspending RIF and the INH-loaded enterosoluble multiparticulates. The dry dispersible multiparticulate system may be reconstituted immediately prior to administration to the patient for once-daily dosing as a compliance- promoting tool. The design of a novel anti-TB drug delivery system hinged on preformulatory investigations and preliminary experimental activities to yield a sufficient database to allow for the selection of the qualitative composition of a prototype formulation. The aforementioned activities initiated the systematic identification of an innovative method for formulating enterosoluble multiparticulates demonstrating the required enteric-release properties. The novelly-formed multiparticulates, referred to as ?enterospheres?, were obtained by inducing separation (?salting-out?) of a pH-sensitive poly (methacrylic acid-co-ethylacrylate) copolymer as a polymer-rich enteric film and ionotropically cross-linking the internal enterosphere matrix. Rational selection of appropriate suspending agents for design of reconstitutable multiparticulates resolved in the identification of a synergistic hydrophilic sodium starch glycolate-kappa carrageenan combination, duly characterised by physicomechanical analyses. The gel-forming composite system attained ease of dispersal and the formation of a three-dimensional supporting network possessing the essential properties for extemporaneous use. Statistical experimental design, implementing response surface methodology, was pivotally instituted on the multiparticulate forms for the identification of critical formulation and processing variables for the development of the optimum enterosoluble and reconstitutable multiparticulate systems for delivery to the patient as the preferred multiparticulate two-drug FDC. Because there was an unequivocal relationship between the properties of a cross-linked enterospheres and their structure in such a way that both characteristics could not be considered in an isolated way, in-depth analyses on drug-free and drug-loaded enterospheres was systematically undertaken. Of principle concern in this study was the attainment of segregated gastrointestinal delivery of RIF and INH in order to address issues of unacceptable RIF bioavailability on co-administration with INH. The proposed United States Pharmacopoeial (USP) high performance liquid chromatographic (HPLC) and colorimetric method, and a proposed regressional analysis of ultraviolet (UV) spectrophotometric absorbance data were employed to resolve RIF and INH release from the optimum multiparticulate system at simulated gastric pH for comparison with the release profiles of anti-TB FDCs commercially available in South Africa. Ultimately, in keeping up to speed with future trends, this dissertation addressed innovations in nanotechnology, with particular reference to anti-TB nanosystems. The novelly identified method for enterosphere manufacture was adapted with a view to nanosizing the salted-out and cross-linked architecture, for controlled delivery of anti-TB drugs to the patient, in the bid to promote patient adherence. 7 ACKNOWLEDEMENTS In completing this work, the author would like to express her deepest gratitude to all who have offered their much-needed aid and support towards the completion of this study. The author would like to acknowledge the following people for their great contribution: ? Foremost to my parents, sister and grandparents for their endearing love and support, and for lifting my spirits when times were tough. ? To my supervisor, Prof. Michael Paul Danckwerts, for his knowledge and encouragement. ? To my co-supervisor, Prof. Viness Pillay, for his inspiring commitment to the