Tse Pui Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves [191]-216). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.III / ABSTRACT --- p.VIII / 摘要 --- p.X / PUBLICATIONS --- p.XII / TABLE OF CONTENTS --- p.XIII / Chapter CHAPTER 1: --- GENERAL INTRODUCTION / Chapter 1.1 --- Human Immune System and Cancer --- p.1 / Chapter 1.1.1 --- Brief Introduction of the Human Immune System --- p.1 / Chapter 1.1.2 --- Prevalence of Cancer in Hong Kong --- p.4 / Chapter 1.1.3 --- The Role of the Immune System in Tumorigenesis --- p.4 / Chapter 1.1.4 --- Cancer Treatment --- p.5 / Chapter 1.1.5 --- Cancer Prevention --- p.5 / Chapter 1.2 --- Mushroom Polysaccharides --- p.6 / Chapter 1.2.1 --- General Aspects of Mushroom Polysaccharides --- p.6 / Chapter 1.2.2 --- Structure of Mushroom Polysaccharides --- p.9 / Chapter 1.2.2.1 --- Beta (P)-D-glucans --- p.9 / Chapter 1.2.2.2 --- Heteroglucans and Protein-bound Polysaccharides --- p.10 / Chapter 1.2.2.3 --- Structure-Function Interactions of Polysaccharides --- p.12 / Chapter 1.2.3 --- Molecular Interactions of Polysaccharides --- p.14 / Chapter 1.2.4 --- Biological Activities of Polysaccharides --- p.15 / Chapter 1.2.4.1 --- Anti-tumor Activities of Polysaccharides --- p.15 / Chapter 1.2.4.2 --- Immunomodulatory Activities of Polysaccharides --- p.16 / Chapter 1.3 --- Yun Zhi (Coriolus versicolor) --- p.17 / Chapter 1.3.1 --- General Features of Yun Zhi --- p.17 / Chapter 1.3.2 --- Traditional Uses of Yun Zhi --- p.20 / Chapter 1.3.3 --- Active Ingredients of Yun Zhi --- p.20 / Chapter 1.3.3.1 --- "Origin, Properties and Composition of PSK" --- p.21 / Chapter 1.3.3.2 --- "Origin, Properties and Composition of PSP" --- p.22 / Chapter 1.3.4 --- Pharmacological Actions of PSP and PSK --- p.25 / Chapter 1.3.4.1 --- Immunomodulatory Activities --- p.25 / Chapter 1.3.4.2 --- Anti-tumor Activities --- p.32 / Chapter 1.3.4.2 --- Antiviral and Antimicrobial Activities --- p.35 / Chapter 1.3.4.3 --- Antioxidant Activities --- p.36 / Chapter 1.3.5 --- Human Clinical Studies on Yun Zhi --- p.36 / Chapter 1.3.6 --- Toxicology of Yun Zhi --- p.42 / Chapter 1.4 --- Danshen (Salvia miltiorrhiza) --- p.43 / Chapter 1.4.1 --- General Features of Danshen --- p.43 / Chapter 1.4.2 --- Traditional Uses of Danshen --- p.46 / Chapter 1.4.3 --- Active Ingredients of Danshen --- p.47 / Chapter 1.4.4 --- Pharmacological Actions of Danshen --- p.50 / Chapter 1.4.4.1 --- Cardiovascular Effects --- p.50 / Chapter 1.4.4.2 --- Scavenging Effects on Free Radicals --- p.52 / Chapter 1.4.4.3 --- Hepatoprotective Effects --- p.54 / Chapter 1.4.4.4 --- Anti-tumor Effects --- p.56 / Chapter 1.4.4.5 --- Renal Protective Effects --- p.56 / Chapter 1.4.5 --- Human Clinical Studies --- p.57 / Chapter 1.4.6 --- Toxicity of Danshen --- p.59 / Chapter 1.5 --- Aims and Scopes of This Investigation --- p.60 / Chapter CHAPTER 2: --- MATERIALS AND METHODS / Chapter 2.1 --- Normal Subjects --- p.62 / Chapter 2.1.1 --- Inclusion and Exclusion Criteria of Recruitment --- p.62 / Chapter 2.1.2 --- Study Design and Procedure --- p.63 / Chapter 2.1.3 --- Treatment and Blinding --- p.65 / Chapter 2.1.4 --- Blood Sampling --- p.66 / Chapter 2.1.5 --- Blood Processing for Assessment of Immunological Functions --- p.67 / Chapter 2.2 --- Materials --- p.69 / Chapter 2.2.1 --- Endotoxin Assay --- p.69 / Chapter 2.2.2 --- Reagents for Whole Blood Assay --- p.69 / Chapter 2.2.2.1 --- Plain RPMI 1640 Medium --- p.69 / Chapter 2.2.2.2 --- Phosphate-Buffered Saline (PBS) --- p.69 / Chapter 2.2.2.3 --- Mitogens --- p.70 / Chapter 2.2.3 --- Reagents for Total RNA Extraction --- p.70 / Chapter 2.2.3.1 --- Ficoll-Paque Density Gradient Solution --- p.70 / Chapter 2.2.3.2 --- RNA Extraction Kit --- p.70 / Chapter 2.2.3.3 --- RNase-Free DNase Set --- p.71 / Chapter 2.2.3.4 --- β-Mercaptoethanol (β-ME) Solution --- p.71 / Chapter 2.2.4 --- Reagents for Flow Cytometric Analysis of T/B/NK Cell Ratios --- p.71 / Chapter 2.2.4.1 --- MultiTEST IMK Kit with TruCOUNT Tubes --- p.71 / Chapter 2.2.4.2 --- FACSFlo´wёØ Sheath Fluid --- p.74 / Chapter 2.2.4.3 --- CaliBRITE 3 and APC Beads --- p.74 / Chapter 2.2.5 --- Immunoassay Kits for Measuring Cytokines Level --- p.75 / Chapter 2.2.5.1 --- Enzyme-linked Immunosorbent Assay (ELISA) Kits of Cytokines --- p.75 / Chapter 2.2.5.2 --- Human Thl/Th2 Cytokine Cytometric Bead Array (CBA) Kit-II --- p.75 / Chapter 2.2.6 --- Reagents and Buffers for Gel Electrophoresis --- p.78 / Chapter 2.2.6.1 --- Ethidium Bromide (EtBr) --- p.78 / Chapter 2.2.6.2 --- Gel Loading Solution (5X) --- p.78 / Chapter 2.2.6.3 --- Tris-Acetate-EDTA (TAE) Buffer --- p.78 / Chapter 2.2.6.4 --- Agarose Gel --- p.78 / Chapter 2.2.6.5 --- 100 base pair DNA Ladder --- p.79 / Chapter 2.2.7 --- Kits and Reagents for Messenger RNA (mRNA) Expression Array --- p.79 / Chapter 2.2.7.1 --- Human Inflammatory Cytokine/Receptor GEArraýёØ Q Series Kit --- p.79 / Chapter 2.2.7.2 --- Deoxynucleoside Triphosphates (dNTPs) --- p.84 / Chapter 2.2.7.3 --- Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) --- p.84 / Chapter 2.2.7.4 --- Rnasin Ribonuclease Inhibitor --- p.84 / Chapter 2.2.7.5 --- Biotin-16-2'-deoxy-uridine-5'-triphosphate (Biotin-16-dUTP) --- p.85 / Chapter 2.2.7.6 --- Salmon Sperm DNA Solution --- p.85 / Chapter 2.2.7.7 --- 100 % Sodium Dodecyl Sulfate (SDS) Solution --- p.86 / Chapter 2.2.7.8 --- 20X SSC --- p.86 / Chapter 2.2.7.9 --- ECL Films (Hyperfilm 226}0ёØ ECL 226}0ёØ) --- p.86 / Chapter 2.3 --- Methods / Chapter 2.3.1 --- Endotoxin Assay --- p.87 / Chapter 2.3.2 --- Whole Blood Assay (WBA) --- p.88 / Chapter 2.3.3 --- Isolation and Preparation of Plasma and Peripheral Blood Mononuclear Cells (PBMC) from EDTA Blood --- p.88 / Chapter 2.3.4 --- Total RNA extraction --- p.89 / Chapter 2.3.5 --- Flow Cytometric Analysis of T/B/NK Cell Ratios --- p.90 / Chapter 2.3.6 --- Immunoassays of Plasma Samples or Culture Supernatant in WBA --- p.92 / Chapter 2.3.6.1 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.92 / Chapter 2.3.6.2 --- Human Thl/Th2 Cytokine Cytometric Bead Assay (CBA) --- p.93 / Chapter 2.3.7 --- mRNA Expression Study --- p.94 / Chapter 2.3.7.1 --- Agarose Gel Electrophoresis --- p.94 / Chapter 2.3.7.2 --- cDNA Expression Array Analysis --- p.95 / Chapter 2.3.8 --- Statistical Analysis --- p.96 / Chapter CHAPTER 3: --- ENDOTOXIN LEVEL OF YUN ZHI-DANSHEN CAPSULES & SAFETY MEASURES ON STUDY POPULATION IN THE CLINICAL TRIAL / Chapter 3.1 --- Introduction --- p.98 / Chapter 3.2 --- Results --- p.101 / Chapter 3.2.1 --- Endotoxin Level of the Yun Zhi and Danshen Active Capsule --- p.101 / Chapter 3.2.2 --- Study Population --- p.103 / Chapter 3.2.3 --- Dropout Cases --- p.103 / Chapter 3.2.4 --- Safety Parameters --- p.104 / Chapter 3.2.5 --- Compliance Rates --- p.104 / Chapter 3.3 --- Discussion --- p.109 / Chapter CHAPTER 4: --- FLOW CYTOMETRIC ANALYSIS OF T/B/NK CELL RATIOS OF HEALTHY SUBJECTS TAKING YUN ZHI-DANSHEN CAPSULES / Chapter 4.1 --- Introduction --- p.112 / Chapter 4.2 --- Results --- p.118 / Chapter 4.2.1 --- The Percentage and Absolute Count of T Lymphocytes (CD3+) --- p.118 / Chapter 4.2.2 --- The Percentage and Absolute Count of T Helper (TH) Lymphocytes (CD3+ CD4+) --- p.121 / Chapter 4.2.3 --- The Percentage and Absolute Count of Cytotoxic T (CTL) and T Suppressor (Ts) Lymphocytes (CD3+ CD8+) --- p.124 / Chapter 4.2.4 --- The Ratio of T Helper Lymphocytes (CD3+ CD4+) and Cytotoxic T (CTL) and T Suppressor (Ts) Lymphocyes (CD3+ CD8+) --- p.127 / Chapter 4.2.5 --- The Percentage and Absolute Count of B Lymphocytes (CD19+) --- p.129 / Chapter 4.2.6 --- The Percentage and Absolute Count of NK Lymphocytes (CD3- CD 16+ and/or CD56+) --- p.132 / Chapter 4.2.7 --- The Absolute Count of Lymphocytes (CD45+) --- p.135 / Chapter 4.3 --- Discussion --- p.138 / Chapter CHAPTER 5: --- PLASMA CONCENTRATION OF SOLUBLE CYTOKINE RECEPTOR AND EX VIVO CYTOKINE PRODUCTION OF HEALTHY SUBJECTS TAKING YUN ZHI-DANSHEN CAPSULES / Chapter 5.1 --- Introduction --- p.142 / Chapter 5.2 --- Results --- p.147 / Chapter 5.2.1 --- Plasma Concentration of Soluble IL-2 Receptor --- p.147 / Chapter 5.2.2 --- Ex vivo Cytokine Production --- p.147 / Chapter 5.2.3 --- Mitogen Induced IL-6 Production --- p.150 / Chapter 5.2.4 --- Mitogen Induced IFN- γ Production --- p.150 / Chapter 5.2.5 --- Mitogen Induced TNF- a Production --- p.153 / Chapter 5.2.6 --- Mitogen Induced IL-10 Production --- p.153 / Chapter 5.3 --- Discussion --- p.156 / Chapter CHAPTER 6: --- "GENE EXPRESSION OF CYTOKINES, CHEMOKINES AND RECEPTORS OF PBMC OF HEALTHY SUBJECTS TAKING YUN ZHI- DANSHEN CAPSULES" / Chapter 6.1 --- Introduction --- p.162 / Chapter 6.2 --- Results --- p.165 / Chapter 6.2.1 --- Gene Expression of IL-2 Receptor β chain --- p.165 / Chapter 6.2.2 --- Gene Expression of IL-2 Receptor γ chain --- p.165 / Chapter 6.2.3 --- Gene Expression of IL-6 Receptor --- p.166 / Chapter 6.2.4 --- "Gene Expression of Other Cytokines, Chemokines and Receptors" --- p.169 / Chapter 6.3 --- Discussion --- p.172 / Chapter CHAPTER 7: --- CONCLUDING REMARKS AND FUTURE / PERSPECTIVES --- p.176 / APPENDICES --- p.184 / REFERENCES --- p.192

Zhang Li. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 148-159). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.II / Abstract (in Chinese) --- p.IV / Publications --- p.V / List of Abbreviations --- p.VI / List of Tables --- p.VII / List of Figures --- p.VIII / Table of Contents --- p.XIII / Chapter Chapter One. --- Introduction --- p.1 / Chapter 1.1 --- Flavonoids --- p.1 / Chapter 1.2 --- Tea --- p.4 / Chapter 1.2.1 --- Composition of green tea catechins (GTC) --- p.4 / Chapter 1.2.2 --- Pharmacological activity --- p.6 / Chapter 1.2.2.1 --- Anticarcinogenic activity --- p.6 / Chapter 1.2.2.2 --- Antioxidative activity --- p.7 / Chapter 1.2.2.3 --- Radical scavenge --- p.7 / Chapter 1.2.2.4 --- Cardiovascular activity --- p.8 / Chapter 1.2.3 --- Pharmacokinetics of GTC --- p.8 / Chapter 1.2.3.1 --- Absorption --- p.10 / Chapter 1.2.3.2 --- Distribution --- p.11 / Chapter 1.2.3.3 --- Elimination --- p.11 / Chapter 1.2.3.4 --- Metabolism --- p.12 / Chapter 1.2.3.4.1 --- Metabolism in the small intestine --- p.12 / Chapter 1.2.3.4.2 --- Metabolism in the liver --- p.13 / Chapter 1.2.3.5 --- Summary of the pharmacokinetics of GTC --- p.13 / Chapter 1.3 --- Hawthorn --- p.14 / Chapter 1.3.1 --- Composition of hawthorn --- p.14 / Chapter 1.3.2 --- Pharmacological activity --- p.16 / Chapter 1.3.2.1 --- Inotonic activity --- p.16 / Chapter 1.3.2.2 --- Antiarrhythmic activity --- p.17 / Chapter 1.3.2.3 --- Hypolipidemic activity --- p.17 / Chapter 1.3.2.4 --- Antihypertensive activity --- p.18 / Chapter 1.3.2.5 --- Antioxidative activity --- p.18 / Chapter 1.3.3 --- Pharmacokinetics of HF --- p.18 / Chapter 1.3.3.1 --- Absorption --- p.19 / Chapter 1.3.3.2 --- Distribution and elimination --- p.21 / Chapter 1.3.3.3 --- Summary of pharmacokinetic of HF --- p.22 / Chapter 1.4 --- Mechanisms of intestinal absorption --- p.22 / Chapter 1.4.1 --- Passive transcellular transport --- p.23 / Chapter 1.4.2 --- Paracellular transport --- p.23 / Chapter 1.4.3 --- Carrier-mediated transport --- p.23 / Chapter 1.5 --- ABC transporters --- p.24 / Chapter 1.5.1 --- Cellular location and tissue distribution --- p.25 / Chapter 1.5.2 --- Substrates and inhibitors of ABC transporters --- p.26 / Chapter 1.6 --- Oral absorption models --- p.31 / Chapter 1.6.1 --- Ussing chamber --- p.31 / Chapter 1.6.2 --- In situ intestinal perfusion model --- p.33 / Chapter 1.6.3 --- Cell culture model --- p.34 / Chapter 1.7 --- Aims of the study --- p.36 / Chapter Chapter Two. --- Transport mechanism of green tea catechins --- p.37 / Chapter 2.1 --- Introduction --- p.37 / Chapter 2.2 --- Materials --- p.38 / Chapter 2.2.1 --- Chemicals --- p.38 / Chapter 2.2.2 --- Materials for cell culture --- p.38 / Chapter 2.2.3 --- Instruments --- p.39 / Chapter 2.3 --- Methods --- p.39 / Chapter 2.3.1 --- Analytical methods --- p.39 / Chapter 2.3.1.1 --- Analytical methods for validation of Caco-2 model --- p.39 / Chapter 2.3.1.1.1 --- Fluorescence analysis of lucifer yellow --- p.39 / Chapter 2.3.1.1.2 --- HPLC analysis of propranolol --- p.39 / Chapter 2.3.1.1.3 --- HPLC analysis of verapamil --- p.40 / Chapter 2.3.1.1.4 --- HPLC analysis of quinidine --- p.40 / Chapter 2.3.1.2 --- Analytical methods for samples contained GTC --- p.41 / Chapter 2.3.1.2.1 --- HPLC analysis for each GTC --- p.41 / Chapter 2.3.1.2.2 --- Preparation of calibration curves for each GTC --- p.42 / Chapter 2.3.1.2.3 --- HPLC/MS analysis of samples containing mixtures of four GTC --- p.42 / Chapter 2.3.1.2.4 --- Preparation of calibration curves for samples containing GTC mixture --- p.43 / Chapter 2.3.1.2.5 --- Validation of the HPLC methods --- p.43 / Chapter 2.3.1.3 --- Identification of metabolites with HPLC/MS --- p.44 / Chapter 2.3.2 --- Determination of stability profile of GTC in phosphate buffer --- p.44 / Chapter 2.3.3 --- Cell culture --- p.45 / Chapter 2.3.4 --- Validation of Caco-2 cell monolayer model --- p.46 / Chapter 2.3.4.1 --- Integrity of Caco-2 cell monolayer at pH 6.0 --- p.46 / Chapter 2.3.