AN ANALYTICAL EVALUATION OF IN VITRO DRUG-DRUG

INTERACTION STUDIES FOR FIXED ARTESUNATE

COMBINATION THERAPY

by

VANESSA SHALINI A/P DANIEL JAYARAJ

Thesis submitted in fulfillment of the requirement for the degree of

Master of Science

April 2010

i

ACKNOWLEDGEMENT

First and foremost, I would like to record my gratitude to Universiti Sains Malaysia

(USM) and the Institute of Postgraduate Studies (IPS) for allowing me the full use of

their facilities for the duration of my studies. I extend my profound appreciations to

have received the support of the IPS Postgraduate Fellowship Scheme for two years.

I gratefully thank Drugs for Neglected Disease initiative (DNDi) for their

collaborative efforts and financial aid during the course of this project.

My sincere thanks and appreciation to my main supervisor Prof Emeritus Dato’ (Dr.)

V Navaratnam, and my co-supervisors Prof Sharif Mahsufi Mansor and Assoc. Prof

Abas Hj. Hussin, who throughout my project, have given me their patience and

knowledge whilst allowing me the room to work independently. My deep gratitude to

Prof N. Nair, whose constructive comments and guidance from the initial to the final

level enabled me to develop an understanding of the subject.

My special thanks to Mr. Asokan Muniandy and Mr. Salam Abdullah for their

expertise in the laboratory, without which my work would not have progressed. I

extend collective and individual acknowledgements to my colleagues at the Centre

for Drug Research and the School of Pharmacy for constructive scientific

discussions, moral support and above all, for giving me such a pleasant time while

working together. Words fail to express my appreciation to my parents Daniel and

Regina, my siblings Angeline and Benedict, and my fiancé John Bosco, who have

unselfishly supported me and believed in me all this time.

Finally, I would like to thank everybody who was important to the successful

realization of thesis, and I apologize for being unable to mention each individual

personally.

ii

Table of Contents

List of Figures ........................................................................................................... viii

List of Tables.............................................................................................................. xii

List of Symbols and Abbreviations ............................................................................ xv

ABSTRAK ................................................................................................................ xix

ABSTRACT ............................................................................................................... xx

CHAPTER 1: INTRODUCTION ............................................................................... 1

1.1. Global Malaria Scene ........................................................................................ 1

1.2. Malaria in Malaysia........................................................................................... 5

1.3. The Mechanism of Malaria ............................................................................. 10

1.4. Methods in Malaria Control ............................................................................ 16

1.5. Treatment of Malaria....................................................................................... 18

1.5.1. Quinolines ....................................................................................................... 19

1.5.1(a) Quinine..................................................................................................... 20

1.5.1(b) Chloroquine (CQ) .................................................................................... 21

1.5.1(c) Piperaquine (PQ)...................................................................................... 22

1.5.1(d) Amodiaquine (AQ) .................................................................................. 23

1.5.1(e) Mefloquine (MQ) ..................................................................................... 24

iii

1.5.2. Antifolates ....................................................................................................... 25

1.5.3. Artemisinin Derivatives .................................................................................. 27

1.5.3(a) Artemesinin .............................................................................................. 27

1.5.3(b) Arteether (Artemotil) ............................................................................... 29

1.5.3(c) Artemether ............................................................................................... 29

1.5.3(d) Artesunate ................................................................................................ 29

1.5.3(e) Dihydroartemisinin (dHA) ....................................................................... 32

1.6. Resistance of Malaria Parasite against Artemisinins ...................................... 33

1.7. Artemisinin- Combination Therapy (ACT) .................................................... 34

1.8. Drug Metabolism ............................................................................................ 35

1.8.1. Cytochrome P450 Enzymes ............................................................................ 36

1.8.2. Enzyme Inhibition and Induction .................................................................... 44

1.8.3. Drug-Drug Interaction ..................................................................................... 49

1.8.4. In vitro Drug Interaction Systems ................................................................... 50

1.8.4(a) Human liver microsomes (HLM) ............................................................ 51

1.8.4(b) cDNA-expressed enzymes ....................................................................... 52

1.8.4(c) Cell-based systems ................................................................................... 53

1.8.5. Enzyme Kinetics ............................................................................................. 54

1.9. Sample Preparation and Analysis.................................................................... 61

iv

1.9.1. Sample Preparation ......................................................................................... 61

1.9.2. Sample analysis ............................................................................................... 63

1.10. Problem Statement .......................................................................................... 70

1.11. Aim of Study ................................................................................................... 70

CHAPTER 2: APPARATUS AND METHODOLOGY ........................................... 72

2.1. Silanisation ...................................................................................................... 72

2.2. Analytical Method ........................................................................................... 72

2.2.1. Chromatographic Conditions .......................................................................... 73

2.2.2. Preparation of Standard Stock Solutions and Dilutions .................................. 75

2.2.3. Calibration Curve (CC) ................................................................................... 76

2.2.4. Method Precision and Quality Control (QC) .................................................. 76

2.2.5. Limit of Detection (LOD) and Limit of Quantification (LOQ) ...................... 77

2.3. Difficulties in Application of Product- Formation Method ............................ 77

2.4. Preparation of Chemicals for Incubation Media ............................................. 78

2.4.1. Tris Buffer pH 7.4 ........................................................................................... 79

2.4.2. Magnesium Chloride (MgCl2)......................................................................... 79

2.4.3. Glucose-6-Phosphate (G6P) ............................................................................ 79

2.4.4. Glucose-6-Phosphate Dehydrogenase (G6PDH) ............................................ 79

2.4.5. Nicotinamide Adenine Dinucleotide Phosphate (NaDP+) .............................. 80

v

2.4.6. Human Liver Microsomes............................................................................... 80

2.4.7. Drug Standards ................................................................................................ 80

