African Potato (Hypoxis hemerocallidea), (AP) is an African traditional medicine (TM) that is commonly used for various nutritional/medicinal purposes and also by people infected with the human immuno deficiency virus HIV and AIDS patients as an immune booster. The use of AP has also been recommended by the former Minister of Health of South Africa for use by HIV positive people. The main phytochemical component of AP is a norlignan glucoside, hypoxoside, and other relatively minor components have also been reported. A recent in vitro study reported the effects of AP extracts, hypoxoside and rooperol (the metabolite of hypoxoside) on human metabolic enzymes such as the cytochrome P450 (CYP450) group of enzymes and also on the transporter protein, p-glycoprotein (P-gp). This research focussed on investigating the clinical significance of those in vitro effects on the pharmacokinetics of efavirenz (EFV) in humans. EFV was chosen as the substrate drug because it is in first-line regimen of treatment of HIV/AIDS in South Africa, and also has been reported to be a substrate for the specific CYP isozymes, 3A4 and 2B6, in common with APs metabolic involvement with 3A4. A high performance liquid chromatography method with ultra-violet detection (HPLC-UV) for the quantitative determination of EFV in plasma was developed and successfully validated according to international standards with good reproducibility, accuracy, recovery, linear response and requisite sensitivity. The preparation of the plasma samples for analysis was effected by using a simple and rapid precipitation method, and the mobile phase consisted of readily available solvents. EFV in plasma samples was found to be stable under the relevant storage conditions studied. The oral dose of AP, administered as a freshly prepared traditional decoction, was standardised based on the hypoxoside content, and the quality of all the AP decoctions was analysed immediately prior to administration, using a validated HPLC-UV method. A single dose, two-phase sequential study was conducted over a period of 31 days in 10 healthy volunteers. The clinical study was approved by the Rhodes University Ethical Standards Committee, and all the participants agreed to the conditions of the study by giving their informed consent. On day 1 of the study, human subjects were administered a 600 mg EFV tablet and blood samples were collected before dosing and at various intervals over a period of 48 hr post dosing. From day 16, a traditionally prepared AP decoction was administered daily at a standardized dose of 15 mg/kg/day per subject until day 30. On day 29, volunteers were administered a single 600 mg dose of EFV as was done on day 1. Plasma samples were harvested immediately after blood sample collection and frozen at -80 ºC until assayed. Geometric mean ratios of relevant pharmacokinetic parameters, Cmax (maximum plasma concentration achieved following dosing) and AUC0-48 (area under the curve of a plot of drug plasma concentrations versus time representing the extent of absorption) of EFV before and after co-administration of 14 successive daily doses of AP were compared and evaluated to determine whether an interaction had occurred. All subjects completed the study and the geometric mean ratios of Cmax and AUC0-48 were 97.30 and 102.82 with corresponding 90% confidence intervals (CIs) of 78.81-120.14% and 89.04-118.80%, respectively. Whereas the acceptance criteria for the ratios of the AUCs fell within the preset 90% CIs indicating no interaction, the Cmax ratios fell outside the limits. Although the protocol was developed in accordance with the United States of America Food & Drug Administration’s Guidance for Drug Interactions, a priori stating that both criteria need to fall within the acceptance limits to indicate no interaction, an argument is presented to waive the Cmax requirement for the declaration of an interaction. As a result, the pharmacokinetic data generated during this study indicated that the effect of AP on the pharmacokinetics of EFV is not clinically significant. Hence, co-administration of AP is unlikely to affect the clinical use of EFV. In summary the objectives of this project were: 1. To develop and validate a suitable HPLC-UV method for the quantitative determination of EFV in plasma. 2. To perform a mini-validation of the determination of hypoxoside for use as a marker in the quality control and standardisation of AP decoctions. 3. To conduct a clinical interaction study in order to determine whether AP affects the pharmacokinetics of EFV following concurrent administration. 4. To apply the validated HPLC-UV method to determine plasma concentrations of EFV in plasma of human subjects. 5. To use appropriate statistical methods and treatments such as a non-compartmental pharmacokinetic analysis to determine the occurrence of an interaction.

