DETERMINANTS OF RESPONSES TO ANTIMALARIAL 
DRUGS IN CHILDREN WITH UNCOMPLICATED 
PLASMODIUM FALCIPARUM MALARIA 
 
BY 
 
 
RY
SIJUADE Abayomi Olusola  
RA
BSc (Chemistry) Ilorin, MSc (Pharmacology & TherapIeButics) Ibadan 
Matric No. 108845  L
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A thesis in the DAeDpartment of  
PHARMACOLOFG YI
B AND THERAPEUTICS 
 O  
Submitted to the IFTacYulty of Basic Medical Sciences in partial fulfillment S of the requirements for the degree of 
ER  
IV DOCTOR OF PHILOSOPHY N OF THE U  UNIVERSITY OF IBADAN 
        
 
March 2011 
1 
 
 
ABSTRACT 
Drug resistance is a challenge to malaria control efforts and several factors 
including parasite genetics, host factors and pharmacokinetics may contribute to the 
selection of drug resistant Plasmodium falciparum.  Understanding the role of these 
factors in patient response to antimalarial drugs is therefore essential in the management 
of malaria.  The aim of the study was to determine the factors contributing to delay  in 
malaria Parasite Clearance (PC) in children and evaluating the effects of pharmRacoYkinetic 
variability on treatment outcome.  A
Children (n=2,752), aged 6 months -12 years, with falcBiparRum malaria, were 
enrolled over a period of eight years and treated with stan dLardI doses of Chloroquine 
(CQ), Sulphadoxine-Pyrimethamine (SP)  or AmodiaNquine (AQ) given alone or in 
combination with artemisinin. Each patient was folloAwed up for at least 14 days.  Age, 
axillary temperature, parasite density and AgamDetocytaemia were assessed for their 
potential association with delay in P CI Band treatment outcomes.  Filter-paper blood 
samples were collected from some Fof the children (n=148) before treatment and on days 
1-7, 14, 28 and 35 after  tOreatment for determination of CQ and sulphadoxine 
concentrations.  In anIoTtheYr subset of patients (n=7), treated with amodiaquine, blood and 
saliva samples wSere collected over 35 days.  High performance liquid chromatographic 
techniqueEs wRere used to determine concentrations of sulphadoxine in whole blood as well 
as AIQV and Desethyl amodiaquine (DEAQ) in saliva.  Mean maximum drug concentration 
(NCmax), half-life (t1/2) and area under concentration-time curve (AUC0-28d) were assessed Ufor their association and predictive value for treatment outcomes. Data were analyzed 
using descriptive statistics, ANOVA, Chi-square, Students’ t-test, Kruskal-Wallis and 
multiple regressions at p = 0.05.  
Age ≤ 2 years (Adjusted odds ratio [AOR] = 2.13), presence of fever (AOR = 
1.33) and parasitaemia > 50,000/µl (AOR = 2.21) at enrolment were independent risk 
2 
 