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.46 / Chapter 2.3.4.3 --- Validation of the existence of P-glycoprotein (P-gp) transporterin Caco-2 monolayer model --- p.46 / Chapter 2.3.4.4 --- Cytotoxicity test --- p.47 / Chapter 2.3.5 --- Transport study of GTC using Caco-2 cell monolayer model --- p.48 / Chapter 2.3.5.1 --- Bi-directional transport experiment --- p.48 / Chapter 2.3.5.2 --- Preparation of different dosing formulations of GTC --- p.48 / Chapter 2.3.5.2.1 --- Preparation of individual pure GTC solutions --- p.48 / Chapter 2.3.5.2.2 --- Preparation of cocktail 1 solution --- p.49 / Chapter 2.3.5.2.3 --- Preparation of green tea extract solution --- p.49 / Chapter 2.3.5.2.4 --- Preparation of cocktail 2 solution --- p.50 / Chapter 2.3.5.3 --- Sample treatment --- p.50 / Chapter 2.3.5.3.1 --- Samples for direct analysis --- p.50 / Chapter 2.3.5.3.2 --- Samples for enzymatic hydrolysis treatment --- p.51 / Chapter 2.3.5.4 --- Further investigation of the transport mechanism of GTC --- p.51 / Chapter 2.3.5.4.1 --- Inhibition transport of EC and EGC --- p.51 / Chapter 2.3.5.4.2 --- Transport mechanism of metabolites of EC and EGC --- p.52 / Chapter 2.3.5.4.3 --- Metabolic competition between EGC and the other GTC --- p.52 / Chapter 2.3.6 --- Calculation --- p.53 / Chapter 2.3.7 --- Data analysis --- p.54 / Chapter 2.4 --- Results --- p.55 / Chapter 2.4.1 --- Validation of the HPLC methods --- p.55 / Chapter 2.4.2 --- Stability of the GTC --- p.55 / Chapter 2.4.3 --- Extract of green tea leaves --- p.55 / Chapter 2.4.4 --- Validation of Caco-2 model --- p.59 / Chapter 2.4.4.1 --- Integrity of Caco-2 cell monolayer --- p.59 / Chapter 2.4.4.2 --- Permeability of paracellular and transcellular markers at pH 6.0 --- p.59 / Chapter 2.4.4.3 --- Validation of P-glycoprotein --- p.60 / Chapter 2.4.4.4 --- Cytotoxicity test --- p.61 / Chapter 2.4.5 --- Transport study of GTC --- p.63 / Chapter 2.4.5.1 --- Bi-directional transport of individual pure GTC --- p.63 / Chapter 2.4.5.2 --- Bi-directional transport of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.2.1 --- Absorption transport profile of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.2.2 --- Secretion transport profile of GTC in different dosing formulations --- p.66 / Chapter 2.4.5.3 --- Identification of metabolites of each GTC formed during the transport in Caco-2 cell model --- p.71 / Chapter 2.4.6 --- Further investigation of the transport mechanism of GTC --- p.82 / Chapter 2.4.6.1 --- Inhibition transport of EC and EGC --- p.82 / Chapter 2.4.6.2 --- Transport mechanism of metabolites of EC and EGC --- p.82 / Chapter 2.4.6.3 --- Metabolic competition between EGC and the other GTC --- p.85 / Chapter 2.4.6.4 --- Contribution of GTC on the metabolism of EGC --- p.89 / Chapter 2.5 --- Discussion --- p.92 / Chapter 2.5.1 --- Stability of the four GTC --- p.92 / Chapter 2.5.2 --- Validation of Caco-2 cell model --- p.92 / Chapter 2.5.3 --- Bi-directional transport of GTC --- p.93 / Chapter 2.5.4 --- Structure related efflux --- p.97 / Chapter 2.5.5 --- Metabolism of GTC --- p.98 / Chapter 2.5.6 --- Relationship between metabolism and efflux transport of GTC --- p.99 / Chapter 2.5.7 --- Bi-directional transport of GTC in different dosing formulations …… --- p.100 / Chapter 2.5.7.1 --- Absorption transport profile of different dosing formulations --- p.100 / Chapter 2.5.7.2 --- Secretion transport profile of different dosing formulations --- p.101 / Chapter 2.6 --- Conclusion --- p.105 / Chapter Chapter Three. --- Transport mechanism of hawthorn flavonoids --- p.106 / Chapter 3.1 --- Introduction --- p.106 / Chapter 3.2 --- Materials --- p.107 / Chapter 3.2.1 --- Chemicals --- p.107 / Chapter 3.2.2 --- Materials for cell culture --- p.108 / Chapter 3.2.3 --- Instruments --- p.108 / Chapter 3.3 --- Methods --- p.109 / Chapter 3.3.1 --- Analytical methods for HF --- p.109 / Chapter 3.3.1.1 --- Analytical methods of individual pure compound of HF --- p.109 / Chapter 3.3.1.1.1 --- HPLC analysis of HP and IQ --- p.109 / Chapter 3.3.1.1.2 --- HPLC analysis of EC --- p.109 / Chapter 3.3.1.2 --- Preparation of calibration curves for individual pure HF --- p.109 / Chapter 3.3.1.3 --- HPLC/MS analysis of three HF in mixture --- p.110 / Chapter 3.3.1.4 --- Preparation of the calibration curves of three HF in mixture --- p.111 / Chapter 3.3.1.5 --- Validation of HPLC methods --- p.111 / Chapter 3.3.2 --- Analytical methods for identification of metabolites with HPLC/MS --- p.111 / Chapter 3.3.3 --- Cell culture --- p.112 / Chapter 3.3.4 --- Cytotoxicity test --- p.113 / Chapter 3.3.5 --- Transport studies of HF using Caco-2 monolayer model --- p.113 / Chapter 3.3.5.1 --- Bi-directional transport experiment --- p.113 / Chapter 3.3.5.2 --- Preparation of loading solutions in different dosing formulations of HF for Caco-2 cell model --- p.114 / Chapter 3.3.5.2.1 --- Preparation of individual pure HF solutions --- p.114 / Chapter 3.3.5.2.2 --- Preparation of cocktail 1 solution --- p.114 / Chapter 3.3.5.2.3 --- Preparation of hawthorn extract solution --- p.114 / Chapter 3.3.5.2.4 --- Preparation of cocktail 2 solution --- p.114 / Chapter 3.3.5.3 --- Sample treatment --- p.115 / Chapter 3.3.5.4 --- Further study of the transport mechanism of HF --- p.115 / Chapter 3.3.5.4.1 --- "Inhibition transport of EC, IQ, and HP" --- p.115 / Chapter 3.3.5.4.2 --- "Transport mechanisms of the metabolites of EC, HP, IQ" --- p.116 / Chapter 3.3.6 --- Calculation --- p.116 / Chapter 3.3.7 --- Data analysis --- p.117 / Chapter 3.4 --- Results --- p.118 / Chapter 3.4.1 --- Validation of the HPLC methods --- p.118 / Chapter 3.4.2 --- Cytotoxicity test --- p.118 / Chapter 3.4.3 --- Transport study of HF --- p.122 / Chapter 3.4.3.1 --- Bi-directional transport of individual pure HF --- p.122 / Chapter 3.4.3.2 --- Bi-directional transport of the HF in different formulations --- p.123 / Chapter 3.4.3.2.1 --- Absorption transport of different formulations of HF --- p.123 / Chapter 3.4.3.2.2 --- Secretion transport of different dosing forms --- p.123 / Chapter 3.4.3.3 --- Identification of metabolites of each HF formed during their transport in Caco-2 model --- p.126 / Chapter 3.4.4 --- Further study on the transport mechanism --- p.136 / Chapter 3.4.4.1 --- "Inhibition transport of EC, HP, IQ" --- p.136 / Chapter 3.4.4.2 --- Transport mechanism of metabolites of HF --- p.136 / Chapter 3.4.4.3 --- Transport profiles of HF metabolites upon the loading of different dosing formulations of HF --- p.138 / Chapter 3.5 --- Discussion --- p.140 / Chapter 3.5.1 --- Bi-directional transport of each HF --- p.140 / Chapter 3.5.2 --- Bi-directional transport of HF in different formulations --- p.141 / Chapter 3.6 --- Conclusion --- p.142 / Chapter Chapter Four. --- Limitations of the current study --- p.143 / Chapter Chapter Five. --- Overall conclusions --- p.146 / References --- p.148 / Appendices --- p.160