by Lam Tin Lun. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 131-151). / Abstracts in English and Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of Tables --- p.IX / List of Figures --- p.XI / Abbreviation --- p.XIII / Abstract --- p.XIV / 論文摘要 --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome (AIDS) --- p.1 / Chapter 1.1.1 --- History of AIDS --- p.1 / Chapter 1.1.2 --- Definition of AIDS --- p.2 / Chapter 1.1.3 --- HIV/AIDS Around the World --- p.4 / Chapter 1.1.4 --- HIV/AIDS in Hong Kong --- p.4 / Chapter 1.1.4.1 --- Hong Kong AIDS Update --- p.4 / Chapter 1.1.4.2 --- AIDS Transmission --- p.6 / Chapter 1.1.4.3 --- Main AIDS Complications Occur in Hong Kong --- p.6 / Chapter 1.2 --- Human Immunodeficiency Virus (HIV) --- p.7 / Chapter 1.2.1 --- Classification of HIV --- p.7 / Chapter 1.2.2 --- The Structure of HIV Virion --- p.9 / Chapter 1.2.3 --- The HIV Genome --- p.11 / Chapter 1.2.4 --- The Life Cycle of HIV --- p.12 / Chapter 1.2.4.1 --- Invasion of the Cells --- p.12 / Chapter 1.2.4.2 --- Integration into cell genome --- p.13 / Chapter 1.2.4.3 --- Protease and assembly to the virus --- p.13 / Chapter 1.2.5 --- Three Essential Enzymes for HTV-1 Replication --- p.16 / Chapter 1.2.5.1 --- HIV-1 Reverse Transcriptase (HIV-1 RT) --- p.16 / Chapter 1.2.5.2 --- HIV-1 Integrase (HIV-1 IN) --- p.17 / Chapter 1.2.5.3 --- HIV-1 Protease (HIV-1 PR) --- p.18 / Chapter 1.2.6 --- The Different Stages of HIV Infection --- p.19 / Chapter 1.3 --- AIDS therapy --- p.23 / Chapter 1.3.1 --- Drugs Approved by US Food and Drug Administration (FDA) --- p.23 / Chapter 1.3.2 --- Vaccine --- p.26 / Chapter 1.3.3 --- Chemokine Receptor Inhibitor --- p.27 / Chapter 1.3.4 --- Antisense Oligonucleotides Therpay --- p.28 / Chapter 1.3.5 --- Traditional Chinese Medicine (TCM) --- p.29 / Chapter 1.4 --- Objective of My Project --- p.32 / Chapter CHAPTER 2 --- SCREENING OF TRADITIONAL CHINESE MEDICINAL PLANTS FOR HIV-1 PROTEASE INHIBITION --- p.33 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials and Methods --- p.35 / Chapter 2.2.1 --- Materials --- p.35 / Chapter 2.2.2 --- Extraction Methods --- p.36 / Chapter 2.2.2.1 --- Aqueous Extraction --- p.36 / Chapter 2.2.2.2 --- Methanol Extraction --- p.37 / Chapter 2.2.3 --- Preparation of Recombinant HIV-1 Protease --- p.37 / Chapter 2.2.3.1 --- Selection of Appropriate Clone --- p.37 / Chapter 2.2.3.2 --- Large-scale Expression of Recombinant HIV-1 Protease --- p.38 / Chapter 2.2.2.3 --- Purification of Recombinant HIV-1 Protease by DEAE Sepharose CL-6B Chromatography --- p.38 / Chapter 2.2.3.4 --- Purification of Recombinant HIV-1 Protease by Mono-S Cation Chromatography --- p.39 / Chapter 2.2.3.5 --- Refolding of Purified Recombinant HIV-1 Protease --- p.40 / Chapter 2.2.3.6 --- Protein Concentration Determination --- p.41 / Chapter 2.2.3.7 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.41 / Chapter 2.2.4 --- Characterization of HTV-1 Protease --- p.42 / Chapter 2.2.4.1 --- HIV-1 PR Fluorogenic Assays --- p.42 / Chapter 2.2.4.2 --- HIV-1 PR Assay by Reverse Phase HPLC Separation of Cleavage Products of the Synthetic Peptide Substrate --- p.43 / Chapter 2.3 --- Results --- p.44 / Chapter 2.3.1 --- Functional Analysis of Recombinant HIV-1 PR Activity --- p.44 / Chapter 2.3.2 --- Screening of Crude Extracts for Inhibition of HIV-1 PR Activity --- p.48 / Chapter 2.4 --- Discussion --- p.53 / Chapter CHAPTER 3 --- ISOLATION AND CHARACTERIZATION OF ACTIVE CONSTITUENTS FROM METHANOL