2.4.8. Stopping Reagent ............................................................................................ 80

2.4.9. Incubation Procedure ...................................................................................... 81

2.5. Determination of Km Value with 0.5 mg Microsomal Protein ........................ 81

2.6. Optimization and Validation of the Incubation Media ................................... 82

2.6.1. Incubation Period ............................................................................................ 82

2.6.2. Microsomal Protein Content ........................................................................... 82

2.6.3. Concentration of G6PDH ................................................................................ 83

2.6.4. Stability of AS in Incubation Media ............................................................... 83

2.6.5. Stability of α and β dHA Epimers Ratios in Incubation Media ...................... 84

2.6.6. Stability of Aliquots during Storage ............................................................... 84

2.6.7. Day-To-Day Precision of Determination of the Rates of AS Depletion ......... 84

2.6.8. Testing the Incubation Media with Risperidone ............................................. 85

2.6.9. Obtaining Km Value with 1.0 mg Microsomal Protein ................................... 85

2.7. Drug-Drug Interactions ................................................................................... 86

2.7.1. Testing the System with Probe Inhibitor Methoxpsoralen .............................. 86

2.7.2. AS-MQ Co-Incubation .................................................................................... 86

2.7.3. AS-AQ Co-incubation ..................................................................................... 87

vi

CHAPTER 3: RESULTS ........................................................................................... 88

3.1 Analytical Method ........................................................................................... 88

3.1.1 Chromatographic Conditions .......................................................................... 88

3.1.2 Calibration Curve (CC) ................................................................................... 93

3.1.3 Method Precision ............................................................................................ 94

3.1.4 Limit of Detection (LOD) and Limit of Quantification (LOQ) ...................... 95

3.2 Difficulties in Application of Product- Formation Method ............................ 95

3.3 Substrate Depletion Method .......................................................................... 103

3.4 The Stability of AS in Incubation Media ...................................................... 105

3.5 Determination of KM Value with 0.5 mg/mL Microsomes ........................... 110

3.6 Testing the Incubation Media with Risperidone ........................................... 112

3.7 Optimization and Validation of the Incubation Media ................................. 115

3.7.1 Incubation Period .......................................................................................... 115

3.7.2 Microsomal Content ...................................................................................... 115

3.7.3 Optimal concentration of G6PDH in incubation media ................................ 118

3.7.4 Presence and Absence of NADPH Regenerating System ............................. 120

3.7.5 Day-To-Day Precision of Determination of the Rates of AS Depletion ....... 123

3.8 Determination of KM Value with 1.0 mg/mL Microsomes ........................... 125

3.8.1 Determining KM and VMAX Values for AS using 1.0 mg/mL Microsomes ... 125

vii

3.9 Comparison between Microsomal Enzyme-Mediated (EM) and Non-Enzyme

Mediated (NEM) AS Hydrolysis to dHA ..................................................... 127

3.10 Drug- Drug Interaction Experiments ............................................................ 131

3.10.1 Testing the Incubation Media with Probe Inhibitor Methoxypsoralen ......... 131

3.10.2 AS-MQ Co-Incubation .................................................................................. 134

3.10.3 AS-AQ Co-Incubation .................................................................................. 137

CHAPTER 4: DISCUSSION ................................................................................... 140

CHAPTER 5: SUMMARY AND CONCLUSION ................................................. 158

REFERENCES ......................................................................................................... 163

Appendix I: List of standards ................................................................................... 212

Appendix II: List of Chemicals ................................................................................ 213

Appendix III: List of Apparatus ............................................................................... 215

Appendix IV: Operational Definitions ..................................................................... 217

Appendix V: List of Non-artemisinin Based Combination Malaria Treatments ..... 218

Appendix VI: List of Artemisinin Based Combination Malaria Treatments ........... 219

Appendix VII: Raw Data ......................................................................................... 220

viii

List of Figures

Figure 1. 1.Malaria-free and malaria-endemic countries in phases of control, pre-

elimination, elimination, and prevention of re-inroduction ...................... 3

Figure 1. 2. Malaria Incidence from 1961 until May 2007 .......................................... 7

Figure 1. 3. Malaria Cases and Incidence Rate in from 1995- May 2007 ................... 8

Figure 1. 4. Malaria Cases and Incidence Rate in Malaysia by State until May 2007

....................................................................................................................9

Figure 1. 5. Malaria Parasite Distribution in Malaysia until May 2007 .................... 10

Figure 1. 6. Lifecycle of Plasmodium falciparum ..................................................... 12

Figure 1. 7. A Feeding female Anopheles mosquito .................................................. 13

Figure 1. 8. Different Life Stages of Plasmodium falciparum ................................... 13

Figure 1. 9. Structures of Major Quinoline Drugs ..................................................... 21

Figure 1. 10. Chemical Structures of Common Antifolates ....................................... 26

Figure 1. 11. Structures of Artemisinin-Type compounds ......................................... 28

Figure 1. 12. The relationship between enzyme, substrate and product .................... 57

Figure 1. 13. The Basic Mechanism of Enzymatic Reactions ................................... 57

Figure 2. 1. Schematic diagram of the HPLC components………………………… 74

Figure 3. 1. Chromatogram of a mixture of 5 µg/mL AS, 5 µg/mL dHA and 3 µg/mL

QHS prepared in ethanol-water (50:50, v/v) with flow rate of 1.0 ml/min.

(RT: AS 3.7 mins, α-dHA 5.5 mins, β-dHA 7.2 mins, and QHS 9.2 mins)

................................................................................................................. 89

Figure 3. 2. Chromatogram of ethanol-water (50:50, v/v) with flow rate of 1.0

ml/min................................................................................................. 90

ix

Figure 3. 3. Chromatogram of blank incubation media with flow rate of 1.0 ml/min

...............................................................................................................91

Figure 3. 4. Chromatogram of a mixture of incubation media spiked with 10 ug/mL

AS, 5 µg/mL dHA, and 3 µg/mL QHS with flow rate of 1.0 ml/min

(RT: AS 3.7 mins, α-dHA 5.5 mins, β-dHA 7.2 mins, and QHS 9.2

min) ....................................................................................................... 92

Figure 3. 5. A typical calibration curve of AS ........................................................... 93

Figure 3. 6. The ratio of α-dHA to β-dHA in 1000µL incubation media with 1mg/mL

microsomes, 0.4 I.U/mL G6PDH, and NADPH Regenerating System at

370C at various time points for 30 minutes. .......................................... 97

Figure 3. 7. Chromatogram of an incubation media spiked with 2 ug/mL AS and 3

µg/mL QHS with flow rate of 1.0 ml/min at 10 min. (RT: AS 3.7 mins,

α-dHA 5.5 mins, β-dHA 7.2 mins, and QHS 9.2 mins) ........................ 99

Figure 3. 8. Ln Percentage of 10 µg/mL dHA remaining in 1000µL incubation media

with 0 mg/mL microsomes, 0.4 I.U/mL G6PDH and NADPH

Regeneration System at 370C for 30 minutes ..................................... 101

Figure 3. 9. Chromatogram of 10 g/mL dHA in incubation media with 0 mg/mL

microsomes, 0.4 I.U/mL G6PDH and NADPH Regeneration System at

370C at time point 10 minutes. (RT (min): - dHA 7.3, -dHA 9.2 and

unknown compound 4.8 and 9.9) ........................................................ 102

Figure 3. 10. A typical AS depletion profile in an incubation media with 1 mg/mL

microsome, 0.8 I.U/mL G6PDH, and NADPH Regenerating System at

370C for 30 minutes. ........................................................................ 104

Figure 3. 11. The segment of the depletion profile from Figure 3.10 that was taken to

determine kdep ................................................................................... 104

x

Figure 3. 12. The Ln Percentage of 2µg/mL AS remaining in 1000µL incubation

media with 0 mg/mL microsomes, 0.8 I.U/mL G6PDH, and NADPH

Regenerating System at 370C for 30 minutes (n=3) ......................... 108

Figure 3. 13. The Ln percentage of 4 µg/mL AS remaining in a aliquots of 1000µL

incubation media with 1.0 mg/mL microsomes, 0.8 I.U/mL G6PDH,

and NADPH Regenerating System at 40C and -20

0C at various time

points up to 72 hours (n=3). ............................................................. 108

Figure 3. 14. Michealis-Menten Profile of Various Concentrations of AS with 0.5

mg/mL microsomes, 0.4 I.U/mL G6PDH, and NADPH Regeneration

System in incubation media as recommended by manufacturer at 370C

for 20 minutes. .................................................................................. 111

Figure 3. 15. Concentration of 9-hydroxyrisperidone (9-OH RSP) formed in

manufacturer-recommended incubation media with 0.5 mg/mL

microsomes, 0.4 I.U/mL G6PDH, and NADPH Regenerating System

at 370C for 75 minutes (n=3) .......................................................... 114