field; thirst for knowledge, which is permeating; and for his constant support. ? To the students and staff of the Department of Pharmacy and Pharmacology, University of the Witwatersrand: their contributions, great and small, have aided me in numerous ways. ? To Dr. Clem Penny, for his advisory support with the scanning electron microscopy. ? This research would also not have been possible without the financial assistance of the Medical Research Council (MRC) and the National Research Foundation (NRF) of South Africa, their support is greatly appreciated. ? To the Almighty Father, without Whom none of this would be possible. 8 DEDICATION This work is dedicated to my nephew, Liam Matthew du Toit, an unexpected, yet most wonderful gift. This dissertation is also dedicated to my country, South Africa. 9 TABLE OF CONTENTS Page 1. Introduction 1.1. Tuberculosis ? Delineating the Disease 1 1.2. Current Anti-tuberculosis Chemotherapy 5 1.3. Novel Drug Delivery Systems for the Treatment of TB 13 1.4. Statement of the Problem 19 1.5. Approach to the Problem 23 1.6. Aims and Objectives: Description of the Oral Multiparticulate Drug Delivery System 27 1.7. Overview of the Dissertation 32 2. Theoretical Considerations for the Design of a Dispersible System Incorporating the Enterosoluble and Dispersible Multiparticulate Entities 2.1. Introduction 36 2.2. Site-Specific Drug Delivery of Multiparticulate Dosage Forms 37 2.3. Selection of an Appropriate Enteric Polymer 39 2.4. Gastrointestinal Anatomy and Physiology Relative to Enteric-Coating Functioning and Design Rationale 45 2.4.1. The Significance of pH 46 2.4.2. Gastric Motility, Emptying and Residence Time 47 2.4.3. Considerations for Drug Release Testing and Development of an Isoniazid Assay Method 50 10 2.5. Theoretical Design of an Enterosoluble Multiparticulate System 51 2.5.1. Microencapsulation Technology 51 2.5.2. Fabrication of Enterosoluble Multiparticulates by the Air Suspension Method 52 2.5.3. Fabrication of Enterosoluble Multiparticulates by Solvent Evaporation Emulsification 55 2.5.4. Fabrication of Enterosoluble Multiparticulates Employing the Principles of Phase Separation (?Salting-Out?) 56 2.6. Delivery of the Enterosoluble Multiparticulates 57 2.6.1. Rationalising Dispersible Multiparticulates as a Dosage Form 57 2.6.2. Ability of a Suspending Agent to Form an Extemporaneous Gel in Tepid Water: Formulation Considerations 58 2.7. Concluding Remarks 62 3. Preliminary Design of an Enterosoluble Multiparticulate System Incorporating Isoniazid 3.1. Introduction 63 3.2. Development of a Methodology for the Fabrication of Enterogranules by the Air Suspension Method 64 3.2.1. Materials and Methods 64 3.2.1.1. Materials 64 3.2.1.2. Equipment 64 3.2.1.3. Identification of Processing Conditions for Successful Operation 65 3.2.1.3.1. Control of Airflow 65 11 3.2.1.3.2. Fluid Application Rate 66 3.2.1.3.3. Drying Time and Temperature 66 3.2.1.4. Formulation of Enterogranules 66 3.2.1.5. Enteric-Film Coating of Enterogranules 67 3.2.1.6. Particle Size Analysis 69 3.2.1.7. Construction of Calibration Curves for Spectrophotometric Determination of Isoniazid Release from the Enterosoluble System 70 3.2.1.8. Drug Content of Enterogranules 71 3.2.1.9. In Vitro Release Studies on Enterogranules 71 3.2.2. Results and Discussion 72 3.3. Development of a Methodology for the Fabrication of Microenterospheres by the Polar Organic-in-Oil Emulsification Solvent Evaporation Method 77 3.3.1. Materials and Methods 77 3.3.1.1. Materials 77 3.3.1.2. Formulation of Microenterospheres 77 3.3.1.3. Microenterosphere Diameter Analysis 78 3.3.1.4. Encapsulation Efficiency of Microenterosphere 79 3.3.1.5. In Vitro Release