 
factors for delay in PC, while a body temperature >38OC and parasitaemia >20,000/µl 
were predictors a day after treatment regardless of the drug used.  Day 3 concentration ≤ 
1750ng/ml and AUC0-28d ≤ 950ng/ml.h were associated with chloroquine treatment 
failure. In a multivariate analysis, a terminal elimination t½ ≤ 220h (AOR = 0.28) and 
AUC0-28d ≤ 950ng/ml.h (AOR = 4.12) were identified as independent pharmacokinetic 
predictors of chloroquine treatment failure. Age stratified analysis showed that SD X 
concentrations were significantly higher in children > 5years compared tRo cYhildren 
<5years: Cmax; 295 vs 125µg/ml, AUC0-28d; 1562 vs 812µg/ml.d.  InA patients who 
received AQ, there was a rapid conversion of AQ to DEAQ, whBichR was detectable in 
plasma and saliva within 40 minutes of administration.  The mLeanI day 7 concentration of 
DEAQ was significantly higher in plasma than in salivaN (24 7.8 vs 125.1ng/ml).  The t1/2 
of DEAQ were similar in plasma (167.25±43.4h) andA saliva (146.12±17.2h). The decline 
phases of DEAQ in saliva concentration-timeA cuDrves were approximately similar to that 
in plasma.  IB
Delay in parasite clearance Fis specific and related to drug resistance.  In addition 
pharmacokinetic variability o f Osulphadoxine in children has potential impact on dosage 
regimen and treatmenItT outYcome.  
 S
KeywordsE: RUncomplicated malaria, Pharmacokinetics, Antimalarials, 
PlasImVodium falciparum. 
WNord Count = 499 U
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DEDICATION 
To the children who participated in the studies used for this thesis.
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ACKNOWLEDGMENT 
I express my profound gratitude to my supervisor, Dr. Grace O. Gbotosho for 
allowing me to draw from her wealth of experience and standing by me through her 
concerns as well as critical reading of my thesis. I also thank Professor A. Sowunmi 
for co-supervising this work. I appreciate his concerns and effort during the course of 
my programme.  
 I appreciate Professor AMJ Oduola for his guidance and support. IR thaYnk the 
Head of Department, Dr. Catherine O. Falade for her support and RadvAise during the 
course of this programme. I thank Dr. T.C. Happi for his assistIanBce at all time. I must 
record my indebtedness to Dr O.A.T. Ogundahunsi for  hLis support at all times. I 
sincerely appreciate the moral support of ProAfessNor O.O. Akinyinka and Dr. 
Olanrewaju Sowunmi.  D
To all my Lecturers in the FBacuAlty and all staff of the Department of 
Pharmacology and Therapeutics, FUni vIersity of Ibadan. My colleagues at the Malaria 
Research Laboratories have  Obeen quite inspiring and they include: Drs. Onikepe 
Folarin, Folusho FalaTdeY, Oyindamola Abiodun, Sogo Olalubi, Micheal Obaro and Thomas AnyorigSiyaI.  I thank Mrs Amoo and Adeola Alabi for running the clinic from 
which all theR children enrolled in the studies were recruited. I appreciate all staffs of 
PostIgVradu
Eate Institute for Medical Research and Training, for accommodating me at 
tNhe Institute for carrying out all my research work.    U I also appreciate the contribution of my dear ones, the SIJUADES: Femi & 
Kemi, Funmilola & Bola, Oyetoso & Moji, Olanrewaju & Seyi, Dupe & Akin and 
Opeyemi; who have been sources of encouragement, financial and moral support for 
me at all times. They have made the dream of completing this research a reality. 
Thank you for been a wonderful family. I thank God for being part of you. 
5 
 
 
I sincerely appreciate the support of my in-laws Dr. and Mrs I.O. Ayeni, 
Tunde & Funmilola Oni-Obasa, Dr & Mrs Ambi Rukewe, and kikelomo.  Late Engr. 
Dare Ayeni for his priceless encouragement. To my friends; Olutunde, Remi, 
Tubosun, Lowo, Adelekan, Pastor Tunde Adisa, Soji Adesina and Ayo Olalekan, I 
appreciate you. I say big thank you to all the children who have participated in these 
studies. I appreciate the support I received from Pastors Oyedokun and Samu el. 
Thank you all.  RY
To my dear wife, Oluwatoyin for her unending love at all tiRmesA, patience and 
understanding. I appreciate my lovely children FiyinfOluwa, FoIgBofOluwa and Ibukun 
for their understanding during my absentness at home.    L
Chief and Deaconess S.A.O. Sijuade; DaAd aNnd Mum I appreciate you for 
standing by me and encouraging me at all timesD. Thank you.   
I acknowledge the University of BAdoA-Ekiti for the support and time to prepare 
parts of this thesis despite our busFy sc hIedule. 
Finally, I give all gl orOy and honour to the Most High God for this privilege 
and being my sustaineTr, pYrovider and for giving me Christ Jesus, my friend and lover of my soul. ThanSk yIou FATHER. 
The sRtudies were supported by grants from UNDP/World Bank/WHO/TDR to 
Dr. GIVrace
E O. Gbotosho.  
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CERTIFICATION 
I certify that this work was carried out by Mr SIJUADE, Abayomi Olusola of the 
Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan. 
 