Figure 3. 16. The Percentage of 2 µg/mL AS remaining at different time points in

1000µL incubation media with 1 mg/ml microsomes, 0.4 I.U/mL

G6PDH, and NADPH Regenerating System at 370C for 60 mins

(n=3). ................................................................................................ 116

Figure 3. 17. The rate of 2 µg/mL AS depletion in 1000µL incubation media with 0.4

I.U/mL G6PDH, NADPH Regeneration System, and various

concentrations of microsomes at 370C for 30 minutes. .................... 117

Figure 3. 18. The rate of 4 µg/mL AS depletion in 1000µL incubation media with

1mg/mL microsomes, NADPH Regeneration System and various

concentrations of G6PDH at 370C for 30 minutes. .......................... 119

xi

Figure 3. 19. The percentage of 2 µg/mL AS remaining in 1000µL incubation media

with 1 mg/mL microsomes and 0.8 I.U/mL G6PDH in the presence

and absence of the NADPH Regenerating System at 370C for 30

minutes. ............................................................................................ 122

Figure 3. 20. Michealis-Menten Profile of AS in 1000 µL incubation media with 1.0

mg/mL microsomes, 0.8 I.U/mL G6PDH, and NADPH Regeneration

System at 370C for 30 minutes ......................................................... 126

Figure 3. 21. First Order Kinetics of AS in 1000 µL incubation media with

1mg/mL microsomes, 0.8 I.U/mL G6PDH and NADPH Regeneration

System at 370C for 60 minutes ......................................................... 129

Figure 3. 22. Percentage of 2.0 µg/mL AS remaining in the presence of 10 µg/mL

probe inhibitor MOP in 1000 µL incubation media with 1 mg/mL

microsomes, 0.8 I.U/ mL G6PDH, and NADPH Regenerating System

at 370C for 30 minutes. ..................................................................... 133

Figure 3. 23. Percentage of 2.0 µg/mL AS remaining in the presence of 0.1, 1.0 and

10 µg/mL MQ in 1000 µL incubation media with 1 mg/mL

microsomes, 0.8 I.U/mL G6PDH, and NADPH Regenerating System

at 370C for 30 minutes. ..................................................................... 136

Figure 3. 24. Percentage of 2.0 µg/mL AS remaining in the presence of 1, 10 and 100

ng/mL AQ in 1000 µL incubation media with 1 mg/mL microsomes,

0.8 I.U/ml G6PDH, and NADPH Regenerating System at 370C for 30

minutes. ............................................................................................ 139

xii

List of Tables

Table 1. 1 Summary of Major CYP450 Enzymes and Their Substrates .................... 40

Table 1. 2. Summary of Major CYP Enzyme Inhibitors ........................................... 46

Table 1. 3. Summary of Major CYP Enzymes Inducers ............................................ 48

Table 1. 4. A Summary of Various Chromatographic Methods for Simultaneous

Detection of AS and dHA in Biological Matrices. ................................. 67

Table 3. 1. Mean regression equations of the calibration curves of AS…………….93

Table 3. 2. Precisions for the determination of AS within-day and day-to-day

variation coefficients .............................................................................. 94

Table 3. 3. The ratio of α-dHA to β-dHA in 1000µL incubation media with 1 mg/mL

microsomes, 0.4 I.U/mL G6PDH, and NADPH Regenerating System at

370C at various time points for 30 minutes. ............................................ 97

Table 3. 4. Percentage of 10 µg/mL dHA remaining in 1000µL incubation media

with 0 mg/mL microsomes, 0.4 I.U/mL G6PDH and NADPH

Regeneration System at 370C for 30 minutes........................................ 100

Table 3. 5. The Percentages of 2µg/mL AS remaining in 1000µL incubation media

with 0 mg/mL microsomes, 0.8 I.U/mL G6PDH, and NADPH

Regeneration System at 370C for 30 minutes........................................ 107

Table 3. 6. The percentages of 4 µg/mL AS remaining in 100 µL aliquots from

1000µL incubation media with 1.0 mg/mL microsomes, 0.8 I.U/mL

G6PDH, and NADPH Regenerating System at 40C and -20

0C at various

time points up to 72 hours ..................................................................... 109

xiii

Table 3. 7. Michealis-Menten Profile of Various Concentrations of AS with 0.5

mg/mL microsomes, 0.4 I.U/mL G6PDH and NADPH Regeneration

System in incubation media as recommended by manufacturer at 370C

for 20 minutes........................................................................................ 111

Table 3. 8. Concentration of 9-hydroxyrisperidone (9-OH RSP) formed in

manufacturer-recommended incubation media with 0.5 mg/mL

microsomes, 0.4 I.U/mL G6PDH, and NADPH Regenerating System at

370C for 75 minutes ............................................................................ 113

Table 3. 9. The Percentages of 2 µg/mL AS remaining at different time points in

1000 µL incubation media with 1 mg/mL microsomes, 0.4 I.U/mL

G6PDH, and NADPH Regenerating System at 370C for 60 mins (n=3).

............................................................................................................ 116

Table 3. 10. The rate of 2 µg/mL AS depletion in 1000µL incubation media with 0.4

I.U/mL G6PDH, NADPH Regeneration System, and various

concentrations of microsomes at 370C for 30 minutes. ...................... 117

Table 3. 11. The rate of 4 µg/mL AS depletion in 1000µL incubation media with

1mg/mL microsomes, NADPH Regeneration System and various

concentrations of G6PDH at 370C for 30 minutes. ............................. 119

Table 3. 12. The percentage of 2 µg/mL AS remaining in 1000µL incubation media

with 1 mg/mL microsomes and 0.8 I.U/mL G6PDH in the presence and

absence of the NADPH Regenerating System at 370C for 30

minutes. ............................................................................................... 121

Table 3. 13. The percentage of AS remaining and the kdep in 1000µL incubation

media with 1 mg/mL microsomes, 0.8 I.U G6PDH, and NADPH

xiv

Regenerating System at 370C for 30 minutes, done in 3 different

days. .................................................................................................... 123

Table 3. 14. Michealis-Menten Profile of AS in 1000 µL incubation media with 1.0

mg/mL microsomes, 0.8 I.U/mL G6PDH, and NADPH Regeneration

System at 370C for 30 minutes ........................................................... 126

Table 3. 15. EM and NEM hydrolysis of 2 µg/mL AS to dHA in an incubation media

with 1 mg/mL microsomes, 0.8 I.U/mL G6PDH and NADPH

Regeneration System in pH 7.4 Tris buffer (final volume 1000µL)...130

Table 3. 16. Percentage of 2.0 µg/mL AS remaining in the presence of 10 µg/mL

probe inhibitor MOP in 1000 µL incubation media with 1 mg/mL

microsomes, 0.8 I.U/ mL G6PDH, and NADPH Regenerating System at

370C for 30 minutes. ........................................................................... 132

Table 3. 17. Percentage of 2.0 µg/mL AS remaining in the presence of 0.1, 1.0 and

10 µg/mL MQ in 1000 µL incubation media with 1 mg/mL

microsomes, 0.8 I.U/mL G6PDH, and NADPH Regenerating System at

370C for 30 minutes. ........................................................................... 135