Studies on Microenterospheres 79 3.3.2. Results and Discussion 80 3.4. Modifications to Overcome Burst Release from Microenterospheres 83 3.4.1. Materials and Methods 84 3.4.1.1. Materials 84 3.4.1.2. Double Entrapment of Methacrylic Acid Ethyl Acrylate Copolymer 85 12 3.4.1.3. Double Entrapment in Ethylcellulose 85 3.4.1.4. In Vitro Release Studies on Reservoir Systems 86 3.4.2. Results and Discussion 86 3.5. Development of a Methodology for the Fabrication of Enterospheres by Novel Salting-Out and Ionotropic Cross-linking of Methacrylic Acid Ethyl Acrylate 88 3.5.1. Materials and Methods 89 3.5.1.1. Materials 89 3.5.1.2. Formulation of Enterospheres 89 3.5.1.3. Enterosphere Diameter Analysis 93 3.5.1.4. Encapsulation Efficiency of Enterospheres 94 3.5.1.5. In Vitro Release Studies on Enterospheres 94 3.5.2. Results and Discussion 95 3.6. Treatment of Dissolution Data for Selection of a Candidate Enterosoluble System 101 3.6.1. Methodology 101 3.6.2. Results and Discussion 103 3.7. Concluding Remarks 105 4. Fabrication and Statistical Optimisation of Salted-Out and Internally Cross-linked Anti-Tuberculosis Polymeric Enterospheres Employing an Experimental Design Strategy 4.1. Introduction 107 13 4.1.1. Development of an Experimental Design Strategy for Enterosphere Fabrication and Optimisation 109 4.2. Materials and Methods 112 4.2.1. Materials 112 4.2.2. Formulation of Enterospheres 112 4.2.3. Experimental Design 113 4.2.4. Surface Morphology and Shape Analysis of Enterospheres 114 4.2.5. Determination of Molar Amount of Zinc Incorporated within the Cross- linked Matrix 115 4.2.6. Drug Content and Entrapment Efficiency 115 4.2.7. In Vitro Drug Release Studies 116 4.2.8. Textural Profile Analysis 117 4.2.9. Optimisation of Formulation Ingredients 119 4.3. Results and Discussion 119 4.3.1. Surface Morphology of the Enterospheres 119 4.3.2. Measured Responses for the Experimentally-Synthesised Enterospheres 120 4.3.3. Analysis of the Box-Behnken Response Surface Design 127 4.3.4. Response Surface Analysis 132 4.3.4.1. Response Surface Analysis for Molar Amount of Zinc 132 4.3.4.2. Response Surface Analysis for Drug Entrapment Efficiency 136 4.3.4.3. Response Surface Analysis for Mean Dissolution Time 138 4.3.5. Response Optimisation 141 4.4. Concluding Remarks 144 14 5. Evaluation of Innovatively-Formed Enterospheres for Targeted Delivery of Isoniazid 5.1. Introduction 146 5.1.1. Characterisation of Vibrational Transitions by Fourier Transform Infrared Spectroscopy 147 5.1.2. Characterisation of Degree of Crystallinity by X-Ray Powder Diffraction 148 5.1.3. Characterisation of Thermal Transitions by Differential Scanning Calorimetry 148 5.1.4. Characterisation of Cross-link Density by Atomic Absorption Spectroscopy 149 5.1.5. Mechanisms of Polymer Dissolution and Biodegradation 150 5.2. Materials and Methods 155 5.2.1. Materials 155 5.2.2. Enterosphere Formulations 155 5.2.3. Characterisation of Vibrational Transitions by Fourier Transform Infrared Spectrooscopic Analysis 156 5.2.4. Characterisation of Degree of Crystallinity by X-Ray Powder Diffraction 156 5.2.5. Characterisation of Thermal Transitions by Differential Scanning Calorimetry 156 5.2.6. Characterisation of Cross-linking Cation: MAEA Stoichiometry by Atomic Absorption Spectroscopy 157 15 5.2.7. Characterisation of Morphological Transitions by Scanning Electron Microscopy 158 5.2.8. Characterisation of Enterosphere Erosion by Gravimetric Transitions 158 5.2.9. Characterisation of Enterosphere Erosion by Volume Reduction 159 5.2.10. Characterisation of Enterosphere Erosion from Drug Release Data 160 5.3. Results and Discussion 160 5.3.1. Vibrational Transitions 160 5.3.2. Degree