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……………………………………… …L……… 
Supervisor N
Grace O. GboDtosAho 
B. Pharm (Hons) (Ife), MSc, PhD PharAmacology and Therapeutics (Ibadan) 
Sen iIoBr Lecturer  
DepartmenOt of FPharmacology and Therapeutics 
Y University of Ibadan IT Ibadan 
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Table of Contents 
 
 
           Page 
Title          i 
Abstract         ii 
Dedication         iv  
Acknowledgements        v Y
Certification               A    vRii Table of Contents                 viii 
List of Tables        B R         xiii 
List of Figures        L I           xvii Glossary of Abbreviations     N            xx  
Chapter 1: INTRODUCTION   DA   1 
  Objectives of the research  A    5 
 IB
Chapter 2: LITERATURE REVFIE W     6 
2.0 Malaria the D isOease      6 2.1  Life cycleY of malaria      8 2.2 MalaIriTa control methods     10 2.3 CShemotherapy of malaria     11   R2.3.1  Classification of antimalarial drugs   12 
  E 2.3.2 Pharmacology of antimalarial drugs   14 
 IV2.4 Review of studies on pharmacokinetics of chloroquine,  N sulphadoxine-pyrimethamine and amodiaquine  23 U 2.5 Antimalarial drug resistance     36 
 
Chapter 3: MATERIALS AND METHODS      
Study 1: Studies on risk factors contributing to delay 
 in parasite clearance in acute uncomplicated malaria. 
3.0 Patients and drug treatment     40 
 
8 
 
 
 
3.1 Sample collection      40 
3.2 Assessment of parasitaemia     42 
3.3 Evaluation of response to treatment    42 
 
         Study 2: Pharmacokinetic determinants of response to treatment  
with chloroquine in children with acute uncomplicated 
  Plasmodium falciparum malaria    44  
3.4. Study Population      4R6 Y
 3.5 Drug Analysis       A46 
  3.5.1 Reagent for drug analysis    R 46 
3.5.2 Preparation of stock and working solutioInB  47 
  3.5.3 Analysis of chloroquine from filter p aLper samples 48 
Study3: Development of High Performance LiNquid  
Chromatographic Analytical mDethAod for  measurement of sulphadoxAine concentration in filter  paper sample.        52 
  3.6 Analysis of sul pIhaBdoxine from filter paper samples 52 
Study 4: Pharmacokinetic Fdisposition of sulphadoxine in children  
with acute un cOomplicated malaria treated with standard 
dose oTf suYlphadoxine-pyrimethamine.   56 Study 5: EvalIuation of the use of saliva for therapeutic drug  
RmSonitoring in children with uncomplicated Plasmodium E falciparum Malaria treated with amodiaquine-artesunate. 
IV         57 N 3.7 Patients enrolled in the study and sample collection  57 U 3.8 Analysis of amodiaquine or desethyl amodiaquine from plasma   or saliva samples      58 
3.9 Statistical analysis for all studies    60 
 
9 
 
 
Chapter 4: RESULTS 
Study 1: Risk factors contributing to delay in parasite clearance in  
children with acute uncompleted malaria.    63 
4.0 Study population       63 
 4.1 Drug treatment and delay in parasite clearance   63 
4.2 Risk factors for delay in parasite clearance at enrolment  69 
 4.3 Risk factors for delay in parasite clearance following initiation 
 of treatment        6 9 
 4.4 Delay in parasite clearance and treatment failure   
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Study 2: Pharmacokinetic determinants of response to trReatment  
with chloroquine in children with acute uncoImBplicated       
Plasmodium falciparum malaria.   L   73 
 4.5 Study population    N    78 
 4.6 Pharmacokinetic parameters of aDll pAatients enrolled in the study 78 4.7 Comparison of blood chloroqAuine concentrations and  pharmacokinetic parameItBers in children with early or delay in  parasite clearance.        81 
4.8 Comparison of phaFrmacokinetic parameters in children with  
or without pre tOreatment chloroquine concentration.    84 
4.9 CompaTrisYon of clinical parameters and treatment outcome in  childIren with or without pre-treatment chloroquine concentration. 87 
4.10 RCSomparison of blood chloroquine concentration in children  E with sensitive or resistance responses to chloroquine.  87 
IV4.11 Comparison of pharmacokinetic parameters in children  N whose infection responded or failed to respond to  U chloroquine treatment.       90 4.12 Pharmacokinetic risk factors associated with chloroquine 
 treatment failure.        90 
Study 3: Development of a simple cost effective high performance liquid 
chromatography assay of sulphadoxine in whole blood spotted on 
filter paper for field studies.     96 
4.13 Chromatographic techniques      96 
 4.14 Recovery, calibration curves and reproducibility   99 
10 
 