Table 3. 18. Percentage of 2.0 µg/mL AS remaining in the presence of 1, 10 and 100

ng/mL AQ in 1000 µL incubation media with 1 mg/mL microsomes,

0.8 I.U/ml G6PDH, and NADPH Regenerating System at 370C for 30

minutes. ............................................................................................... 138

xv

List of Symbols and Abbreviations

ACT- artemisinin combination therapy

AE- arteether

APCI- atmospheric pressure chemical ionization

AS – artesunate

AQ- amodiaquine

AVQ – atovaquone

C- concentration

CC- calibration curve

cDNA – complimentary DNA

Cmax – peak plasma concentration

CMQ- carboxymefloquine

CQ- chloroquine

CRPF – chloroquine-resistant Plasmodium faciparum

CYP- cytochrome

deAQ- desetylamodiaquine

DDT – dichlorodiphenyltrichloroethane

DFN- diclofenac

xvi

dHA – dihydroartemisinin

DHER- dihydrofolate reductase

DMSO- dimethyl sulfoxide

DNDi – Drugs for Neglected Diseases initiave

DNA- deoxyribonucleic acid

E- enzyme

EAC- enzyme activity change

EM- enzyme mediated

EC- electrochemical

ES- enzyme-substrate complex

ESI- electrospray ionization

FMO- flavin monooxygenase

FDA- food and drug administration

G6P- glucose-6-phosphate

G6PDH- G6P dehydrogenase

HLM- human liver microsome

HPLC- high pressure liquid chromatography

IC50 - half maximal inhibitory concentration

IRS – indoor residual spraying

xvii

ITN- insecticide-treated net

Kdep – sunstrate depletion rate constant

Ki - dissociation constant for inhibitor binding

KM – Michealis Menten constant

LCMS – liquid chromatography-mass spectrometry

LOD- limit of detection

LOQ- limit of quantification

MgCl2- magnesium chloride

MM- Michealis Menten

MOP- methoxypsoralen

MQ- mefloquine

NaCl- sodium chloride

NaOH- sodium hydroxide

NADP+- nicotinamide adenine dinucleotide phosphate

NADPH- reduced nicotinamide adenine dinucleotide phosphate

NCE- new chemical entity

NEM- non-enzyme mediated

NME- new molecular entity

NTR – narrow therapeutic range

xviii

P- product

PG- proguanil

pI- isoelectric point

PLC- pilocarpine

PQ- piperaquine

PR- primaquine

PYR- pyrimethamine

QHS – artemisinin

RSP- risperidone

RSP-OH - dihydroxyrisperidone

S- substrate

SA – sulphonamide

TCP- tranylcypromine

TNF- tumour necrosis factor

TRP- tryptamine

UMMC- Universiti Malaya Medical Centre

UV- ultraviolet

VMAX – maximum reaction velocity

WHO- World Health Organisation

xix

PENILAIAN ANALITIKAL INTERAKSI DRUG-DRUG SECARA IN VITRO

BAGI TERAPI KOMBINASI ARTESUNATE TETAP: ABSTRAK

Plasmodium falciparum yang rintang ubat kini merupakan suatu ancaman serius,

terutamanya di kawasan-kawasan tertentu dunia di mana malaria adalah endemik.

Oleh itu, pihak WHO mengesyorkan regimen kombinasi seperti artesunat-

meflokuina dan artesunate-amodiakuina sebagai rawatan utama bagi penyakit

malaria terutamanya di kawasan-kawasan di mana parasit falciparum adalah paling

rintang. Walau bagaimanapun, kajian interaksi drug in vitro bagi kombinasi

artesunat-meflokuina dan artesunate-amodiakuina belum pernah dijalankan.

Teknik experimen in vitro mikrosom hepar manusia digunakan untuk mengkaji

metabolisme artesunat secara bersendirian dan juga dengan kehadiran mefloquina

dan amodiakuina. Penentuan serentak artesunat, dihidroartemisinin dan artemisinin

dalam sampel mikrosom dijalankan menggunakan kaedah kromatografi cecair

tekanan tinggi yang dilengkapi dengan pengesan elektrokimia. Kajian ini

menunjukkan bahawa apabila diinkubasi secara in vitro dengan mikrosom, artesunat

dimetabolisme kepada dihidroartemisinin, dimangkin oleh enzim CYP450 .

Kajian in vitro ini juga menunjukkan bahawa tindak balas metabolisme artesunat

tidak dipengaruhi oleh tindak balas metabolisme meflokuina ataupun amodiakuina

pada kepekatan terapeutik. Ketiadaan interaksi di antara drug-drug ini memberi

kesimpulan bahawa kombinasi artesunat-meflokuina dan artesunate-amodiakuina

adalah rawatan anti-malaria yang berkesan dan selamat apabila digunakan pada

kepekatan terapeutik. Walau bagaimanpun, kajian klinikal bagi kedua-dua kombinasi

ini perlu dijalankan bagi menyokong penemuaan kajian ini.

xx

AN ANALYTICAL EVALUATION OF IN VITRO DRUG-DRUG

INTERACTION STUDIES FOR FIXED ARTESUNATE COMBINATION

THERAPY: ABSTRACT

Drug-resistant Plasmodium falciparum has become a serious threat, especially in

malaria-endemic regions of the world. In light of this, the WHO has recommended

artemisinin combination therapy such as artesunate-mefloquine and artesunate-

amodiaquine as first line treatment for uncomplicated falciparum malaria especially

in regions harbouring the most resistant isolates. However, the in vitro drug

interaction studies have not been conducted for both these combinations.

An in vitro experimental technique, utilizing human liver microsomes was used to

study the metabolism of AS in the presence and absence of mefloquine and

amodiaquine. Simultaneous determination of artesunate, dihydroartemisinin and

artemisinin in microsomal sample was performed using high performance liquid

chromatography- electrochemical (HPLC-EC) method. The study shows that

artesunate is metabolised to dihydroartemisinin in the in vitro microsomal assay. The

reaction is mediated by CYP450 enzymes present in the microsomal assay.

The metabolism of artesunate was not affected by mefloquine or amodiaquine at

therapeutic concentrations using the in vitro microsomal technique. The absence of

interactions between these drugs led to the conclusion that the artesunate-mefloquine

and artesunate-amodiaquine combinations are ideal and safe treatments when used in

therapeutic concentrations. However, further clinical studies must be done to confirm

these findings.

1

CHAPTER 1: INTRODUCTION

1.1. Global Malaria Scene

Malaria is a vector-borne infectious disease caused by protozoan parasites. It is

widespread in tropical and subtropical regions of the world, including parts of the

Americas, Asia, and Africa (Figure 1.1). Still, malaria continues to receive

insufficient attention since industrial and financial powers are concentrated in

temperate countries where malaria is no longer a threat. For this reason, malaria is

considered a neglected disease (Carmargo, de Oliveira, Basano, & Garcia, 2009).

It has been documented that malaria pre-dates the evolution of man and the malarial

parasite is known to have been a human pathogen for the entire history of its species

(Joy, et al., 2003); (Hayakawa, Culleton, Otani, Horii, & Tanabe, 2008). The first

evidence of malaria dates back to as far as 30 million years ago (Poinar, 2005) with

historical records of the disease dating back to more than 3000 years ago (Sherman,

1998a); (Harrison, 1979); (Bruce-Chwat, 1988). However, the discovery of the

causative agent of malaria only took place in 1880 by a French army doctor working

in the military hospital of Constantine in Algeria , Charles Louis Alphonse Laveran

(1845-1922). Laveran observed parasites for the first time, inside the erythrocytes of

patients suffering from malaria and was awarded the Nobel Prize for his discovery.