of Crystallinity 164 5.3.3. Thermal Transitions 172 5.3.4. Cross-linking Cation: MAEA Stoichiometry 176 5.3.5. Morphological Transitions 177 5.3.6. Gravimetric and Volumetric Transitions 180 5.4. Concluding Remarks 185 6. Preliminary Investigation and Characterisation of a Gel-Forming Suspending Agent for Extemporaneous Dispensing 6.1. Introduction 186 6.2. Preliminary Evaluation of Appropriate Suspending and Gelling Agents for Extemporaneous Dispensing 193 6.2.1. Materials and Methods 193 6.2.1.1. Materials 193 6.2.1.2. Preliminary Suspension Preparation and Evaluation 193 6.2.2. Results and Discussion 194 16 6.3. Textural Profiling of a Sodium Starch Glycolate-kappa-Carrageenan Combination: Demonstration of the Functional Synergism 196 6.3.1. Materials and Methods 196 6.3.1.1. Materials 196 6.3.1.2. Preparation of odium Starch Glycolate-kappa-Carrageenan Sol and Gel Systems 196 6.3.1.3. Textural Analysis of Sol and Gel Systems 197 6.3.1.4. Gelation and Viscosity Analysis of Sol and Gel Systems 198 6.3.1.5. Statistical Analysis 198 6.3.2. Results and Discussion 199 6.3.2.1. Selection of Processing Conditions for Gel Systems 199 6.3.2.2. Formation of a Three-Dimensional Network in Sol and Gel Systems 199 6.3.2.3. Textural Analysis of Sol and Gel Systems 202 6.3.2.4. Viscosity Analysis of Sol and Gel Systems 206 6.4. Concluding Remarks 208 7. A Face-Centred Central Composite Design to Optimise the Extemporaneous Use Properties of the Suspension System Delivered as Reconstitutable Multiparticulates 7.1. Introduction 210 7.2. Materials and Methods 212 7.2.1. Materials 212 7.2.2. Experimental Design 213 7.2.3. Evaluation of Granule Flow Properties 214 17 7.2.3.1.Angle of Repose 214 7.2.3.2.Compressibility Index 215 7.2.4. Evaluation of Extemporaneous Granule Formulations 215 7.2.4.1. Measurement of Sedimentation Volume 215 7.2.4.2. Viscosity Analysis 216 7.2.4.3. Theoretical Sedimentation Rate 217 7.2.4.4. Importance of the Hydrocolloid Stoichiometric Coefficient in Predicting Gel-Formation Time of the Extemporaneous Granules 218 7.3. Results and Discussion 219 7.3.1. Evaluation of Granule Flow Properties 219 7.3.2. Evaluation of Suspensions 219 7.3.3. Response Surface Analysis for Sedimentation Volume Ratio 223 7.3.4. Response Surface Analysis for Final Viscosity 224 7.3.5. Response Surface Analysis for Gelation Rate 225 7.3.6. Response Surface Analysis for Sedimentation Rate 226 7.3.7. Correlation of the Hydrocolloid Stoichiometric Coefficient with Measured Responses for Reconstitutable Granule Formulations 227 7.3.8. Identification of the Optimum Reconstitutable Granule Formulation 228 7.4. Concluding Remarks 229 8. Methodological Approaches for the Simultaneous In Vitro Analysis of Rifampicin and Isoniazid 8.1. Introduction 230 18 8.1.1. Spectrophotometric Analysis of Rifampicin and Isoniazid 232 8.1.2. High Performance Liquid Chromatographic Analysis of Rifampicin and Isoniazid 233 8.2. Materials and Methods 236 8.2.1. Materials ? Commercially Available Fixed-Dose Combinations 236 8.2.2. In Vitro Drug Release Testing 236 8.2.3. Quantitative Analytical Determination of Rifampicin and Isoniazid by Regressional Analysis of Spectrophotometric Data 237 8.2.4. Quantitative Analytical Determination of Rifampicin and Isoniazid by Colorimetry and High Performance Liquid Chromatography 238 8.3. Results and Discussion 240 8.3.1. Spectrophotometric Analysis 240 8.3.2. Colorimetric and High Performance Liquid Chromatographic Analysis 248 8.4. Concluding Remarks 251 9. Approaches to Formulating Anti-Tuberculosis Nanosystems Embodying A Salted-Out and Cross-linked Architecture 9.1. Introduction 252 9.2. Materials and Methods 262 9.2.1. Materials 