 
 4.15 Interference                 102 
 4.16 Clinical application                102 
  
Study 4: Pharmacokinetic disposition of sulphadoxine in children with 
acute uncomplicated malaria treated with standard dose of 
sulphadoxine-pyrimethamine.              105 
4.17 Study population                105 
4.18 Pharmacokinetic parameters of sulphadoxine in children with  
uncomplicated falciparum malaria after administration of staRndaYrd 
dose of sulphadoxine-pyrimethamine.   A          107 
4.19 Comparison of blood sulphadoxine concentration andR          
pharmacokinetic parameters in children with earIlyB parasite clearance 
or delay in parasite clearance.    L            109 
4.20 Comparison of disposition of sulphadoxNine in children with and 
without detectable blood levels of suAlphadoxine at enrolment  
before treatment.  A D              112 4.21 Pharmacokinetic of 61 children without pretreatment  
sulphadoxine at enrolm eInBt.               115 
4.22 Comparison of blooFd sulphadoxine concentrations in children  
with sensitive  aOnd resistant responses to sulphadoxine- 
pyrimeTthaYmine                117 4.23 CompIarison of disposition of sulphadoxine in children  
RwShose infection responded or failed to respond to standard  E dose of sulphadoxine-pyrimethamine.                        117 
IV4.24 Relationship between age and sulphadoxine pharmacokinetic N  parameters in children treated with standard oral dose U  of sulphadoxine-pyrimethamine.              121  
Study 5: Evaluation of the use of saliva for therapeutic drug monitoring 
in Children with Uncomplicated Plasmodium falciparum malaria treated 
with amodiaquine.               
4.25 Patients enrolled in the study                       130 
4.26 Pharmacokinetic parameters of desethyl amodiaquine in children with 
uncomplicated falciparum malaria after administration of standard doses of 
11 
 
 
artesunate-amodiaquine.                      132 
Chapter 5: DISCUSSION                                139 
CONCLUSION AND RECOMMENDATION              154 
REFERENCES                  155 
APPENDICES                  174 
 
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List of Tables 
 
Table Title Page 
 
2.1 Classification of antimalarial drugs. 13 
 
3.1 Antimalrial drugs and their treatment regimens with time of 41 
 study administered to children with acute uncomplicated malaria  
   
3.2 Evaluation of clinical response of children to treatment with 43  
 antimalarial drugs  
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4.1 Baseline clinical and parasitological parameters of 2752 children R65 
 with acute uncomplicated falciparum malaria enrolled Rin tAhe  study. 
 
4.2 Proportion of children with delay in parasite clearancIe Bfollowing 66 
treatment with standard doses of selected antimal arLial drugs. 4.3 Proportion of children with falciparum malNaria who failed to 67 respond to treatment with standard doses of antimalarial drugs. 
4.4 Predictors of delay in parasite clearanAce at presentation in 70 
children with acute falciparum malaria
 D 
4.5 Predictors of delay in parasiteI Bclea
Arance on day 1 after treatment 71 
in children with acute falcip arum malaria.    
4.6 Percentage recovery of Fchloroquine from whole blood spotted 75 
on filter paper using  Othe analytical method for chloroquine.  
4.7 Precision ofT anYalytical method for extraction of chloroquine 76 from wholeI blood spotted on filter paper.  
4.8 BasRelinSe clinical and demographic characteristics of seventy 79 four children with acute uncomplicated malaria treated with Estandard doses of chloroquine. 
4.9 IV  Pharmacokinetic parameters of chloroquine determined in 80 N children with falciparum malaria and treated with standard doses U of chloroquine.  
4.10 Pharmacokinetic parameters of chloroquine in children with 83 
acute uncomplicated malaria who had delay in parasite clearance 
after treatment with standard doses of chloroquine. 
 
4.11 Pharmacokinetic parameters of chloroquine in children with and 86 
without pretreatment chloroquine levels following treatment 
with standard doses of chloroquine (n=74). 
 
4.12 Comparison of clinical parameters and treatment outcome in 88 
13 
 
 
children with acute uncomplicated malaria who had or without 
pretreatment chloroquine concentration (n=74). 
 
4.13 Whole blood chloroquine concentrations in children with 89 
uncomplicated falciparum malaria with sensitive or resistant 
response after administration of 25mg/kg chloroquine base 
(n=74). 
 