Malaria causes about 250 million cases of fever and approximately 1 million deaths

annually (World Health Organisation (WHO), 2008). Most of these cases occur in

children under 5 years of age (Greenwood, Bojang, Whitty, Targett, & GA., Malaria,

2005). Approximately one infant dies of malaria every 30 seconds (Carmargo, de

Oliveira, Basano, & Garcia, 2009). Pregnant women are also especially vulnerable.

Pregnancy reduces a woman’s immunity to malaria, making her more susceptible to

2

malaria parasite infections and other illnesses including severe anaemia and in

extreme cases, even death.

3

Figure 1. 1. Malaria-free and malaria-endemic countries in phases of control, pre-elimination, elimination, and prevention of re-

introduction

Source: World Malaria Report 2008, World Health Organisation, 2008

4

For the foetus, maternal malaria is one of the leading causes of child mortality, with

higher risks of spontaneous abortion, stillbirth, premature delivery and low birth

weight (http://www.who.int/features/2003/04b/en/, 2003).

Uncomplicated malaria is the mild form of the disease which occurs as a febrile

illness with headache, tiredness, muscle pains, abdominal pains, rigors (severe

shivering), and nausea and vomiting. If left untreated, P. falciparum malaria can

rapidly develop into severe malaria with anaemia (low haemoglobin in the blood),

hypoglycaemia (low blood sugar), renal failure (kidney failure), pulmonary oedema

(fluid in the lungs), convulsions, coma, and eventually death (World Health

Organization (WHO), 2006).

Despite efforts to reduce transmission and increase treatments, there has been little

change in malaria high-risk areas since 1992 (Hay, Guerra, Tatem, Noor, & Snow,

2004). If the prevalence of malaria continues, the death rate could double in the next

twenty years (Bremen, 2001). Precise statistics are unknown because many cases

occur in rural areas where people do not have access to hospitals or the means to

afford health care. As a consequence, the majority of cases are undocumented

(Bremen, 2001).

Malaria is presently endemic around the equator, in areas of the Americas, many

parts of Asia, and much of Africa (Figure 1.1). In fact, in sub-Saharan Africa,

malaria fatalities are about 85– 90% (Layne, 2005). Malaria is more common in rural

areas than in cities; this is in contrast to dengue fever where urban areas present the

greater risk (Van Benthem, et al., 2005). For example, the cities of Vietnam, Laos

and Cambodia are essentially malaria-free, but the disease is present in many rural

5

regions (Trung H, 2004). By contrast, malaria in Africa is present in both rural and

urban areas, though the risk is lower in the larger cities (Keiser J, 2004).

1.2. Malaria in Malaysia

Malaria is still the most important endemic disease in Malaysia, especially in remote

areas. The first documented case of malaria fever (known then as ‘Pinang fever’) in

Malaysia was in 1830 in Penang Island (Ward & Grant, 1830). Today, in spite of

more than 30 years of active malaria control and eradication activities, the disease

remains prevalent.

In 1901, the first organized anti-malarial campaign in Malaysia (then known as

Malaya until 1957) was launched, spearheaded by Sir (Dr.) Malcolm Watson and it

went on to be described as the first successful anti-malarial effort in the British

Empire (Watson M. S., 1935). The campaign brought about a major reduction in

malaria-caused deaths in Klang and Port Swettenham, from 368 deaths in 1901 to 59

deaths in 1902. A key activity that contributed significantly to the success of the

campaign was the introduction of the drainage system, devised to control the

Anopheles mosquitoes. This eventually led to other, better devised methods such as

agitation ponds, automatic siphons and flush gates, some of which are still used

today.

Prior to 1904, the management of malaria in Malaya was based on vector-control.

However, with the growing global use of the anti-malarial drug quinine to treat

malaria after its formal discovery by the Jesuit priests, drug therapy using quinine

was used on a large scale. The discovery of synthetic anti-malarial drugs for

treatment and prophylaxis from 1930 onwards propelled the malaria control efforts

6

further ahead. With the discovery of the effective insecticide

dichlorodiphenyltrichloroethane (DDT) by Muller and Weisman in 1936 and further

advances in chemotherapy, malaria eradication programmes were introduced

worldwide. In Malaysia, the malaria eradication pilot project was launched in 1967

with the help of the WHO. Although the programme did not completely eradicate

malaria in Malaysia, it successfully reduced annual death numbers of 250,000 in

1961 to 40,000 by the 1970es.

By 1980es however, difficulties in obtaining the large financial resources required

for successful malaria eradication, development of successful insecticides and

emergence of drug-resistant malarial parasites all led to malaria control programmes

being established in place of malaria eradication programmes. Subsequently these

programmes were integrated with other control programmes for diseases such as

dengue, filariasis, Japanese encephalitis, typhus, plague and yellow fever. Among the

strategies adopted in the malaria control programme were:

Improving early diagnosis of malaria disease and its treatment

Creating greater awareness of the seriousness of malaria

Promoting the use of pyrethroid-treated mosquito nets

Improving epidemiological data collection

Improving and strengthening management and supervision

Improving national and international training capabilities

Operational research

Development of malaria control staff

Integration with other disease control programmes

7

In 1990, the number of reported malaria cases was 50,500 but this value decreased

by 75% by year 2000 (Figure 1.2). Despite high numbers of reported cases, the

number of malaria-caused deaths in 1990 was only 43 and only 35 in 2000 (World

Health Organisation (WHO), 2008). With the development of anti-malarial drugs and

the application of various methods of vector control, the number of reported malaria-

related cases and incidence rates continues to undergo a steady decline (Figure 1.3).

Figure 1. 2. Malaria Incidence from 1961 until May 2007

Source: Department of Public Health, Ministry of Health Malaysia, 2008

8

Figure 1. 3. Malaria Cases and Incidence Rate in from 1995- May 2007

Source: Department of Public Health, Ministry of Health Malaysia, 2008

Today, malaria is still a significant public health problem in Malaysia (Lim, 1998);

(Ministry of Health Malaysia, 2002); (Ministry of Health Malaysia, 2004); (Jamaiah,

Anuar, Najib, & Zurainee, 1998). The location of Malaysia at the equator, combined

with optimal temperatures and humidity facilitates rapid growth and transmission of

malaria parasites and vector (Rahman, Che’ Rus, & Ahmad, 1997).

Although malaria incidence rates are higher in rural areas (86.5%) such as Sabah and

Sarawak (Figure 1.4) and among the Orang Asli (33.1%) (Ministry of Health

Malaysia, 2004), a recent study reported that migrant workers, namely from malaria-

endemic countries such as Indonesia, India, Vietnam, Myanmar, and Pakistan (Lim,

1998); (Vijayakumari, 2006) are mainly responsible (up to 60% contribution) for the

spread of malaria in Malaysian urban areas (Masitah, Nor aini, & Mas Ayu, 2008).

9

Figure 1. 4. Malaria Cases and Incidence Rate in Malaysia by State until May 2007

Source: Department of Public Health, Ministry of Health Malaysia, 2008

Another study reported that malaria recurrently occurred more often among foreign

patients (57%) in University Malaya Medical Centre (UMMC) than Malaysian

patients (43%) (Jamaiah I, 1998). Between the years 1983 to 1992, the number of

malaria cases among foreigners in Malaysia had increased by 33%. With the annual

increase of foreign workers influx into Malaysia, this percentage increase is not

surprising.