262 9.2.2. Formulation of Nanosystems by Emulsion-Based Salting-Out 263 9.2.3. Formulation of Nanosystems by Aqueous-Based Salting-Out 264 9.2.4. Vibrational Transitions in Nanosystems 266 9.2.5. Elucidation of Particulate Architecture 266 19 9.2.6. Plackett-Burman Experimental Design for Critical Factor Identification in Nanosystem Manufacture 266 9.2.7. Nanosystem Recovery 268 9.2.8. Determination of Drug Incorporation Efficiency of Nanosystems 268 9.2.9. In Vitro Release Behaviour of Nanosystems 268 9.3. Results and Discussion 269 9.4. Concluding Remarks 279 10. Conclusions and Recommendations 10.1. Conclusions 280 10.2. Recommendations for Further Development and Implementation of the Anti-Tuberculosis Drug Delivery System 282 References 287 20 LIST OF FIGURES Page 1.1. Estimated TB incidence and mortality 2 1.2. Pathogenesis of TB 5 1.3. Action of anti-TB drugs 8 1.4. Novel anti-TB drug delivery systems 18 1.5. Proposed mechanisms for interaction between RIF and INH 22 1.6. Diagrammatic representation of: (a) the formulation strategy to attain differentiated gastrointestinal delivery in a single dose and (b) the final anti- TB drug delivery system 29 1.7. Organogram of a rational approach to the design of an anti-TB drug delivery system 35 2.1. Candidate enteric polymer 44 2.2. Schematic depicting proposed mechanism for targeted delivery of INH from enterosoluble matrices 51 2.3. Schematic of the film-coating process 54 3.1. Experimental set-up for the air-suspension coating process 65 3.2. Feret?s diameters (df) 69 3.3. INH calibration curve at 265nm in 0.1M HCl (pH 1.2) 72 3.4. INH calibration curve at 263nm in PBS (pH 6.8) 73 3.5. Stereomicrographs of enterogranules 75 3.6. Feret?s diameter of the preliminary enterogranule formulations 75 3.7. Drug release profiles of preliminary enterogranules in acidic media (0.1M HCl, pH 1.2) 75 3.8. Stereomicrographs of representative samples of microenterospheres 81 3.9. Drug release profiles of preliminary microenterosphere formulations in acidic media (0.1M HCl, pH 1.2) 82 21 3.10. Schematic of reservoir and multireservoir enterosphere representing double entrapment of INH 84 3.11. Representative stereomicrographs of representative reservoir enterospheres 87 3.12. Drug release profiles of reservoir systems in acidic media (0.1M HCl, pH 1.2) 87 3.13. Schematic of salting-out process for enterosphere fabrication 92 3.14. Stereomicrographs of representative enterospheres 95 3.15. Drug release profiles of preliminary enterosphere formulations in acidic media (0.1M HCl, pH 1.2) 100 3.16. Composite drug release profiles for A7, B8 and R1 representative of the degree of similarity between R1, and the candidate formulation, B8 104 4.1. Hydration shell of sulphate anions 111 4.2. Two-dimensional schematic of proposed non-stereo-specific (a) inter- and (b) intra-molecular ionic interactions (?salt-bridges?) between the anionic poly(methacrylic acid-co-ethylacrylate) copolymer and cationic agent 111 4.3. Typical textural profiles for the measurement of (a) deformation energy (upward gradient) and matrix hardness (AUC) and (b) resilience 118 4.4. Stereomicrographs of enterosphere formulations 120 4.5. Composite release profiles of the enterosphere formulations in acidic (0.1M HCl, pH 1.2) and phosphate buffered media (pH 6.8) 122 4.6. Variable resilience of enterosphere formulations in the dry and hydrated state 125 4.7. Relationship between fractional drug release and acid-hydrated resilience 126 4.8. 