Ng Chau Wah Stephen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 120-135). / Abstracts in English and Chinese. / Abstract --- p.i / Acknowledgements --- p.viii / Publications --- p.ix / Abbreviations --- p.x / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Traditional Chinese Medicine --- p.1 / Chapter 1.2 --- Danshen --- p.7 / Chapter 1.2.1 --- Chemical constituents of Danshen --- p.7 / Chapter 1.2.2 --- Pharmacology of Danshen --- p.10 / Chapter 1.3 --- Vascular system --- p.13 / Chapter 1.3.1 --- Physiology of blood vessels --- p.13 / Chapter 1.3.2 --- Vascular smooth muscle contraction --- p.14 / Chapter 1.3.3 --- Mechanism of vascular smooth muscle contraction --- p.15 / Chapter 1.3.3.1 --- Adrenoceptor in vascular system --- p.19 / Chapter 1.3.3.2 --- Muscarinic receptor in vascular system --- p.20 / Chapter 1.3.3.3 --- Synthesis and release of Nitric Oxide (NO)in vascular system --- p.22 / Chapter 1.3.3.4 --- Synthesis and release of postanoidsin vascular system --- p.25 / Chapter 1.3.3.5 --- Synthesis and release of histaminein vascular system --- p.28 / Chapter 1.3.3.6 --- Synthesis and release of Calcitonin gene-related peptide in vascular system --- p.29 / Chapter 1.4 --- Aims of the studies --- p.33 / Chapter Chapter 2 --- Materials and methods / Chapter 2.1 --- Materials --- p.35 / Chapter 2.2 --- Methods - General procedures --- p.35 / Chapter 2.2.1 --- Preparations of drug solutions --- p.35 / Chapter 2.2.2 --- Animals used and anaesthetization --- p.36 / Chapter 2.2.3 --- Cannulation of carotid artery and jugular vein --- p.37 / Chapter 2.2.4 --- Blood pressure measurement --- p.37 / Chapter 2.2.5 --- Knee joint denervation --- p.38 / Chapter 2.2.6 --- Knee joint blood flow measurement --- p.39 / Chapter 2.3 --- Methods - Specific procedures --- p.41 / Chapter 2.3.1 --- Validation of Laser Doppler Imaging (LDI) measurements --- p.41 / Chapter 2.3.2 --- Actions of topical administration of Danshen --- p.42 / Chapter 2.3.2.1 --- Studies of the mechanism(s) of action of Danshen --- p.43 / Chapter 2.3.2.2 --- Investigation for α-adrenoceptor antagonist activity --- p.44 / Chapter 2.3.2.3 --- Investigation for neural involvement --- p.44 / Chapter 2.3.3 --- Actions of intravenous administration of Danshen --- p.45 / Chapter 2.3.4 --- Data analysis --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Validation of LDI measurement --- p.47 / Chapter 3.2 --- Actions of intravenous administration of Danshen --- p.53 / Chapter 3.3 --- Actions of topical administration of Danshen --- p.53 / Chapter 3.4 --- Muscarinic receptor antagonist on Danshen --- p.61 / Chapter 3.5 --- β-adrenoceptor antagonist on Danshen --- p.67 / Chapter 3.6 --- Danshen on α-adrenoceptor agonist-induced vasoconstriction --- p.74 / Chapter 3.7 --- Nitric oxide synthase inhibitor on Danshen --- p.79 / Chapter 3.8 --- Cyclo-oxygenase (COX) inhibitor on Danshen --- p.83 / Chapter 3.9 --- Histamine receptor antagonists on Danshen --- p.87 / Chapter 3.10 --- CGRP receptor antagonist on Danshen --- p.92 / Chapter 3.11 --- Effect of denervation on Danshen --- p.92 / Chapter Chapter 4 --- Discussion --- p.100 / Reference --- p.120

Objetivo: Avaliar a influência da infecção por Pseudomonas aeruginosa formadora de biofilme na penetração pulmonar da tobramicina através da modelagem populacional dos dados de plasma e microdialisado em animais sadios e infectados. Metodologia: A pneumonia foi desenvolvida através de inoculação de P. aeruginosa (cepa PA14) pela via intratraqueal (109 UFC/mL) a ratos Wistar. Sete dias após a inoculação os animais infectados (n = 5) receberam tobramicina 10 mg/kg i.v. bolus. Animais saudáveis (n = 6) foram utilizados como controle. As concentrações livres pulmonares foram coletadas por microdiálise (sonda CMA/20). As sondas de microdiálise foram calibradas in vitro através de diálise e retrodiálise e in vivo utilizando retrodiálise. A ligação da tobramicina às proteínas plasmáticas foi determinada por microdiálise. As concentrações do fármaco nas amostras foram determinadas por cromatografia líquida em tandem com espectrometria de massas (CLAE-EM/EM) utilizando metodologia validada. Os parâmetros farmacocinéticos foram determinados por abordagem não-compartimental (Phoenix®) e modelagem populacional (popPK) (Monolix®). Resultados e Discussão: A recuperação relativa (RR) das sondas foi independente da concentração de tobramicina e inversamente proporcional ao fluxo de perfusão. A RR determinada in vivo foi de 27,64 % ± 7,70 para animais sadios e 24,47 % ± 1,66 para animais infectados. A ligação às proteínas plasmáticas foi de 11,3 ± 1,9%. A infecção com formação de biofilme não alterou a farmacocinética plasmática da tobramicina, entretanto reduziu em cerca de 70% a penetração pulmonar do fármaco. As concentrações plasmáticas e teciduais foram simultaneamente descritas por um modelo farmacocinético populacional de dois compartimentos, tanto em animais sadios como infectados. A infecção, utilizada como covariável categórica, permitiu descrever as alterações no volume do compartimento periférico e na constante de eliminação do compartimento central devido à infecção. Conclusões: As concentrações plasmáticas da tobramicina, utilizadas para ajuste posológico, superestimam as concentrações ativas no pulmão infectado. O modelo popPK descrito permite a previsão das concentrações livres pulmonares da tobramicina em pulmão infectado, podendo auxiliar na otimização da terapia de pneumonias com P. aeruginosa formadora de biofilme. / Objective: To evaluate the influence of biofilm-forming Pseudomonas aeruginosa infection on tobramycin lung penetration by population pharmacokinetic modeling of plasma and microdialysate data in healthy and infected rats. Methodology: The infection was developed by intratracheal inoculation (109 CFU/mL) of P. aeruginosa (PA14 strain) to Wistar rats. In order to determine plasma and tissue concentrations, seven days after the inoculation the infected animals (n = 5) received tobramycin 10 mg/kg i.v. bolus dose via femoral vein. A healthy group (n = 6) was used as control. Free lung concentrations were determined in microdialysate samples obtained using CMA/20 probes. Microdialysis probes were calibrated in vitro by dialysis and retrodialysis and in vivo by retrodialysis. Tobramycin plasma protein binding was determined by microdialysis. Plasma and tissue concentrations were quantified by a developed and validated liquid chromatography in tandem with mass spectrometry (LC-MS/MS) method. Compartmental and non-compartmental analyses were carried out by Monolix™ and Phoenix™ software, respectively. Results and Discussion: Microdialysis probes relative recovery was independent of the tobramycin concentration and is inversely proportional to the perfusion flow rate investigated. The in vivo probe recovery was 27.64 % ± 7.70 (healthy rats) and 24.47 % ± 1.66 (infected rats). The plasma protein binding was 11.3 ± 1.9%. The biofilm-forming lung infection did not alter tobramycin plasma pharmacokinetics, however, reduced lung penetration in about 70%. The plasma and tissue concentrations-time profiles were simultaneously described by a two compartment popPK model in healthy and infected animals. The infection process, used as categorical covariate allowed describing the changes observed in the volume of the peripheral compartment and in constant rate of elimination from the central compartment. Conclusions: Tobramycin plasma concentrations, used for dosing adjustments, overestimate active concentrations in infected lung. The described popPK model allows predicting free tobramycin lung concentrations in infected lung and could be useful to optimize the treatment of pneumonia caused by biofilm-forming P. aeruginosa with this drug.