4.14 Pharmacokinetic parameters of chloroquine determined in 91 
children with acute uncomplicated falciparum malaria who had 
sensitive or resistant responses after treatment with standard  
doses of chloroquine (n=74). 
 Y
4.15 Pharmacokinetic predictors of treatment failure in children wAith R93 uncomplicated malaria treated with standard doses of 
chloroquine. R
 
4.16 Pharmacokinetic risk factors of treatment failure in chIilBdren with 94 
 uncomplicated malaria treated with comp leLte doses of chloroquine (when considering P value 0.3). 
  N  
4.17 Percentage recovery of sulphadoxine fromA whole blood samples 100 
collected on filter paper. 
 D  
4.18 Precision of analytical method foAr sulphadoxine determination 101 
using whole blood spotted o nI fBilter paper.  
4.19 Demographic and pharmFacokinetic parameters of a patient who 104 
received a standard  dOose of sulphadoxine-pyrimethamine.  
4.20 Baseline cliTnicYal and demographic characteristics of children 106 with acuteI uncomplicated falciparum malaria treated with standarSd dose of sulphadoxine-pyrimethamine (n=74). 4.21 PhaRrmacokinetic parameters of sulphadoxine in seventy-four 108 children treated with standard dose of sulphadoxine-Epyrimethamine. 
IV  4.22 Comparison of pharmacokinetic disposition of sulphadoxine in 111 N children with and without delay in parasite clearance. U  4.23 Pharmacokinetic parameters of sulphadoxine in children with 113 
and without sulphadoxine in pretreatment whole blood samples. 
 
4.24 Clinical and laboratory data at enrolment of children with acute, 116 
uncomplicated falciparum malaria treated with standard dose of 
sulphadoxine-pyrimethamine without pre-treatment 
sulphadoxine levels. 
 
4.25 Whole blood sulphadoxine concentration in children with 118 
uncomplicated falciparum malaria with sensitive or resistant 
14 
 
 
response after oral administration of a standard dose of 
sulphadoxine-pyrimethamine. 
  
4.26 Pharmacokinetic parameters of sulphadoxine in children without 119 
pre-treatment sulphadoxine with uncomplicated falciparum 
malaria who had sensitive or resistant responses to oral 
administration of sulphadoxine-pyrimethamine.  
 
4.27 Whole blood sulphadoxine concentration in children with 123 
uncomplicated falciparum malaria with sensitive or resistant 
responses after administration of standard oral dose  
sulphadoxine-pyrimethamine. 
 Y
4.28 Relationship between age and pharmacokinetic parameters of R124 
sulphadoxine in children with uncomplicated falciparum malaAria 
treated with standard sulphadoxine-pyrimethamine. R
   
4.29 Baseline clinical and demographic characteristics Lof IsBeven with 131 acute uncomplicated falciparum malaria enrolled  in the study.  
4.30 Saliva-plasma ratio of desethyl amodiaqAuinNe concentrations in 134 children with falciparum malaria treaDted with standard doses of artesunate-amodiaquine.  
4.31 Pharmacokinetic disposition IofB de
Asethyl amodiaquine in plasma 136 
after standard oral doses o f artesunate-amodiaquine (30mg/kg amodiaquine) in five chilFdren with uncomplicated malaria.  
4.32 Pharmacokinetic d isOposition of desethyl amodiaquine in saliva 137 after standard oYral doses of artesunate-amodiaquine (30mg/kg amodiaquine) in five children with uncomplicated malaria  
4.33 Comparison of pharmacokinetic parameters of desethyl 138 
amodiaSquinIe
T after standard oral doses of artesunate-
amoRdiaquine (30mg/kg amodiaquine) in children with uncomplicated malaria. E       4.aI VStandard curve table for extrapolation of unknown concentration of  chloroquine in whole blood on filter paper.        176  
UN4.b Standard curve table for extrapolation of unknown concentration of sulphadoxine  in whole blood on filter paper.      177 
 
4.c  Standard curve table for extrapolation of unknown concentration 
 of desethyl amodiaquine in plasma       178 
 
   4.d  Standard curve table for extrapolation of unknown concentration of 
 desethyl amodiaquine in saliva.        179 
15 
 