3-D scatter plot of matrix hardness vs. molar amount of Zn (nZn) vs. formulation 127 4.9. Residual plots 128 4.10. Interaction plots 134 4.11. Main effects plots 135 4.12. Response surface plots 136 22 4.13. Stereomicrographs and corresponding scanning electron micrographs of enterosphere formulation 22 139 4.14. Optimisation plots delineating factor settings and desirability values for optimal formulations 142 4.15. Composite release profile of INH from optimum formulation 144 5.1. Relationship between structural variables and properties for the cross-linked enterosphere 146 5.2. Controlled release of drug molecules from a matrix diffusion-controlled diffusion-type drug delivery device in which solid drug is homogenously dispersed in the polymer matrix 153 5.3. Proposed interaction between MAEA and INH 162 5.4. FTIR spectra 163 5.5. XRPD patterns 167 5.6. DSC thermograms 168 5.7. Scanning electron micrographs of DL2% enterospheres 178 5.8. Scanning electron micrographs of DL8% enterospheres 179 5.9. Relationship between drug release and erosional transitions (weight and volume) 183 5.10. Erosional behaviour of representative enterosphere formulations 184 6.1. Gel formation in carrageenan 190 6.2. Conical probe configuration illustrating the compression and decompression phases 197 6.3. (a) Structural formulae of SSG and ?C repeating units and (b) schematic of proposed effect of SSG on ?C due to (A) exclusion effect (B) Coulombic interactions 201 6.4. Stereomicrographs of SSG-?C sol systems 201 6.5. Representative textural profiles depicting the AUC 203 6.6. Effect of increasing ?C concentrations on compressibility 204 23 6.7. Effect of ?C levels on the change in viscosity following the sol-gel transition 206 7.1. Residual plots 221 7.2. Agreement between experimental and predicted values 222 7.3. Response surface plot for sedimentation ratio (V/Vo) versus SSG, ?C 224 7.4. Response surface plot for final viscosity versus SSG, ?C 225 7.5. Response surface plot for gelation rate (?V/?t) versus SSG, ?C 226 7.6. Response surface plot for sedimentation rate (V) vs SSG, ?C 227 8.1. UV spectra 241 8.2. PLS analysis of spectrophotometric absorbance data for Rifanah 150 242 8.3. PLS analysis of spectrophotometric absorbance data for Rifanah 300 243 8.4. PLS analysis of spectrophotometric absorbance data for optimum RIF-INH dispersible multiparticulate system 244 8.5. Drug release from FDCs following regressional analysis of UV spectrophotometric absorbance data 247 8.6. Drug release from FDCs following chromatographic determination of INH and colorimetric determination of RIF 250 9.1. Comparison of complexity as a function of molecular architecture, strategy, quantised building block and technological age highlighting the intricate role of polymers in supramolecular chemistry 254 9.2. The array of nanosystems encompassed by nanopharmaceuticals 255 9.3. Fabrication of nanosystems by the emulsion-based salting-out method 261 9.4. Experimental system set-up for aqueous salting-out by spray atomisation 265 9.5. SEMs of nanosystems generated by the emulsion-based salting-out approach 270 9.6. SEMs of nanosystems generated by the aqueous-based salting-out approach 271 9.7. FTIR spectra of nanosystems 272 24 9.8. (a) Schematic of zinc oxide crystal formation and (b) Stereomicrographs depicting tubular morphological transition in NS fabricated by aqueous- based salting-out 274 9.9. Selected drug release profiles from emulsion-based salted-out nanosystems in PBS (pH 7.0) 275 9.10. Drug release profiles from aqueous-based salted-out nanosystems in PBS (pH 7.0) 275 9.11. Surface plots derived from P-B design for emulsion-based nanosystems and aqueous-based nanosystems 276 9.12. Proposed mechanism for targeted pulmonary delivery of nanosystems via the inhalation route 278 10.1. Spray-dryer configuration and process staging for enterosphere manufacture 285 25 