ABC運輸蛋白的過度表達是多重抗藥性（MDR）的重要機制之一，癌細胞會同時對結構上無關的抗癌藥物產生抗藥性。避免癌細胞的多重抗藥性有不同方法，其中用聚合物納米載體來攜帶易受多重抗藥性影響的抗癌藥物近年來獲得了很大的關注。本研究的目標在使用一個相對新穎的納米開發技術，被稱為瞬時納米沉澱（FNP），去開發一種運載著易受多重抗藥性影響的抗癌藥物的聚合物納米粒子系統。為此，我們使用專門設計的四流多進旋渦混合器（MIVM），把阿黴素（DOX），一種屬於蒽環類的抗癌藥物，亦同時作為P糖蛋白（P-gp）底物的藥物，包進在二嵌段共聚物內。 / 目的：本研究的目的是:（一）通過MIVM，利用瞬時納米沉澱去配製運載DOX的聚合物納米粒子；（二）辨别和優化納米粒子的大小，物理性能和運載DOX聚合物納米粒子的體外釋放速率；（三）檢查納米粒子的表面元素和化學組成;(四）評估優化納米粒子在抗藥性癌症細胞模型的抗腫瘤能力和抵抗多重抗藥性的能力。 / 方法：不同藥物（DOX)對聚合物比例的瞬時納米沉澱是通過在四流MIVM中混合溶在有機溶液二甲基甲酰胺（DMF)或二甲基酮（ACT)的鹽酸阿黴素(DOX.HC1)或阿黴素游離鹼（DOX.FB)和兩親性二嵌段共聚物[聚乙二醇-聚乳酸；分子量2000-10000]和反抗溶劑（含有氫氧化鈉為DOX.HCl或純淨水DOX+FB)來製備的。納米混懸劑的平均粒徑和粒度分佈會通過動態光散射粒度分析法去檢測，表面電荷會通過界達電位測量去檢測。阿黴素的包封率和載藥量會用紫外/可見光譜儀在波長為480 nm時測定。粒子形態將會用原子力顯微鏡（AFM)來去檢測，粒子表面的組合物將會用X-射線光電子能譜（XPS)來去檢測DOX聚合物納米粒子在不同pH值的的體外釋放會通過紫外/可見光譜儀去檢測。DOX聚合物納米粒子的體外細胞毒性會利用橫若丹明B比色法檢定，藥物積累和反轉運會利用流式細胞儀分析來測定。 / 結果：在適當優化鹽酸阿黴素（DOX.HC1)或阿黴素游離鹼（DOX.FB)的聚合物的比例後，我們成功製備了平均粒徑小於100 nm的DOX聚合物納米粒子(DOX.NP)與使用在有機溶液中DOX.HC1和水相的氫氧化鈉中和法相比，通過在有機溶液中的DOX.FB和純水作為反溶劑來製備的DOX.NP表現出類似的平均粒子大小（小於100 nm)，但顯示出更高的藥物包封率（48 %， 而不是中和法的25 %)。用DOX.FB製備的DOX.NP的載藥量可達14 %DOX.NP表現出pH依賴性的藥物釋放曲線，和在酸性pH值時更强的累積釋放率。X-射線光電子能譜顯示沒有阿黴素出現在納米粒子的表面上P-gp過度表達的LCC6抗藥性乳腺癌细胞的細胞毒性作用顯示了 DOX.NP和DOX.HC1在缓衝溶液中的差異並不顯著。相對DOX.HC1，流式細胞儀分析確定了 DOX.NP明顯增加了細胞攝取DOX的能力。此外，在外排後，DOX.NP在細胞內DOX的濃度顯示出了更長的保留時間。 / 結論：一種通過在多進旋過混合器（MIVM)進行反溶劑沉澱，用於配製具有可控的粒子大小運載DOX的聚合物納米粒子的快速，方便，和可重複性的方法已經被開發。配製的納米粒子顯示出pH值依賴性持續的藥物釋放曲線和更強的癌細胞攝取DOX能力。 / Over-expression of ATP-binding cassette (ABC) is one of the most important mechanisms responsible for multidrug resistance (MDR), in which tumor cells exhibit simultaneous resistance to structurally unrelated anticancer drugs. Various approaches have been attempted to circumvent MDR in cancer cells, among which polymeric nanocarrier for delivery of MDR-sensitive anticancer drugs has received considerable attention in recent years. The present project was aimed at developing a polymeric nanoparticle system using a relatively novel nanoparticle technology termed flash nanoprecipitation (FNP) for delivery of MDR-susceptible chemotherapeutic agents. To this end, doxorubicin (DOX), an anthracycline anticancer agent and a P-gp substrate, was incorporated into an amphiphilic diblock copolymer using a specially designed four-stream multi-inlet vortex mixer (MIVM). / PURPOSES: The objectives of the present study are: (a) to formulate DOX-loaded polymeric nanoparticles by FNP using an MIVM; (b) to characterize and optimize the particle size, physical properties and in vitro DOX release rate of the formulated nanoparticles; (c) to examine the surface elemental and chemical compositions of the formulated nanoparticles; (d) to evaluate the anti-tumor activity of the optimized nanoparticles and their ability to combat MDR in resistant cancer cell line models. / METHODS: FNP of DOX was effected in a four-stream MIVM by mixing organic solutions of doxorubicin hydrochloride (DOX.HCl) or doxorubicin free base (DOX.FB) and an amphiphilic diblock copolymer [polyethylene glycol-polylactic acid (PEG-PLA); MW2k-10 ki]n dimethylformamide (DMF) or acetone (ACT) at different drug-to-polymer ratios with an antisolvent (water containing sodium hydroxide for DOX.HCl or pure water for DOX.FB). The resulting nanosuspensions were characterized for mean particle size and size distribution by dynamic light scattering particle size analysis; surface charges by zeta potential measurements; drug encapsulation efficiency and drug loading by UV/visible spectroscopy at 480 nm; particle morphology by atomic force microscopy (AFM); and surface composition by x-ray photoelectron spectroscopy (XPS). In vitro DOX release from the nanoparticles was measured at different pHs by UV/visible spectroscopy. In vitro cytotoxicity was evaluated by Sulforhodamine B colorimetric assay, and drug accumulation and efflux were determined by flow cytometric analysis. / RESULTS: DOX-loaded polymeric nanoparticles (DOX.NP) with mean particle size below 100 nm were obtained after appropriate optimization of the DOX.HCl or DOX.FB to polymer ratio. Compared with the neutralization method using DOX.HCl in the organic phase and sodium hydroxide in the aqueous phase, DOX.NP prepared with DOX.FB in the organic phase and pure water as antisolvent exhibited a similar mean particle size (< 100 nm) but a significantly higher drug encapsulation efficiency (48% as opposed to 25% for the neutralization method). Drug loading of DOX.NP prepared with DOX.FB could reach up to 14%. DOX.NP exhibited a pH-dependent drug release profile with a much higher cumulative release rate at acidic pHs. XPS revealed that no DOX was present on the nanoparticle surface. The cytotoxic effect on P-gp over-expressing LCC6/MDR cell line revealed insignificant differences between DOX.NP and DOX.HCl in buffered aqueous media. DOX.NP exhibited a marked increase in DOX cellular uptake relative to free DOX, as determined by flow cytometric analysis. Furthermore, DOX.NP showed a significant retention of intracellular concentration of DOX after efflux. / CONCLUSION: A rapid, convenient, and reproducible method for generating DOX-loaded polymeric nanoparticles with controllable particle size through antisolvent precipitation in a multi-inlet vortex mixer has been developed. The formulated nanoparticles displayed a pH-dependent sustained drug release profile and an enhanced DOX uptake into cancer cells. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tam, Yu Tong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 119-130). / Abstracts also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.vii / LIST OF FIGURES --- p.x / LIST OF TABLES --- p.xiii / ABBREVIATIONS --- p.xv / Chapter CHAPTER 1. --- Introduction --- p.1 / Chapter 1.1 --- Rationale of the Study --- p.2 / Chapter 1.2 --- Doxorubicin --- p.3 / Chapter 1.2.1 --- Origin --- p.3 / Chapter 1.2.2 --- Physico-chemical properties --- p.6 / Chapter 1.2.3 --- Mechanism of Action --- p.7 / Chapter 1.2.4 --- Multidrug Resistance in Cancer --- p.7 / Chapter 1.2.4.1 --- Mechanisms of Multidrug Resistance --- p.8 / Chapter 1.3 --- Nanoparticles for Cancer Therapy --- p.9 / Chapter 1.3.1 --- Properties of Nanoparticles --- p.9 / Chapter 1.3.1.1 --- Small Particle Size --- p.10 / Chapter 1.3.1.2 --- High Payload Density --- p.11 / Chapter 1.3.1.3 --- Flexible Modification of Surface Properties --- p.11 / Chapter 1.3.2 --- Targeted Cancer Therapy --- p.12 / Chapter 1.3.2.1 --- Passive Tumor Targeting --- p.13 / Chapter 1.3.2.2 --- Active Tumor Targeting --- p.14 / Chapter 1.3.3 --- Reversal of Multidrug Resistance --- p.15 / Chapter 1.3.3.1 --- Endocytosis of Nanoparticles --- p.16 / Chapter 1.3.4 --- Nanoparticle Approaches to Anti-cancer Drug Delivery --- p.17 / Chapter 1.3.4.1 --- Liposomes --- p.18 / Chapter 1.3.4.2 --- Polymeric Nanoparticles --- p.18 / Chapter 1.4 --- Fabrication of Nanoparticles --- p.19 / Chapter 1.5 --- Aims and Scope of the Present Study --- p.21 / Chapter CHAPTER 2. --- Materials & Methods --- p.23 / Chapter 2.1 --- Materials --- p.24 / Chapter 2.1.1 --- Chemicals --- p.24 / Chapter 2.1.2 --- Materials for Cell Culture --- p.25 / Chapter 2.2 --- Methods --- p.26 / Chapter 2.2.1 --- Preparation of Doxorubicin Nanoparticles by Flash Nanoprecipitation --- p.26 / Chapter 2.2.1.1 --- Acid-Base Neutralization during Mixing --- p.26 / Chapter 2.2.1.2 --- Preparation of Doxorubicin Free Base before Mixing --- p.29 / Chapter 2.2.1.2.1 --- Doxorubicin Free Base Preparation --- p.29 / Chapter 2.2.2 --- Determination of Particle Size and Zeta Potential --- p.30 / Chapter 2.2.3 --- Co-stabilizers and Particle Stability --- p.30 / Chapter 2.2.4 --- Chemical Stability of Doxorubicin --- p.31 / Chapter 2.2.5 --- Determination of Encapsulation Efficiency --- p.31 / Chapter 2.2.5.1 --- Calibration Curve of Doxorubicin --- p.33 / Chapter 2.2.5.2 --- Dialysis --- p.33 / Chapter 2.2.5.3 --- Ultrafiltration --- p.35 / Chapter 2.2.6 --- Determination of Drug Loading --- p.35 / Chapter 2.2.6.1 --- Freeze Drying --- p.36 / Chapter 2.2.7 --- Morphological Examination --- p.36 / Chapter 2.2.7.1 --- X-ray Photoelectron Spectroscopy --- p.36 / Chapter 2.2.7.2 --- Atomic Force Microscopy --- p.36 / Chapter 2.2.8 --- In vitro release study --- p.37 / Chapter 2.2.8.1 --- Experimental Protocols --- p.37 / Chapter 2.2.8.2 --- Calculation of Cumulative Drug Release --- p.37 / Chapter 2.2.9 --- In vitro cytotoxicity study --- p.38 / Chapter 2.2.9.1 --- Sulforhodamine B Colorimetric Assay --- p.38 / Chapter 2.2.10 --- Cellular Uptake study --- p.39 / Chapter 2.2.10.1 --- Drug Accumulation Assay --- p.39 / Chapter 2.2.10.1 --- Drug Efflux Assay --- p.39 / Chapter 2.2.11 --- Analytical techniques --- p.40 / Chapter 2.2.11.1 --- UV/Vis Analysis --- p.40 / Chapter 2.2.11.2 --- HPLC Analysis --- p.40 / Chapter 2.2.12 --- Statistical analysis --- p.41 / Chapter CHAPTER 3. --- Results & Discussions --- p.42 / Chapter 3.1 --- Preparation of Doxorubicin Nanoparticles by Flash Nanoprecipitation --- p.43 / Chapter 3.1.1 --- Acid-Base Neutralization during Mixing --- p.44 / Chapter 3.1.1.1 --- Influence of Drug Concentration --- p.44 / Chapter 3.1.1.2 --- Influence of Alkaline Medium --- p.48 / Chapter 3.1.1.3 --- Influence of Drug-to-Polymer Ratios --- p.53 / Chapter 3.1.1.4 --- Particle Stability --- p.54 / Chapter 3.1.1.5 --- Co-stabilizers Tests on Stability --- p.55 / Chapter 3.1.1.5.1 --- Effect of PEG-PLA Co-polymers --- p.55 / Chapter 3.1.1.5.2 --- Effect of Co-stabilizers --- p.56 / Chapter 3.1.2 --- Preparation of Doxorubicin Free Base before Mixing --- p.62 / Chapter 3.1.2.1 --- Influence of Solvent System --- p.62 / Chapter 3.1.2.2 --- Influence of Drug-to-Polymer Ratios --- p.65 / Chapter 3.1.2.3 --- Drug Loading --- p.65 / Chapter 3.1.2.4 --- Particle Stability --- p.68 / Chapter 3.1.2.4.1 --- Concentrated Particle Stability --- p.73 / Chapter 3.2 --- Stability Studies on Doxorubicin Nanoparticle at Physiological and Cancer Cell pHs --- p.75 / Chapter 3.2.1 --- Chemical Stability --- p.75 / Chapter 3.2.2 --- Physical Stability --- p.77 / Chapter 3.3 --- In vitro Release Study --- p.79 / Chapter 3.4 --- Morphological Examination --- p.86 / Chapter 3.4.1 --- Zeta Potential --- p.92 / Chapter 3.5 --- In vitro Cellular Study --- p.93 / Chapter 3.5.1 --- Cellular Uptake Study --- p.93 / Chapter 3.5.1.1 --- Drug Accumulation and Drug Efflux --- p.93 / Chapter 3.5.2 --- Cytotoxicity of Blank Nanoparticles --- p.98 / Chapter 3.5.3 --- Cytotoxicity of DOX loaded Nanoparticles --- p.100 / Chapter CHAPTER 4. --- Conclusions --- p.106 / APPENDIX --- p.109 / REFERENCES --- p.118

Orientador: Celso Antônio Rodrigues / Banca: Anatonio Carlos Paes / Banca: Rosângela Gonçalves Peccinini / Resumo: Os equinos são acometidos frequentemente por processos sépticos na porção distal dos membros. Dentre as mais variadas condutas terapêuticas, destaca-se a perfusão regional intravenosa de antimicrobianos (PRIVA) utilizada como opção para combater a infecção local. A técnica objetiva proporcionar uma concentração elevada do fármaco no local acometido. O procedimento se baseia no posicionamento de um torniquete na região proximal ao local infectado, proporcionando a oclusão do fluxo sanguíneo venoso e arterial, permitindo que o fármaco permaneça na região acometida por no mínimo 30 minutos. Dentre os antimicrobianos indicados para a PRIVA, destaca-se o sulfato de gentamicina. Objetivou-se com este estudo avaliar e comparar a farmacocinética da gentamicina, no plasma e líquido sinovial da articulação do carpo de equinos, submetidos a PRIVA, utilizando dois modelos de torniquetes. Foram utilizados dez equinos hígidos, divididos aletoriamente em dois grupos de cinco animais, os torniquetes foram aplicados no terço médio do rádio, sempre pelo mesmo aplicador. O grupo GTT recebeu torniquete de látex tubular, enquanto que o grupo GTE, faixa de Esmarch. Após o posicionamento dos torniquetes, acessou-se a veia cefálica e administrou-se 2,2 mg/kg de sulfato de gentamicina, adicionado à solução fisiológica q.s.p. 40 mL. Previamente ao posicionamento do torniquete, procedeu-se avaliação ultrassonográfica color Doppler no membro tratado para visualização da veia cefálica e artéria mediana. Amostras de líquido sinovial do carpo e sangue foram obtidas nos momentos M0, M15', M30', M1h, M2h, M4h, M6h, M8h, M12h, M24h e M48h para a realização da análise de concentração de gentamicina por cromatografia líquida de alta eficiência (CLAE), bem como para os cálculos dos parâmetros farmacocinéticos em modelo monocompartimental. Não foram encontradas diferenças significativas dos parâmetros ... / Abstract: The horses are often affected by septic processes in the distal portions of the limbs. Among the various therapeutic approaches, there is intravenous regional perfusion of antimicrobials (IRPA) used as an option to combat local infection. The technique aims to provide a high concentration of the drug in the affected area. The procedure is based on placing a tourniquet proximal to the infected area in the region of providing the venous and arterial blood flow occlusion, allowing the drug to remain on the affected area at least 30 minutes. Among the antimicrobial agents indicated for IRPA, highlight the gentamicin sulfate. The objective of this study was to evaluate and compare the pharmacokinetics of gentamicin in plasma and synovial fluid of carpal joint of horses undergoing IRPA using two models of tourniquets. Were used ten healthy equines, randomly divided into two groups of five animals, the tourniquets were applied in the middle third of the radius, always by the same applicator. The tourniquet GTT group received latex tube, while the GTE group Esmarch bandage. After positioning the tourniquets, the cephalic vein had been accessed and administered 2.2 mg / kg gentamicin sulfate, added to 40 mL saline q.s.. Prior to placement of the tourniquet, proceeded color Doppler ultrasonographic evaluation in the treated limb for viewing the median artery and cephalic vein. Samples of synovial fluid and blood carpal were obtained in the moments M0, M15 ', M30', M1h, M2h, M4h, M6h, M8h, M12h, M24h and M48h to perform the analysis of concentration of gentamicin by high-performance liquid chromatography (HPLC), as well as calculations of pharmacokinetic parameters in monocompartiment model. No significant differences in pharmacokinetic parameters were found between treatment groups (P>0.05). The results showed that the two types of tourniquets in IRPA can be used in a proximal region of forelimb in horses, if applied properly, both are capable of ... / Mestre

No description available.