In many malaria-endemic countries outside the African continent such as Malaysia,

P. vivax coexists with P. Falciparum (Korsinczky, Fischer, Chen, Baker, Rieckmann,

& Cheng, 2004). Plasmodium vivax is the most common strain of malaria parasite in

Malaysia, followed by P. Falciparum (Figure 1.5). A study reported that P. vivax

10

was the most common infection in northern peninsular Malaysia, bordering Thailand,

but P. falciparum and mixed infections also occurred (Rahman, Abu Hassan,

Adanan, & Rashid, 1993).

Figure 1. 5. Malaria Parasite Distribution in Malaysia until May 2007

Source: Department of Public Health, Ministry of Health Malaysia, 2008

1.3. The Mechanism of Malaria

The causative agent of malaria is a protozoan parasite of the genus Plasmodium

(phylum Apicomplexa). In humans, the etiologic agents of malaria are the multistage

P. falciparum, P. malariae, P. ovale, P. vivax and P. knowlesi (Mueller, Zimmerman,

& Reeder, 2007); (Singh, et al., 2004). P. falciparum is the most common cause of

infection and is responsible for about 80% of all malaria cases and 90% of malaria-

caused deaths (Mendis, Sina, Marchesini, & Carter, 2001). The Plasmodium species,

with the exception of P.malariae (which may affect the higher primates) are

exclusively parasites of man.

11

The life cycle of Plasmodium is shown in Figure 1.6. The parasite's primary

(definitive) hosts and transmission vectors are adult female mosquitoes of the

Anopheles genus (Ross, 1897); (Holt, et al., 2002). Only female Anopheles

mosquitoes (Figure 1.7) feed on blood, thus males do not transmit the disease. Out of

the 380 species of Anopheles mosquito, only 60 can transmit malaria. Malaria

parasites can also be transmitted by blood transfusions, although this is rare

(Marcucci, Madjdpour, & Spahn, 2004).

Uninfected mosquitoes first ingest the malaria parasite by feeding on an infected

human (host) carrier. During ingestion of a blood meal from an infected host, the

parasites enter the female Anopheles’ digestive system in the male and female sexual

forms called gametocytes. Both male and female gametocytes then undergo rapid

cellular division to form male and female gametes called microgametes and

macrogametes respectively. The flagellated microgametes then fertilize the

macrogametes by fusing with it in the mid-gut of the female Anopheles. The

fertilization process produces an ookinete that penetrates the gut lining and produces

an oocyst in the gut wall. When the oocyst ruptures, it releases sporozoites that

migrate through the mosquito's body to the salivary glands, where they are then

ready to infect a new human host. When the mosquito takes a subsequent blood

meal, the motile, infective sporozoites are injected into the victim’s skin capillaries,

with the Anopheles’ saliva and pass into the human’s bloodstream. This type of

transmission is occasionally referred to as anterior station transfer (Talman, Domarle,

McKenzie, Ariey, & Robert, 2004).

12

Figure 1. 6. Lifecycle of Plasmodium falciparum

Source: http://www.nap.edu/books/0309092183/xhtml/images/p2000af94g137001.jpg

13

Figure 1. 7. A Feeding female Anopheles mosquito

Source: http://www.arbovirus.health.nsw.gov.au

Figure 1. 8. Different Life Stages of Plasmodium falciparum

Source: http://img.medscape.com

14

Once in the human body, the malaria parasite develops via two phases: an

exoerythrocytic and an erythrocytic phase. The exoerythrocytic phase involves

infection of the hepatic system, or liver, whereas the erythrocytic phase involves

infection of the erythrocytes, or red blood cells. When an infected mosquito pierces a

person's skin to take a blood meal, sporozoites in the mosquito's saliva enter the

bloodstream and migrate to the liver. Within 30 minutes of being inoculated into the

human host, the sporozoites disappear from the blood. Many are destroyed by the

human body’s phagocytes but some enter the hepatocytes (parenchyma cells of the

liver) directly or via the Kupffer cells. In the hepatocytes, the sporozoites multiply

asexually and asymptomatically in a process called exoerythrocytic schizogony for a

period of 6–15 days. During this process, multinucleated schizonts are formed. The

cytoplasm of each schizont divides in a manner that results in the encapsulation of

each nucleus, such that each encapsulated nucleus forms a merozoite. About 2000 to

40,000 merozoites can be formed in the liver depending on the species of

Plasmodium. The merozoites then undergo maturation in the hepatocytes. The

merozoite-containing hepatocytes eventually rupture, releasing the mature

merozoites into the blood. Despite the numerous defence mechanisms present in the

internal body systems, the merozoites escape from the liver undetected. This is done

by wrapping themselves in the cell membrane of the infected host liver cell (Sturm,

et al., 2006). The released merozoites then invade the erythrocytes, thus beginning

the erythrocytic stage of the life cycle (Bledsoe, 2005). Figure 1.8 shows the

different life stages of the Plasmodium falciparum.

In the erythrocytic stage, the released merozoites invade the erythrocytes present in

the sinusoids of the liver while some are phagocytised. Within the erythrocytes, the

merozoites undergo a trophic period, in which they enlarge and again multiply

15

asexually, periodically breaking out of their host erythrocyte to invade fresh

erythrocytes. Several such amplification cycles occur, significantly destroying many

erythrocytes in order to release mature merozoites. Following the lysis of

erythrocytes to release mature merozoites, a glycolipid with many properties of

bacterial endotoxins is released, causing a cascade reaction. The glycolipid activates

cytokines such as tumour necrosis factor (TNF) and interleukins II-1, II-6 and II-8

(Stein, 1983) and other unidentified pyrogenic substances that leads to paroxysms of

malarial fever and malaise Thus, classical descriptions of waves of fever arise from

simultaneous waves of merozoites escaping and infecting red blood cells.

Some P. vivax and P. ovale sporozoites do not immediately develop into

exoerythrocytic-phase merozoites, but instead produce hypnozoites that remain

dormant for periods ranging from several months (6–12 months is typical) to as long

as three years. After a period of dormancy, they reactivate and produce merozoites.

Hypnozoites are responsible for long incubation and late relapses in these two

species of malaria (Cogswell, 1992).

The Plasmodium parasite is relatively protected from attack by the body's immune

system because for most of its human life cycle it resides within the liver and

erythrocyes and is relatively invisible to immune surveillance. However, circulating

infected erythrocytes are destroyed in the spleen. To avoid this fate, the P.

falciparum parasite displays adhesive proteins on the surface of the infected

erythrocytes, causing the erythrocytes to adhere to the walls of small blood vessels,

thereby sequestering the parasite from passage through the general circulation and

the spleen (Chen, Schlichtherle, & Wahlgren, 2000). This "adherence" is the main

factor that gives rise to hemorrhagic complications of malaria. High endothelial

venules (the smallest branches of the circulatory system) can be blocked by the

16

attachment of masses of these infected erythrocytes. The blockage of these vessels

causes symptoms such as in placental and cerebral malaria. In cerebral malaria the

sequestrated erythrocytes can breach the blood brain barrier possibly leading to coma

(Adams, Brown, & Turner, 2002).

