Elucidation of Arctigenin Biotransformation through Its Biopharmaceutics and Pharmacokinetics Profiling GAO, Qiong A Thesis Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy in Pharmacy The Chinese University of Hong Kong September 2014 Thesis Assessment Committee Professor Lawrence William BAUM (Chair) Professor ZUO Zhong (Thesis Supervisor) Professor LIN Ge (Committee Member) Professor TO Kin Wah (Committee Member) Professor MORRIS Marilyn E. (External Examiner) Professor HSIAO Wendy Wen-luan (External Examiner) Table of Contents Table of Contents ................................................................................................................ I Acknowledgements ........................................................................................................... V Publications ...................................................................................................................... VI Abstract ......................................................................................................................... VIII List of Tables .................................................................................................................. XII List of Figures ............................................................................................................... XIII List of Abbreviations .................................................................................................... XVI Chapter One Introduction 1 1.1 Background of arctigenin .......................................................................................... 1 1.1.1 Origin of arctigenin ............................................................................................ 1 1.1.2 Pharmacological effects of arctigenin and its containing herbs .......................... 7 1.1.2.1 Anti-inflammation and anti-oxidation ......................................................... 7 1.1.2.2 Anti-cancer ................................................................................................. 16 1.1.2.3 Anti-virus and anti-bacteria ....................................................................... 25 1.1.2.4 Other miscellaneous therapeutic activities ................................................. 28 1.1.3 Analyses of arctigenin in-vitro and in-vivo ...................................................... 34 1.1.3.1 Identification of arctigenin and arctiin in-vitro .......................................... 34 1.1.3.2 Identification and quantification of arctigenin and arctiin in-vivo ............ 51 1.1.4 Biopharmaceutics and pharmacokinetics studies of arctigenin and arctiin ...... 54 1.1.5 Clinical usage and efficacy of arctigenin, arctiin and Arctium Lappa .............. 57 1.2 Rationale and objectives of the current study ......................................................... 60 Chapter Two Development of UPLC/MS/MS Method for Simultaneous Detection of Arctigenin and Its Metabolites in Rat Plasma 67 2.1 Introduction ............................................................................................................. 67 2.2 Materials and reagents............................................................................................. 69 2.3 Methods ................................................................................................................... 70 2.3.1 Identification of major metabolites of arctigenin in-vivo ................................. 70 2.3.1.1 Metabolites of arctigenin in plasma ........................................................... 70 2.3.1.2 Metabolites of arctigenin in bile ................................................................ 71 2.3.2 Quantification of metabolites and arctigenin in-vivo ........................................ 72 2.3.2.1 Chemical synthesis of AA, the major metabolites found in-vivo .............. 72 2.3.2.2 Quantification of identified arctigenin metabolites ................................... 73 2.3.2.3 Preparation of standard solutions, calibration standards and quality control (QC) samples ............................................................................................. 73 2.3.2.4 Plasma sample preparation ......................................................................... 74 2.3.2.5 Instrumentation and chromatographic conditions ...................................... 75 2.3.2.6 Method Validation ...................................................................................... 76 2.4 Results and discussions ........................................................................................... 79 2.4.1 Identification of arctigenin metabolites in-vivo ................................................ 79 2.4.2 Simultaneous determination of arctigenin and its metabolites in rat plasma .... 82 2.4.2.1 Selection of internal standard ..................................................................... 86 2.4.2.2 Sample preparation .................................................................................... 86 2.4.2.3 Instrumentation and chromatographic conditions ...................................... 87 2.4.3 Validation of the developed UPLC/MS/MS method ......................................... 90 2.5 Conclusions ............................................................................................................. 95 Chapter Three Absorption, Metabolism and Disposition of Arctigenin in Intestine, Plasma and Liver 96 3.1 Introduction ............................................................................................................. 96 3.2 Materials and reagents........................................................................................... 101 3.3 Methods ................................................................................................................. 102 3.3.1 LC/MS/MS method for quantification of arctigenin and its metabolites ........ 102 3.3.2 Absorption, metabolism and disposition of arctigenin in intestine ................. 103 3.3.2.1 Caco-2 cell monolayer model .................................................................. 103 3.3.2.2 Rat in-situ single-pass intestinal perfusion model ................................... 106 3.3.2.3 Intestinal metabolism of arctigenin and arctigenic acid ........................... 108 3.3.2.4 Metabolism of arctigenin and metabolites in gastrointestinal content ..... 109 3.3.3 Hydrolysis of arctigenin in plasma ................................................................. 111 3.3.3.1 Kinetics study of arctigenin hydrolysis in rat plasma .............................. 111 3.3.3.2 Identification of plasma esteraseresponsive for arctigenin hydrolysis .... 112 3.3.3.3 Enzyme kinetics of arctigenin in human recombinant paraoxonase 1 ..... 112 3.3.4 Hepatic metabolism ......................................................................................... 113 3.3.5 Formation pathways of secondary metabolites ............................................... 113 3.3.6 Data Analysis .................................................................................................. 114 3.3.6.1 Caco-2 cell monolayer model .................................................................. 115 3.3.6.2 Rat intestinal perfusion model ................................................................. 116 3.3.6.3 Inhibition studies of arctigenin plasma hydrolysis .................................. 117 3.3.6.4 In-vitro metabolism study ........................................................................ 117 3.4 Results and discussions .......................................................................................... 117 3.4.1 Caco-2 monolayer transport model ................................................................. 117 3.4.2 Rat in-situ single-pass intestinal perfusion model .......................................... 120 3.4.3 Metabolism of arctigenin and metabolites in gastrointestinal content ............ 124 3.4.4 Hydrolysis of arctigenin in plasma ................................................................. 128 3.4.4.1 Kinetics study of arctigenin hydrolysis in rat plasma .............................. 128 3.4.4.2 Identification of plasma esterase responsive for arctigenin hydrolysis and enzyme kinetics of arctigenin in human recombinant paraoxonase 1 ..... 128 3.4.4.3 In-vitro metabolism of arctigenin in rat intestinal and liver microsome .. 133 3.5 Conclusion .............................................................................................................. 137 Chapter Four Pharmacokinetics of Arctigenin after Intravenous and Oral Administration in Rats 138 4.1 Introduction ............................................................................................................ 138 4.2 Materials and reagents ............................................................................................ 142 4.3 Methods .................................................................................................................. 142 4.3.1 UPLC/MS/MS method for quantification of arctigenin and its metabolites in rat plasma ........................................................................................................ 142 4.3.2 Intravenous and oral administration ................................................................ 143 4.3.3 Integrated semi-mechanistic pharmacokinetics model for arctigenin and its metabolites .....................................................................................................