A Política de Saúde no Brasil, que inclui a Política Nacional de Medicamentos, a criação da Agência Nacional de Vigilância Sanitária (ANVISA), a promulgação da Lei de Medicamentos Genéricos, bem como a publicação das Resoluções que estabelecem os critérios técnicos para seu registro, revolucionou o mercado farmacêutico brasileiro na última década, introduzindo vários conceitos como Equivalência Farmacêutica e Terapêutica, Biodisponibilidade e Bioequivalência. Tais conceitos constituem as bases científicas para a implantação dos medicamentos genéricos, aliados à certificação de Boas Práticas de Fabricação e Controle de Qualidade (BPFs). Após cinco anos, os medicamentos genéricos representam cerca de 10% do mercado farmacêutico brasileiro em unidades com redução mínima de 35% no preço do genérico em relação ao medicamento de referência, em função de que o fabricante não necessita investir em estudos clínicos para comprovação da eficácia e segurança, garantidas pela comprovação da equivalência terapêutica com o medicamento de referência. O mercado brasileiro de genéricos é muito atrativo, pois 86% dos fármacos registrados no país não são patenteados e mais de 50% da população brasileira não tem acesso a medicamentos por problemas econômicos. Por outro lado, 70% do mercado farmacêutico brasileiro é composto por medicamentos similares, que somente em 2003 passaram a ter regulamentação técnica específica para comprovação da eficácia e segurança. Dessa forma, apesar de vasta literatura existente, justifica-se a sistematização dos aspectos técnicos e científicos que fundamentam a regulamentação técnica de biodisponibilidade relativa e bioequivalência com aplicabilidade na XXIII capacitação de recursos humanos em Biofarmacotécnica e na área regulatória no país. A análise da implantação e evolução das regulamentações técnicas, bem como, das conclusões dos estudos de bioequivalência e biodisponibilidade relativa avaliados pela ANVISA, torna-se ferramenta essencial para a compreensão dos aspectos regulatórios dos estudos de biodisponibilidade relativa e bioequivalência adotados. Considerando-se, ainda, a importância da racionalização de recursos e a necessidade de manutenção da qualidade dos medicamentos genéricos e similares no Brasil, com base na literatura científica mundial e no Banco de Dados da ANVISA, avaliou-se a viabilidade do emprego do Sistema de Classificação Biofarmacêutica (SCB), proposta elaborada por Amidon et al. (1995), para isenção da necessidade de realização de estudos de biodisponibilidade relativa/bioequivalência para o registro e pós-registro de medicamentos no Brasil. Assim sendo, concluiu-se que: a implantação de medicamentos genéricos no Brasil significou grande avanço técnico-científico para as áreas regulatória, acadêmica e industrial; a implementação e o aprimoramento da regulamentação técnica para medicamentos genéricos ocorreu devido à sua revisão contínua e publicação de quatro novas versões no período de 2.000 a 2.004; a experiência adquirida foi a base para a elaboração da regulamentação para medicamentos similares; a reprovação de estudos de bioequivalência de fármacos da Classe I do SCB é um alerta para que um estudo aprofundado das causas e da aplicação desse sistema na isenção de estudos in vivo visando o registro de medicamentos no Brasil seja realizado. / Health Policy in Brazil, which includes the National Policy on Medicines, the creation of the National Agency for Sanitary Vigilance (ANVISA), the promulgation of the Generic Medicines Law, as well as the publication of Resolutions establishing technical criteria for their registration, has revolutionized the Brazilian pharmaceutical market over the past decade introducing a number of concepts such as Pharmaceutical and Therapeutic Equivalence, Bioavailability and Bioequivalence. Such concepts have comprised the scientific basis for the implementation of generic medicines, in conjunction with the certification of Good Manufacturing and Quality Control Practices (BPFs). Five years on, generic medicines account for around 10% of the Brazilian pharmaceutical market in units, with a price cut in generics of at least 35% compared with the corresponding reference medicine, as a result of manufacturers not having to invest in clinical trials to prove efficacy and safety which are guaranteed by proof of therapeutic equivalence to the reference medicine. The Brazilian generics market is highly attractive since 86% of active principles registered in the country are not patented, and given that more than 50% of the Brazilian population does not have access to medicines for economic reasons. However, 70% of the Brazilian pharmaceutical market is made up of similar medicines, which only gained specific technical regulation for proof of efficacy and safety in 2003. Therefore, despite the vast body of literature available, a systematic approach for technical and scientific aspects underlying the technical regulation of relative bioavailability and bioequivalence is warranted, where this may also apply to both training of human resources in Biopharmaceutics and to the regulatory area in the country. Analysis of the XXVI implementation and evolution of technical regulations, along with the conclusions of ANVISA-assessed bioequivalence and relative bioavailability trials, have become an essential tool in understanding the regulatory aspects of the studies on relative bioavailability and bioequivalence adopted. Furthermore, given the continuing importance of rationalizing resources and the need to maintain the quality of generic medicines and similars in Brazil, the viability of employing the Biopharmaceutical Classification System (SCB) proposed by Amidon et al. (1995) dispensing with the need to run relative bioavailability/bioequivalence studies for the registration and post-registration of medicines in Brazil, has been assessed based on world scientific literature and ANVISAs database. Thus it was concluded that the implementation of generic medicines in Brazil has represented a major technical and scientific step forward for the regulatory, academic and industrial areas. Moreover, the implementation and refining of the technical regulations for generic medicines has taken place as a result of ongoing review and publication of four new versions between 2000 and 2004. The experience gained has provided the foundation in devising technical regulations for similar medicines. Finally, the rejection of bioequivalence studies for medicines from Class 1 SCB may serve as a warning that more in-depth studies into the root causes, and the application of this system in the absence of in-vivo studies for registration of medicines in Brazil, should be undertaken.

