STUDY ON HERB-DRUG INTERACTIONS A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Liping Yang B.Sci. School of Health Sciences College of Science, Engineering and Health RMIT University March 2009 i Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to quality for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; and, any editorial work, paid or unpaid, carried out by a third party is acknowledged. Liping Yang Date ii Acknowledgements I am most grateful to my supervisors, Associate Professor Chun Guang Li, Associate Professor Shufeng Zhou and my consultant Professor Charlie Changli Xue for their inspiration, outstanding intellect, encouragement, guidance, and support during my study for this degree. Sincere thanks also go to my consultant, Professor Hualiang Jiang and Professor Hong Liu, at Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China, for their invaluable support for my in silico studies. Without your support, the thesis would not have been possible. I would like to greatly thank Dr. Mingyue Zheng, Dr. Cheng Luo and Professor Weiliang Zhu for their knowledge, friendship and support when I conducted my project in Shanghai. Special thanks go to Dr. Tony Lin Zhang for his support in my systematic review of relevant literatures. Many thanks also go to Dr. George Lenon for his help in ethnic application. I wish to thank Dr. Shujun Sheng for his valuable suggestion for my project. The help of the staff and students from the Discipline of Chinese Medicine, School of Health Sciences, RMIT University, Victoria, Australia, have also been most helpful. I would like particularly to acknowledge the contribution of Dr. Ye Shen, Mr. Michael Owen, and Dr. Thomas Cheung. I wish to thank Professor Emilio Badoer and Dr Martin Stebbing, School of Medical Sciences, RMIT University, for their help and understanding since the first day of my study in RMIT University. Many thanks also go to Dr. Neil Owens and Dr. Ari Kantzides for their kind support for my study in RMIT University. Most of all I would like to thank my parents, Yirao Yang and Shuqiu Li, who have been a constant source of support – emotional and moral– during my postgraduate years, and this thesis would not have existed without them. Finally, I would like to thank my son, Harry Xin, who is my spiritual source of support to go through all the hard years. iii Summary of Thesis Herbal medicines, such as St John's wort, garlic, gingko, and ginseng, are commonly used complementary therapies. These products are often available over the counter and self-administered along with conventional therapeutic drugs, which raise concerns of potential herb-drug interactions. Most reported herb-drug interactions are pharmacokinetic interactions, through modulation of the activities of cytochrome P450 (CYP), and/or drug transporters. The changes of CYP activities by herbal ingredients may lead to modifications of efficacies of prescribed drugs or result in adverse reactions. Hence, understanding of mechanism of interactions of herbal ingredients with human CYPs is important in evaluating and predicating potential herb-drug interactions and necessary for the safe practice of herbal and conventional medicines. The human CYP enzymes are a superfamily which consists of at least 57 functional CYP genes. Among them, CYP1A2, 2C9, 2C19, 2D6 and 3A4/5 are the most important enzymes responsible for the Phase I metabolism of therapeutic drugs. There is a large variability in the expressions and activities of different CYPs, which are impacted by numerous factors, including genetic (e.g., mutation), host (e.g., diseases), and environmental (e.g., inducers and inhibitors), which makes the metabolism of drugs highly variable in individuals. Inhibition of CYP enzymes is one of the most common causes of harmful drug–drug interactions and some severe adverse reactions due to drug-CYP interactions, which has led to the recent withdrawal of several drugs from the market, such as the nonsedating antihistamine terfenadine. When different compounds (e.g., a drug and herbal compound) are co-administered, they may compete at the same active site of CYPs, resulting in potential inhibition. We hypothesize that the atom-atom interactions between the ligands and the residues at the active site of CYPs determine the substrate and inhibitor specificity of individual CYPs. To test our hypothesis, we conducted a series of experiments including in vitro assays to determine inhibitory actions of a variety of natural compounds on human CYPs, pharmacokinetic-based predication of in vivo situation using the in vitro data; and in silico studies to explore the ligand-CYP interactions using docking and pharmacophore modeling methods. We first determined the inhibitory effects (IC50) of 56 herbal compounds on activities of five human drug metabolising CYPs (CYP1A2, 2C9, 2C19, 2D6 and 3A4) in vitro using a high iv throughput approach. The tested herbal components included a variety of structurally distinct compounds such as triterpenoids of danshen (Salvia miltiorrhiza), flavonoids and their glycoside derivatives, saponine, other glucosides, lactones, alkaloids, and acids. A small number of them are found to significantly inhibit human CYP1A2, 2C9, 2C19, 2D6 and 3A4 with differential potency, including tanshinone I, tanshinone IIA, cryptotanshinone, baicalein, quercetin, silybin, osthole and γ-schisandrin. Based on the in vitro data obtained, we predicted metabolic herb-drug interactions of these compounds in vivo with the application of appropriate pharmacokinetic principles. Some predicting results were consistent with published clinical reports. For example, the prediction of S. miltiorrhiza increasing the AUC value of warfarin is consistent with the results from clinical case reports. However, a marked disparity has been observed when some predictions are compared with results from clinical studies. For example, the prediction of S. mariani (containing silybin) increasing the AUC of indinavir (a CYP3A4 substrate) is not in agreement with the result of a clinical report where the plasma concentration of indinavir was not altered by co-administered silymarin in healthy volunteers. Finally, we studied the interactions of a series of ligands including substrates and inhibitors with CYP1A2 using docking and pharmacophore modeling approaches. We have identified 6 residues at the active site of CYP1A2 which are essential for ligand recognition. Furthermore, the relative potency of potential inhibitors could be predicted through analysis of hydrophobic interactions between the ligand and the 6 essential residues at the active site of CYP1A2. Moreover, we developed a pharmacophore model on the basis of the common features of known CYP1A2 inhibitors. In combination with the docking results, the established pharmacophore model could be applied for screening novel CYP1A2 inhibitors. In conclusion, our in vitro and in silico studies have provided further insights into the interactions of ligands including herbal components with the active site of CYP1A2, which may be useful for the future studies of herb-drug and herb-CYP interactions. Further studies are warranted to explore the mechanisms underlying herb-CYP and herb-drug interactions. v Table of Content STUDY ON HERB-DRUG INTERACTIONS .................................... 错误！未定义书签。 Declaration ....................................................................................................................... ii Acknowledgements ........................................................................................................ iii Summary of Thesis ......................................................................................................... iv Table of Content ............................................................................................................. vi Table of Figures ............................................................................................................ viii Table of Tables ............................................................................................................... ix Publications ..................................................................................................................... x Abbreviations .................................................................................................................. xi Chapter 1 General Introduction ........................................................................................... 1 1.1 An Introduction to Human Cytochrome P450s ...................................................... 1 1.2 Biology and Pharmacology of Human CYPs ......................................................... 4 1.2.1 Human CYP1A2 enzyme ........................................................................... 4 1.2.2 Human CYP2C9 enzyme .......................................................................... 18 1.2.3 Human CYP2D6 enzyme ......................................................................... 22 1.2.4 Human CYP3A4 enzyme ......................................................................... 28 1.2.5 Other CYPs ............................................................................................... 32 1.3 Genetic Mutations of Human