Some merozoites in the blood turn into male and female gametocytes. If a mosquito

pierces the skin of an infected person, it potentially picks up gametocytes within the

blood. Fertilization and sexual recombination of the parasite occurs in the mosquito's

gut, thereby defining the mosquito as the definitive host of the disease. New

sporozoites develop and travel to the mosquito's salivary gland, completing the cycle.

Pregnant women are especially attractive to the mosquitoes due to hormonal,

metabolic, or mechanical (increased expression of adherence factors in placenta

enhancing the conditions for P. falciparum development) changes (Lindsay, Ansell,

Selman, Cox, Hamilton, & Walraven, 2000). Malaria in pregnant women is a major

cause of stillbirths, infant mortality and low birth weight (van Geertruyden, Thomas,

Erhart, & D'Alessandro, 2004), particularly by P. falciparum infections, but also by

other species infection, such as P. vivax (Rodriguez-Morales, et al., 2006).

1.4. Methods in Malaria Control

Methods used to prevent the spread of disease, or to protect individuals in areas

where malaria is endemic, include prophylactic drugs, mosquito eradication, and the

prevention of mosquito bites. The continued existence of malaria in an area requires

a combination of high human population density, high mosquito population density,

and high rates of transmission from humans to mosquitoes and from mosquitoes to

humans. If any of these is lowered sufficiently, the parasite will eventually disappear

17

from that area, which occured in North America, Europe and much of Middle East.

However, unless the parasite is eliminated globally, it could re-establish if conditions

revert to a combination that favours the parasite's reproduction. Many countries are

observing an increasing number of imported malaria cases due to extensive travel

and migration.

Efforts to eradicate malaria by eliminating mosquitoes have been successful in some

areas. In the United States of America, the draining of wetland breeding grounds,

better sanitation, and the use of the pesticides eliminated mosquitoes and led to the

reduction of malaria cases ( http://www.cdc.gov/malaria/history/eradication_us.htm,

2004). Before pesticides were used, malaria was successfully eradicated or controlled

also in several tropical areas by removing or poisoning the breeding grounds of the

mosquitoes or the aquatic habitats of the larva stages.

Sterile insect technique is also emerging as a potential mosquito control method.

Progress towards genetically modified insects suggests that wild mosquito

populations could be made malaria-resistant. Researchers at Imperial College

London in 2000, created the world's first transgenic malaria mosquito (Catteruccia, et

al., 2000) and the first Plasmodium-resistant species was produced by a team at Case

Western Reserve University in Ohio in 2002 (Ito, Ghosh, Moreira, Wimmer, &

Jacobs-Lorena, 2002). Following that, a separate published study found that a

chemical produced by sea cucumber Cucumaria echinata, impaired the development

of the malaria parasites produced by transgenic mosquitoes (Yoshida, et al., 2007).

Indoor residual spraying (IRS) is the practice of spraying insecticides on the interior

walls of homes in malaria-infected areas. After feeding, many mosquito species rest

on nearby surfaces while digesting the blood meal. Therefore, insecticide-coated

18

walls of dwellings could eliminate mosquitoes before they inject another victim,

transferring the malaria parasite. The first and historically most effective insecticide

used for IRS was DDT. Initially it was used exclusively to combat malaria. However,

its use quickly spread to agriculture. This large-scale use led to the evolution of

resistant mosquitoes in many regions of the world. The DDT resistance shown by the

Anopheles mosquitoes is comparable to antibiotic resistance exhibited by bacteria.

Despite its effectiveness, awareness of the negative consequences of DDT overuse

led it to be banned from agricultural applications in many countries. It is likely that

DDT may now be more effective method of malaria control since its use has been

limited or banned for some time.

Mosquito nets hinder direct contact between mosquitoes and people, greatly reducing

the infection and transmission of malaria. Nevertheless, nets are not perfect barriers.

As such, they are often treated with insecticides (insecticide-treated nets, ITN)

designed to kill the mosquito before it has time to search for a way past the net. ITN

are estimated to be twice as effective as untreated nets (Hull, 2006) and offer greater

than 70% protection compared to absence of net (Bachou, Tylleskär, Kaddu-

Mulindwa, & Tumwine, 2006).

1.5. Treatment of Malaria

For most infectious diseases for which there are effective vaccines, a single infection

confers long-standing protective immunity. However, this type of protective

immunity does not exist for malaria. There is currently no licensed vaccine that will

prevent malaria (Plowe, Alonso, & Hoffman, 2009), but this is an active field of

19

research. Vaccines for malaria are under development, with no completely effective

vaccine yet available. However, there are antimalarial medications.

Malaria is treated with antimalarial drugs. Antimalarials can be classified as

prophylactic and therapy drugs. Prophylactic drugs are taken as prevention and

require continuous administration to reduce the risk of infection. Therapy drugs on

the other hand are taken when the person is already infected with Plasmodium.

However, strategies for combating malaria change rapidly, and when drugs are

administered in combination, it can be difficult to identify which agents are

prophylactic and which are therapeutic. Another approach for classifying

antimalarials is to group them by mechanism of action and by chemical structure.

Based on this type of classification, there are the following three groups of

antimalarials: the quinolines, antifolates, and the artemisinin derivatives.

1.5.1. Quinolines

The first quinoline antimalarials were alkaloids extracted from the cinchona tree.

There are 3 groups of quinolines developed as antimalarials. They are the 8-

aminoquinolines, 4-aminoquinolines, and quinolinemethanols. The first synthetic

antimalarial was an 8-aminoquinoline called plasmaquine (later called Pamaquine)

that was found to be very effective but too toxic. A less toxic analogue called

primaquine (PR) was synthesized and is still utilized until today to destroy liver

reservoirs of Plasmodium. Figure 1.9 shows the structure of major quinoline drugs.

20

1.5.1(a) Quinine

Quinine is a natural white crystalline alkaloid with a bitter taste and has antipyretic

(fever-reducing), antimalarial, analgesic (painkilling), and anti-inflammatory

properties. It is a stereoisomer of quinidine. Quinine was the first effective treatment

for malaria caused by P. falciparum. It remained the antimalarial drug of choice until

the 1940s. Since then, many other effective antimalarials have been introduced,

although quinine is still used to treat the disease in certain critical situations.

Quinine is an alkaloid that acts as a blood schizonticidal and weak gametocide

against P. vivax and P. malariae. As an alkaloid, it is accumulated in the food

vacuoles of the Plasmodium species, especially P. falciparum. It acts by facilitating

an aggregation of cytotoxic heme. Quinine is less effective and more toxic as a blood

schizonticidal agent than another aminoquinoline, chloroquine (CQ). However it is

still very effective and widely used in the treatment of acute cases of severe P.

falciparum. It is especially useful in areas where there is known to be a high level of

resistance to CQ, mefloquine (MQ) and pyrimethamine (PYR). Quinine is also used

in post-exposure treatment of individuals returning from an area where malaria is

endemic. Quinidine is a direct derivative of quinine. It has similar anti-malarial

properties to the parent compound. Quinidine is recommended only for the treatment

of severe cases of malaria.