Introdução: O monitoramento plasmático e a avaliação farmacocinética são importantes ferramentas empregadas no controle terapêutico. O sufentanil é responsável pela estabilização hemodinâmica do paciente com melhor supressão da resposta neuroendócrina comparado ao seu análogo o fentanil. Este fármaco tem sido largamente utilizado em cirurgias cardíacas devido também, a sua menor meia vida plasmática em relação ao fentanil o que permite uma rápida recuperação cirúrgica de pacientes submetidos a tais procedimentos. Objetivo: Realizar o monitoramento plasmático do sufentanil em pacientes submetidos à cirurgia cardíaca com e sem circulação extracorpórea (CEC) e posteriormente avaliar a farmacocinética do mesmo. Casuística: Investigaram-se 42 pacientes de ambos os sexos, portadores de insuficiência coronária crônica e candidatos à cirurgia eletiva de revascularização do miocárdio com ou sem circulação extracorpórea, internados na enfermaria clínica do Instituto do Coração do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Métodos - Etapa Clínica: Os pacientes inclusos neste estudo foram preparados para a realização do procedimento cirúrgico. Na indução da anestesia administrou-se 0,5 &#181;g/Kg de sufentanil através de bolus, seguido de infusão de manutenção de 0,5 &#181;g/Kg-h. Coletaram-se amostras seriadas de sangue no intra-operatório pós-indução e no pós operatório até 36 h após administração do sufentanil. A infusão de sufentanil foi suspensa no momento do término da sutura da pele. O plasma foi separado e transferido para tubos de polietileno devidamente identificados e armazenados em temperatura - 20 ºC até a realização da análise. Métodos - Etapa Analítica: As concentrações plasmáticas foram determinadas através do método desenvolvido e validado por cromatografia líquida acoplada a espectrometria de Massas (LC-MS/MS). As amostras biológicas foram extraídas através de extração líquido-líquido em meio alcalino as quais foi adicionado fentanil como padrão interno. A separação cromatográfica foi obtida através de uma coluna C18 e fase móvel constituída por acetonitrila:acetato de amônio 5 mM + ácido fórmico 0,25% (70:30 v/v). O espectrômetro triplo quadro pólo, eletrospray positivo, monitorou as transições de massa entre 387,0>238,0, 285,7>165,1 e 337,0>188,0, para sufentanil, morfina e fentanil respectivamente. Métodos - Etapa Estatística: A modelagem farmacocinética foi realizada através da aplicação do software NonCompartmental Analysis, PK Solutions 2.0. O índice de significância empregado foi de 5% (p<0,05). Utilizou-se o teste Qui-quadrado para avaliação da distribuição do gênero e o teste t-student para os parâmetros idade, peso, altura e IMC. Para os dados de concentração plasmática foi utilizado o teste não paramétrico de Friedman seguido do teste post-hoc de Dunn\'s para comparação dos momentos da cirurgia do grupo que foi submetido a CEC. Para comparação dos momentos entre os grupos (submetido a CEC versus sem CEC) aplicou-se o teste de Wilcoxon. Resultados: Os pacientes cirúrgicos incluídos no protocolo eram adultos de ambos os sexos 9F/33M, com média de idade de 62,48 anos, 68,66 kg e IMC de 25,52 kg/m2. Destes, 30 pacientes referem-se ao Grupo com CEC e 12 são do grupo sem CEC. As doses totais médias de sufentanil administradas ao grupo com CEC e ao grupo sem CEC foram semelhantes, 3,23 ±0,67&#181;g/kg e 3,53 ±0,90&#181;g/kg respectivamente. O método analítico proposto apresentou-se linear no intervalo entre 0,05 - 500 ng/mL para o sufentanil e 10 - 1000 ng/mL para a morfina. Os dados obtidos na validação do método apresentaram especificidade, linearidade, robustez, precisão e exatidão. As concentrações plasmáticas obtidas forma estatisticamente diferentes entre os grupos com CEC e sem CEC. Durante o procedimento de circulação extra-corpórea foi observada intensa flutuação das concentrações plasmáticas de sufentanil. Foi aplicado o modelo tri-compartimental na avaliação cinética do sufentanil. Foram determinados os seguintes parâmetros farmacocinéticos: meia-vida de eliminação (t1/2), alfa (&#945;), beta (&#946;) e gama (&#947;), área sob a curva (ASC), volume de distribuição (VD) e a depuração plasmática total (Cl). Destes, apenas T1/2 (&#947;) apresentou diferença significativa entre os grupos. Conclusões: O método proposto foi empregado satisfatoriamente na avaliação cinética do sufentanil. O protocolo realizado e os limites de quantificação do método analítico desenvolvido oportunizaram o emprego do modelo farmacocinético tricompartimental para o fármaco estudado. As concentrações plasmáticas de sufentanil foram afetadas pela CEC o que implicou na diferença significativa entre as meia-vida de eliminação &#947; calculadas para os grupos com CEC e sem CEC. / Introduction: The plasma monitoring and the pharmacokinetic assessment are important tools employed in therapeutic control. Sufentanil is responsible for the hemodynamic stabilization of the patient with a better suppression of the neuroendocrine response compared to its analogue fentanyl. This pharmaco has been widely used in cardiac surgery also due to its shorter plasma half-life in relation to fentanyl, which allows a fast surgical recovery of patients who have undergone such procedures. Objectives: Perform the plasma monitoring of sufentanil in patients undergoing cardiac surgery with or without extracorporeal circulation and afterwards assess the pharmacokinetics of it. Study design: 42 patients of both genders with chronic coronary disease and candidates to elective surgery of myocardial revascularization with or without extracorporeal circulation were investigated. They were hospitalized in the clinical ward of the Heart Institute Hospital of the Medicine Faculty Clinic of the University of São Paulo. Methods- Clinical phase: the patients included in this study were prepared for the performance of the surgical procedure. In the anesthesia induction 0,5 &#181;g/Kg was administered through bolus, followed by maintenance infusion 0,5 &#181;g/Kg-h. Serial blood samples were collected in the intra-operatory after induction and in the pos-toperatory after 36 h of administration of sufentanil. Sufentanil infusion was suspended just as the skin suture was finished. The plasma was separated and transferred to the identified polyethylene test-tube and stored in a temperature -20ºC until the analyses. Methods - Analytical phase: The plasma concentrations were determined through the developed method and validated by liquid chromatography mass spectrometry (LC-MS/MS). The biological samples were extracted through liquid-liquid extraction in alkaline mean, to which fentanyl was added as an internal pattern. The chromatographic separation was obtained through a C18 column and the mobile phase constituted by acetonitrile: 5 mM ammonia acetate + 0,25% formic acid (70:30 v/v). The triple-quad pole spectrometry, positive electrospray, monitored the mass transitions among 387.0>238.0, 285.7>165.1 and 337.0>188.0, for sufentanil, morphine and fentanyl , respectively. Methods - Statistical phase: The pharmacokinetic modeling was performed through the application of the software NonCompartmental Analysis, PK Solutions 2.0. The significance index employed was 5% (p<0,05). The qui-square test was used for the assessment of gender distribution and the t-student test for the age, weight, height and IMC parameters. The nonparametric test of Friedman was used for the plasma concentration, followed by Dunn´s post-hoc test for the comparison of the surgery moments of the group that was submitted to extracorporeal circulation. The test Wilcoxon was applied for the comparison of the moments between the groups (submitted to extracorporeal circulation versus without extracorporeal circulation). Results: The surgical patients included in the protocol were adults of both genders 9F/33M, with an average age of 62.48 years old, 68.66 kg and IMC of 25.52 kg/m2. 30 patients are from the group with extracorporeal circulation and 12 are from the group without extracorporeal circulation. The average total doses of sufentanil administered to the group with extracorporeal circulation and to the group without extracorporeal circulation were similar, 3.23 ±0.67&#181;g/kg and 3.53 ±0.90&#181;g/kg respectively. The analytical method proposed proved linear in the interval between 0.05 - 500 ng/mL for sufentanil and 10 - 1000 ng/mL for morphine. The data obtained in the validation proved specificity, linearity, robustness, precision and accuracy. The plasma concentrations obtained were statistically different between the groups with extracorporeal circulation and without extracorporeal circulation. During the extracorporeal circulation procedure an intense fluctuation was observed in the plasma concentration of sufentanil. The tri-compartmental model was applied in the kinetic assessment of sufentanil. The following pharmacokinetic parameters were determined: half-life elimination (t1/2), alpha (&#945;), beta (&#946;) and gamma (&#947;), area under the curve, distribution volume and the total plasma depuration. Only T1/2 (&#947;) presented a significant difference between the groups. Conclusions: The proposed method was satisfactorily employed in the kinetic assessment of sufentanil. The protocol carried out and the quantification limits of the analytical method developed opportunized the employment of the tri-compartmental pharmacokinetic model for the pharmaco studied. The plasma concentrations of sufentanil were affected by the extracorporeal circulation, which implied in the meaningful difference between the elimination half-life &#947; calculated for the groups with extracorporeal circulation and without extracorporeal circulation.

Flavonoids are polyphenolic secondary metabolites found in plants that have bioactive properties including antiviral, antioxidant, and anticancer. Two isomeric flavone were extracted from Gnaphalium elegans and Achyrocline bogotensis, plants used by the people from the Andean region of South America as remedies for cancer. 5,7-dihydroxy-3,6,8-trimethoxy-2-phenyl-4H-chromen-4-one (5, 7–dihydroxy- 3, 6, 8 trimethoxy flavone/ flavone A) and 3,5-dihydroxy-6,7,8-trimethoxy-2-phenyl-4H-chromen-4-one (3, 5–dihydroxy-6, 7, 8–trimethoxy flavone/ flavone B) have shown antineoplastic activity against colon cancer cell lines dependent upon their differentiation status. Pharmacokinetic studies reported herein were used to determine dosing for antitumor assays, as well as determine target tissue concentration. These included the development of methods to extract the flavones from plasma or colon tissue and reverse phase high performance liquid chromatography methods for quantification. Quantification methods were linear (r2 ≥ 0.99) with plasma calibration curves ranging from 250 - 2,500 ng/mL and 2,500 - 100,000 ng/mL for both flavones and colon calibration curves ranging from 250 – 100,000 ng/g (flavone A) and 1,000-25,000 ng/g (flavone B). Intravenous administration of a 20 mg/kg dose in rats yielded half-lives of 83.68 ± 56.61 and 107.45 ± 53.31 minutes with clearance values of 12.99 ± 13.78 and 80.79 ± 35.06 mL/min/kg for flavones A and B, respectively. Analysis of colon tissue yielded concentrations of 1639 ± 601 ng/g (flavone A) and 5975 ± 2480 ng/g (flavone B), suggesting both may be good candidate for individual or adjunct therapy for colon cancer due to distribution to the target tissue. Preliminary studies in colon cancer cells CaCo 2 and HCT 116 using either flavone in combination with 5-fluorouracil (5-FU) suggested synergistic activity of these compounds. The combination treatment increased induction of apoptosis by enhancing the DNA damaging mechanism of 5-FU. In vivo, preliminary xenograft experiments using HCT 116 cells showed smaller tumors in mice dosed with flavone B as compared to the 5-FU or combination treatment. Further experiments are warranted to confirm these observations.