LIST OF TABLES Page 1.1. Regimen 1 ? for treatment of new smear positive adult patients 6 1.2. Classes of anti-TB drugs 9 1.3. Synopsis of novel anti-TB drug delivery systems 10 2.1. Commercially available enteric polymers 40 2.2. Microencapsulation methods for the formulation of enterosoluble multiparticulates 52 2.3. Parameters and process variables affecting solvent evaporation 56 2.4. Common suspending and gelling agents 61 3.1. Identified processing conditions 65 3.2. Formulae for Eudragit S? 100 and Eudragit L? 100 dispersions 68 3.3. Preliminary enterogranule formulations 69 3.4. Accuracy determination for INH assay method in 0.1M HCl 73 3.5. Precision determination for INH assay method in 0.1M HCl 73 3.6. Accuracy determination for INH assay method in 0.2M PBS pH 6.8 74 3.7. Precision determination for INH assay method in 0.2M PBS pH 6.8 74 3.8. Preliminary microenterosphere formulations 78 3.9. Particle size, entrapment and release characteristics of preliminary microenterosphere formulations 81 3.10. Solubility and key hydrational properties of electrolytes tested 93 3.11. Preliminary enterosphere formulations 93 3.12. Particle size, entrapment and release characteristics of preliminary enterosphere formulations 96 3.13. Similarity and difference factors of the preliminary enterosoluble formulations 104 26 4.1. Factors and levels of independent variables generated by the 34 Box- Behnken Design 114 4.2. Textural parameters for determination of matrix hardness, deformation energy and matrix resilience 118 4.3. Measured responses for the enterosphere formulations 123 4.4. Measured textural properties of the experimentally synthesised variants 124 4.5. Correlation between experimental and predicted values for nZn, DEE, and MDT 130 4.6. Estimated p-values for the measured responses 131 4.7. Constrained settings for response optimisation 142 4.8. Experimental and predicted response values for the optimised formulations 143 5.1. Band assignments for MAEA-INH physical mixture 161 5.2. Intensity counts of representative samples at the main interplanar distances 166 5.3. Thermodynamically-derived data for enterosphere formulations with relation to cross-linking cation: MAEA stoichiometry 175 6.1. Characteristics of hydrophilic suspending agents for extemporaneous dispensing 195 6.2. TA-XT.plus settings for sol and gel analysis. 198 6.3. Textural properties and viscosity of SSG-?C Systems 204 7.1. Suspending Agents and Levels Derived from the Central Composite Design 213 7.2. Results from analysis of suspension formulations 219 7.3. Estimated regression coefficients and p-values for sedimentation ratio (V/Vo), final viscosity, gelation rate (?V/?t) and sedimentation rate (V) 222 7.4. Constraints for optimal suspension formulation 228 7.5. Experimental and predicted response values for the optimised gelling suspension 228 27 8.1. Specifications for HPLC methodologies 235 8.2. Gradient program prescribed in the USP method 235 8.3. Drug content validation in FDC tablet formulations 245 8.4. Model selection and validation and regression coefficients for RIF release prediction 245 8.5. Model selection and validation and regression coefficients for INH release prediction 245 9.1. Compositions of preliminary formulations for production of nanosystems by the emulsion-based salting-out approach 263 9.2. Compositions of preliminary formulations for production of nanosystems by novel aqueous-based salting-out approach 265 9.3. Plackett-Burman design for emulsion-based salted-out nanosystems 267 9.4. Plackett-Burman design for aqueous-based salted-out nanosystems 267 9.5. Measured yield and DIE responses for Plackett-Burman generated formulations 274