EXTRACTS OF WOODWARDIA UNIGEMMATA AGAINST HIV-1 PROTEASE --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.57 / Chapter 3.2.1 --- Materials --- p.57 / Chapter 3.2.2 --- Methods --- p.58 / Chapter 3.2.2.1 --- Methanol Extraction --- p.58 / Chapter 3.2.2.2 --- Removal of Tannins --- p.60 / Chapter 3.2.2.3 --- Glucosidase Digestion --- p.60 / Chapter 3.2.2.4 --- Analytical Thin Layer Chromatographic (TLC) --- p.61 / Chapter 3.2.2.5 --- A cid Hydrolysis --- p.62 / Chapter 3.2.2.6 --- Electrospray Mass Spectrometry --- p.62 / Chapter 3.2.2.7 --- Dose-response Curve --- p.63 / Chapter 3.2.2.8 --- Kinetic Studies --- p.63 / Chapter 3.2.2.9 --- Activity of the HPLC-purified principle (s) on Other Aspartyl Proteases --- p.63 / Chapter 3.3 --- Results --- p.66 / Chapter 3.3.1 --- Purification of Methanol Extracts of Woocdwardia unigemmata --- p.66 / Chapter 3.2.2 --- Removal of Tannins --- p.70 / Chapter 3.2.3 --- Glucosidase Digestion --- p.73 / Chapter 3.2.4 --- Acid Hydrolysis --- p.73 / Chapter 3.2.5 --- Analytical Thin Layer Chromatography --- p.74 / Chapter 3.2.6 --- Electrospray Mass Spectrometry --- p.80 / Chapter 3.2.7 --- Dose-response Inhibition of HIV-1 Protease --- p.80 / Chapter 3.2.8 --- Kinetic Studies --- p.85 / Chapter 3.2.9 --- Effects of HPLC-purified Active Principle on Other Aspartyl Proteases --- p.87 / Chapter 3.3 --- Discussion --- p.89 / Chapter CHATPER 4 --- IDENTIFICATION OF SELECTIVE RNA APTAMERS AGAINST HIV-1 PROTEASE BY SYSTEMATIC EVOLUTION OF LIGANDS BY EXPONENTIAL ENRICHMENT (SELEX) --- p.95 / Chapter 4.1 --- Introduction --- p.95 / Chapter 4.2 --- Materials and Methods --- p.101 / Chapter 4.2.1 --- Materials --- p.101 / Chapter 4.2.2 --- Methods --- p.102 / Chapter 4.2.2.1 --- PCR Amplification for the Generation of a Double-Stranded DNA Library --- p.103 / Chapter 4.2.2.2 --- Preparation of RNA Pools --- p.104 / Chapter 4.2.2.3 --- In vitro Selection of RNA Ligands --- p.104 / Chapter 4.2.2.4 --- Reverse Transcription Reaction of Selected RNA --- p.108 / Chapter 4.2.2.5 --- Cloning of the Amplified cDNA pools --- p.108 / Chapter 4.2.2.6 --- Subcloning of the digested DNA product into pBluescript® IIKS (-) --- p.108 / Chapter 4.2.2.8 --- RNA Labeling with Digoxigenin (DIG) --- p.109 / Chapter 4.2.2.9 --- Binding Affinity of RNA Ligands for HIV-1 PR --- p.109 / Chapter 4.2.2.10 --- Competition Binding Reactions --- p.111 / Chapter 4.2.2.11 --- HIV-1 PR Inhibitory Activities of the Selected RNA Ligands --- p.112 / Chapter 4.3 --- Results --- p.113 / Chapter 4.3.1 --- In Vitro Selection of RNA Ligands --- p.113 / Chapter 4.3.2 --- Sequences of RNA Ligands --- p.114 / Chapter 4.3.3 --- Binding Affinity of RNA Ligands --- p.114 / Chapter 4.3.4 --- Inhibitory Activity of RNA Ligands --- p.119 / Chapter 4.4 --- Discussion --- p.122 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.128 / REFERENCES --- p.132

by Lam Mei Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 127-137). / Abstract also in Chinese. / Acknowledgment --- p.I / Table of content --- p.II / List of figures --- p.VII / List of tables --- p.IX / Abbreviation --- p.X / Abstract --- p.XII / 論文摘要 --- p.XIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Acquired immunodeficiency syndrome --- p.1 / Chapter 1.1.1 --- Discovery of AIDS --- p.1 / Chapter 1.1.2 --- Definition and symptoms of AIDS --- p.1 / Chapter 1.1.3 --- AIDS transmission --- p.2 / Chapter 1.1.4 --- AIDS epidemic --- p.3 / Chapter 1.2 --- Human immunodeficiency virus --- p.3 / Chapter 1.2.1 --- Discovery of HIV --- p.3 / Chapter 1.2.2 --- The structure of HIV --- p.4 / Chapter 1.2.3 --- Genomic structure of HIV --- p.5 / Chapter 1.2.4 --- Life cycle of HIV --- p.5 / Chapter 1.2.5 --- How HIV is involved in different stages of AIDS --- p.7 / Chapter 1.3 --- Therapeutic targets for treatment of AIDS --- p.8 / Chapter 1.3.1 --- HIV reverse transcriptase (HIV RT) --- p.8 / Chapter 1.3.2 --- HIV integrase (HIV IN) --- p.11 / Chapter 1.3.3 --- HIV protease (HIV PR) --- p.12 / Chapter 1.3.4 --- Chemokine receptors --- p.14 / Chapter 1.3.5 --- Vaccine development --- p.16 / Chapter 1.4 --- AIDS therapy --- p.17 / Chapter 1.4.1 --- Current status of AIDS therapy --- p.17 / Chapter 1.4.1.1 --- Drugs approved by US Food & Drug Administration (FDA) --- p.17 / Chapter 1.4.1.2 --- Combination therapy --- p.19 / Chapter 1.4.1.3 --- Vaccine development --- p.19 / Chapter 1.4.2 --- Alternative treatment --- p.20 / Chapter 1.5 --- Objective of my project --- p.21 / Chapter Chapter 2 --- Screening of traditional Chinese medicinal (TCM) plants for HIV reverse transcriptase inhibition --- p.22 / Chapter 2.1 --- Introduction --- p.22 / Chapter 2.1.1 --- HIV RT structure and function --- p.22 / Chapter 2.1.2 --- Natural product against HIV RT --- p.25 / Chapter 2.1.3 --- Inhibitory activities from plant extracts --- p.27 / Chapter 2.2 --- Materials and Methods --- p.28 / Chapter 2.2.1 --- Materials --- p.28 / Chapter 2.2.2 --- Extraction methods --- p.30 / Chapter 2.2.2.1 --- Methanol extraction --- p.30 / Chapter 2.2.2.2 --- Hot water extraction --- p.30 / Chapter 2.2.2.3 --- Preparation of Prunella vulgaris extract --- p.30 / Chapter 2.2.3 --- Reverse transcriptase assay --- p.31 / Chapter 2.2.4 --- Characterization of active component in extract of Prunella vulgaris --- p.32 / Chapter 2.2.4.1 --- Protease digestion --- p.32 / Chapter 2.2.4.2 --- Glucosidase digestion --- p.32 / Chapter 2.2.4.3 --- Ethanol precipitation --- p.33 / Chapter 2.2.4.4 --- Sodium periodiate oxidization --- p.33 / Chapter 2.2.4.5 --- Polyvinylpyrrolidone (PVP) Precipitation --- p.34 / Chapter 2.2.4.6 --- Polyamide resin binding --- p.34 / Chapter 2.2.5 --- Purification of Prunella vulgaris extract --- p.34 / Chapter 2.2.5.1 --- Polyamide resin column chromatography --- p.34 / Chapter 2.2.5.2 --- Sephadex LH-20 chromatography --- p.35 / Chapter 2.2.5.3 --- Reverse phase HPLC chromatography --- p.36 / Chapter 2.2.6 --- Characterization of purified Prunella vulgaris extract --- p.37 / Chapter 2.2.6.1 --- Paper chromatography --- p.37 / Chapter 2.2.6.2 --- Acid hydrolysis of extract --- p.37 / Chapter 2.2.6.3 --- Thin layer chromatography --- p.38 / Chapter 2.2.6.4 --- Other assays --- p.39 / Chapter 2.2.7 --- Calculation --- p.40 / Chapter 2.3 --- Results --- p.41 / Chapter 2.3.1 --- Screening of Herbs --- p.41 / Chapter 2.3.1.1 --- Screening of methanol extracts --- p.41 / Chapter 2.3.1.2 --- Screening of hot water extracts --- p.41 / Chapter 2.3.2 --- Characterization of active components in Prunella vulgaris crude extracts --- p.44 / Chapter 2.3.2.1 --- Protease digestion --- p.44 / Chapter 2.3.2.2 --- Glucosidase digestion --- p.44 / Chapter 2.3.2.3 --- Ethanol precipitation --- p.44 / Chapter 2.3.2.4 --- Sodium periodate oxidation --- p.48 / Chapter 2.3.2.5 --- Effect of naturally occurring chemicals on inhibition of HIV RT --- p.48 / Chapter 2.3.2.6 --- Effect of removal of polyphenolic components of aqueous extract on inhibition of HTV RT --- p.51 / Chapter 2.3.3 --- Further purification of active components in aqueous extract of Prunella vulgaris --- p.53 / Chapter 2.3.3.1 --- Absorption chromatography by polyamide resin --- p.53 / Chapter 2.3.3.2 --- The Sephadex LH-20 chromatography --- p.53 / Chapter 2.3.3.3 --- Reverse phase high performance liquid chromatography --- p.56 / Chapter 2.3.3.4 --- Recovery of extract --- p.59 / Chapter 2.3.3.5 --- Inhibition from extract of various steps of purification --- p.59 / Chapter 2.3.4 --- Characterization of purified aqueous extract of Prunella vulgaris --- p.62 / Chapter 2.3.4.1 --- Paper chromatography --- p.62 / Chapter 2.3.4.2 --- Dose response curve --- p.62 / Chapter 2.3.4.3 --- Acid hydrolysis of purified extract --- p.68 / Chapter 2.3.4.4 --- Identification of monosaccharide in purified extract by Thin layer chromatography (TLC) --- p.71 / Chapter 2.3.5 --- Specificity of the purified extract on polymerase inhibition --- p.75 / Chapter 2.3.5.1 --- Inhibition of purified Prunella vulgaris extract on Taq polymerase --- p.75 / Chapter 2.3.5.2 --- Inhibition of purified Prunella vulgaris extract on Superscript II --- p.75 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Screening of inhibitory activities from traditional Chinese medicinal (TCM) plants extracts to HIV protease --- p.86 / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.1.1 --- HIV Protease structure and function --- p.86 / Chapter 3.1.2 --- Natural products against HIV Protease --- p.87 / Chapter 3.1.3 --- Plant extracts against HIV Protease --- p.89 / Chapter 3.2 --- Materials and Methods --- p.91 / Chapter 3.2.1 --- Materials --- p.91 / Chapter 3.2.2 --- Expression of HIV protease --- p.92 / Chapter 3.2.2.1 --- Expression and purification of HIV protease --- p.92 / Chapter 3.2.2.2. --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.94 / Chapter 3.2.3 --- Characterization of HIV protease --- p.95 / Chapter 3.2.3.1 --- HIV protease assay by fluorometric measurement --- p.95 / Chapter 3.2.3.2 --- HIV protease assay by using reverse phase high performance liquid chromatography --- p.96 / Chapter 3.3 --- Results --- p.98 / Chapter 3.3.1 --- Expression of HIV protease --- p.98 / Chapter 3.3.2 --- HIV protease assay --- p.98 / Chapter 3.3.2.1 --- Protease assay by using reverse phase HPLC --- p.98 / Chapter 3.3.2.2 --- Protease assay by fluorometric measurement --- p.98 / Chapter 3.3.3 --- Screening of crude Chinese medicinal extracts on inhibition of HIV protease --- p.104 / Chapter 3.3.3.1 --- Methanol extracts --- p.104 / Chapter 3.3.3.2 --- Water extracts --- p.105 / Chapter 3.3.4 --- Characterization of herbal extracts on inhibition of HIV protease --- p.110 / Chapter 3.3.4.1 --- Dose response curve of methanol extract of Woodwardia unigemmata --- p.110 / Chapter 3.3.4.2 --- Dose response curve of hot water extract of Prunella vulgaris --- p.110 / Chapter 3.3.4.3 --- Inhibition mode of methanol extract of Woodwardia unigemmata --- p.113 / Chapter 3.3.4.4 --- Inhibition mode of hot water extract of Prunella vulgaris --- p.113 / Chapter 3.3.4.5 --- Effect of partially purified extracts on HIV protease inhibition --- p.116 / Chapter 3.4 --- Discussion --- p.119 / Chapter Chapter 4 --- General discussion --- p.124 / References --- p.127 / Appendix / Appendix 1 Pictures of herbs used in this study --- p.i / Appendix 2 Mass spectrometry of purified Prunella vulgaris extract --- p.vi / Appendix 3 Calibration curve for determination of HIV PR concentration --- p.viii