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