21

Figure 1. 9. Structures of Major Quinoline Drugs

1.5.1(b) Chloroquine (CQ)

CQ is perhaps one of the earliest and effective 4-aminoquinolines to have been

produced. It is metabolized by hepatic CYPs 2C8, 3A4 and 2D6 into N-

desethylchloroquine (Projean, et al., 2003), which retains half of CQ’s antimalarial

properties (McChesney & Fitch, 1984). CQ until recently was a widely used

antimalarial due to its efficacy and low risk of side effects in prescribed doses. It was

often combined with PR tablets. Since CQ also has a significant anti-pyretic and anti-

inflammatory effect when used to treat P. vivax infections, it may still remain useful

even when resistance is more widespread. A slightly different drug called nivaquine

or CQ phosphate has also been used.

22

However, due to improper use, parasite resistance developed rapidly, rendering the

drug useless in many malaria endemic countries. Although the emergence of drug

resistant parasitic strains is rapidly decreasing its effectiveness, it is still the first-line

drug for malaria treatment in most sub-Saharan African countries. It is now

suggested that it be used in combination with other antimalarial drugs to increase

effectiveness.

1.5.1(c) Piperaquine (PQ)

Piperaquine (PQ), a bisquinoline, was used in China in the late 1970es and 1980es as

malaria prophylaxis and treatment of CQ-resistant falciparum malaria due to its good

tolerability and efficacy. It is structurally similar to CQ and highly lipophilic and

hydrophobic (Sim, Davis, & Ilett, 2005); (Ahmed, et al., 2008). PQ exhibits elevated

absorption and bioavailability in healthy volunteers when taken with moderately

fatty meals (D'alessandro, 2009) maximising its therapeutic effects.

Due to its rather extended elimination half-life (20 -33 days) (Hung, et al., 2004);

(Tarning, et al., 2005); (Nguyen, et al., 2009), it is often co-administered with

dihydroartemisinin (dHA) in ACTs. It is found to be effective against both

falciparum and vivax malaria, with a mechanism that acts through chemical

inhibition of parasite heme detoxification (Davis, Hung, Sim, Karunajeewa, & Ilett,

2005).

23

1.5.1(d) Amodiaquine (AQ)

Amodiaquine (AQ) has been used as falciparum malaria prophylaxis for over 40

years (Foley & Tilley, 1998). It is a 4-aminoquinoline produced as an alternative to

CQ. It is shown that AQ is highly effective in inhibiting growth of P.falciparum in

vitro (Ekweozor, Aderounmu, & Sodeinde, 1987). It is intrinsically more active than

CQ against P. falciparum parasites, which are moderately CQ resistant. The drug is

therefore increasingly being considered as a replacement for chloroquine as a first

line drug in Africa because of widespread CQ resistance.

Because of major side effects, mainly agranulocytosis, observed during prophylactic

use of the drug, AQ is now only recommended for treatment of malaria for which no

serious cases of toxicity have been reported (Laurent, et al., 1993). After oral

administration, AQ is rapidly absorbed and metabolized into mainly N-

desethylamodiaquine (DEAQ) with other minor metabolites, 2-hydroxyl-DEAQ and

N-bisdesethylAQ (bis- DEAQ) (Churchill, Patchen, Campbell, Schwartz, Nguyen-

Dinh, & Dickinson, 1985); (Churchill, Mount, Patchen, & Björkman, 1986); (Mount,

Patchen, Nguyen-Dinh, Barber, Schwartz, & Churchill, 1986). Although the

formation of DEAQ is rapid, its elimination is very slow with a terminal half-life of

over 100 h (Winstanley, Edwards, Orme, & Breckenridge, 1987); (Laurent, et al.,

1993). AQ and DEAQ both have antimalarial activity, but AQ is 3 times more active

than its metabolite DEAQ (Churchill, Patchen, Campbell, Schwartz, Nguyen-Dinh,

& Dickinson, 1985); (Li, Björkman, Andersson, Ridderström, & Masimirembwa,

2002), produced through metabolism catalysed by hepatic enzyme CYP2C8 (Li,

Björkman, Andersson, Ridderström, & Masimirembwa, 2002). However, since AQ is

rapidly cleared and the metabolite DEAQ attains high plasma concentrations for a

24

long time, AQ is considered a prodrug. AQ is currently used in combination with

artemisinin derivatives.

1.5.1(e) Mefloquine (MQ)

Quinolinemethanols, structural analogs of quinine (Hofheinz & Merkli, 1984) are

potent drugs against both P.falciparum and P.vivax. Although some of the earlier

compounds exhibited appreciable photosensitivity (Pullman, Eichelberger, Alving,

Jones, Craige, & Whorton, 1948), a derivative with elevated efficacy and negligible

photosensitivity was successfully developed. This derivative called mefloquine (MQ)

is still utilized to treat malaria.

MQ is an orally-administered antimalarial drug used as a prophylaxis against and for

treatment of malaria. MQ is a very potent blood schizonticide and active against the

erythrocytic stages of Plasmodium species. It is the drug of choice to treat malaria

caused by chloroquine-resistant Plasmodium vivax (Maguire, Krisin, Marwoto,

Richie, Fryauff, & Baird, 2006). MQ interferes with transportation of haemoglobin

products and other substances from the host cell to the parasite’s food vacuole.

However, the drug has no effect against the exoerythrocytic (hepatic) stages of the

parasite.

MQ is metabolized by the hepatic CYP3A4 into carboxymefloquine and

hydroxymefloquine. The rather long half-life of MQ of about 20-30 days (Schwartz,

et al., 1982); (Desjardins, Pamplin, von Bredow, Barry, & Canfield, 1979) and sub-

therapeutic concentrations of the drug which can remain in the blood for months after

treatment, may contribute to emergence of parasite resistance (Karbwang & White,

1990); (Nosten & Price, 1995). Parasite resistance towards MQ is evident especially

25

in Thailand and other parts of Southeast Asia. Therefore, MQ is now used in

combination with artemisinin derivatives such as artesunate (AS) or artemether in

order to stem further development of resistance (Price, et al., 1995). The sustained

use of the AS-MQ combination has, in fact, reduced falciparum malaria transmission

and progression of drug resistance in western Thailand (Nosten, et al., 2000);

(Woodrow, Haynes, & Krishna, 2005).

1.5.2. Antifolates

Antifolates act by inhibiting the enzyme dihydrofolate reductase (DHFR) in the

Plasmodium parasite (Yuthavong, 2002). DHFR is important in folate synthesis that

forms the essential folate cofactor, fully-reduced tetrahydrofolate (Gregson & Plowe,

2005). Tetrahydrofolate is vital for the production of purine and pyrimidine bases,

which are necessary monomers in DNA synthesis. Low levels of tetrahydrofolate are

known to arrest DNA replication in Plasmodium (Triglia & Cowman, 1999).

Sulphonamides (SA), sulfones, pyrimethamine (PRY) and proguanil (PG) are among

the most widely used antifolates (Figure 1.10). However, rapid emergences of

resistance among Plasmodium towards these drugs are reducing its effectiveness

(Olliaro & Yuthavong, 1999).

PYR is used in the treatment of uncomplicated malaria. It is used in cases of CQ-

resistant P. falciparum strains when combined with sulphadoxine (a type of SA). It

acts primarily on the schizonts during the hepatic and erythrocytic phases.

Sulphadoxine acts on the schizonts during the hepatic and erythrocytic phases. It is

mainly used for treating P. falciparum infections and is less active against other

Plasmodium strains. However usage is restricted due to the long half life of the