 
LIST OF FIGURES 
 
Figure Legend Page 
2.1 Global distribution of malaria. 8 
 
2.2 Plasmodium SPP life cycle. 9 
 
2.3 Chemical structure of Quinine 15 
 
2.4 Chemical structure of Mefloquine 17 
  
2.5 Chemical structure of Halofantrine 1Y9  
2.6 Chemical structure of Artemisinin derivatives  AR22  
2.7 Chemical structure of Chloroquine R 23 
 
2.8 Mode of action of Chloroquine. LIB 25  
2.9 Chemical structure of Sulphadoxine. N 29  
2.10 Mode of action of Sulphadoxine-pyrimethAamine. 30 
 
2.11 Chemical structure of PyrimethamAine D 32  
2.12 Structures of amodiaquine a nId BN-desethyl amodiaquine 34  
4.1 Numbers of children wiFth delay in parasite clearance following 65 
treatment with antim Oalarial drugs.  
4.2 The proportTionYs of children treated with various antimalarials 69 who had dIelay in parasite clearance (PC) and subsequently failed treatme
 Snt  
4.3 StanRdard curve of chloroquine for extrapolation of unknown 74 Econcentrations from whole blood spotted on filter paper.  
4.4 IVChromatogram showing chloroquine and desethyl chloroquine 77 N from whole blood sample collected from collected from a patient U and spotted on filter paper on day 0 and day 3.    
4.5 Mean whole blood concentration-time curves of chloroquine 82 
following oral administration of 25mg/kg of chloroquine base 
over 3 days in children infected with Plasmodium falciparum 
malaria with early or delay in parasite clearance (PC). 
 
4.6 Mean whole blood concentration-time curves of chloroquine in 85 
children infected with Plasmodium falciparum malaria treated 
with oral doses of 25mg/kg chloroquine base over 3 days with or 
without chloroquine in their blood at enrolment. 
16 
 
 
 
4.7 Mean concentration-time curve for all patients, those who 92 
responded to treatment and those who failed to respond to oral 
dose of chloroquine base given over 3 days. 
 
4.8 Decline of chloroquine concentration in children with or without 95 
chloroquine in their pre-treatment whole blood samples following 
oral administration of chloroquine base over 3 days. 
 
4.9 Sulphadoxine standard curve for extrapolation of sulphadoxine 97 
concentrations from unknown blood sample spotted on filter  
paper. 
 Y
4.10 Chromatograms showing peaks after extraction of sulphadoxineR 98 
and internal standard from drug free whole blood spiked wAith 
60µg/ml sulphadoxine, day 0 and day 3 of sample collecteRd from 
a patient who was administered with a standaIrdB dose of sulphadoxine/pyrimethamine.  
 L
4.11 Concentration – time curve for sulphadoxine in w hole blood after 103 
single oral dose of sulphadoxine-pyrimethamNine (25mg/kg body 
weight of sulphadoxine and 1.25mgA/kg body weight of 
pyrimethamine. 
 D
4.12 Mean whole blood concentratiBon-AI time curves of sulphadoxine in 110 children infected with Plasm odium falciparum malaria with early or delay in parasite clearance. 
 F
4.13 Mean whole blood  SODX concentration - time curves in children 114 with PlasmodYium falciparum malaria with or without sulphadoxinTe in their blood at enrolment.  4.14 Mean wShoIle blood concentration-time profile of sulphadoxine in 120 chilRdren whose infection responded and whose infection failed to responded to treatment after oral administration of standard dose Eof sulphadoxine-pyrimethamine. 
4.15I V  Mean concentration-time curve of sulphadoxine in capillary 125 N whole blood of children younger than 5 years or older than 5 U years after oral administration of standard dose of sulphadoxine-pyrimethamine. 
 
4.16 Standard curve of desethyl amodiaquine for extrapolation of 127 
unknown concentrations of DEAQ in plasma. 
 
4.17 Standard curve of desethyl amodiaquine for extrapolation of 128 
unknown concentrations of DEAQ in saliva. 
 
4.18 Chromatograms showing amodiaquine (AQ) and 129 
desethylamodiaquine (DEAQ) in plasma and saliva of a patient at 
17 
 
 
4 h after the first dose of oral artesunate-amodiaquine. 
 
4.19 Plasma and Saliva log concentration - time curves of 135 
desethylamodiaquine in children infected with P. falciparum 
malaria who were treated with oral doses of artesunate-
amodiaquine. 
 
 
 
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Glossary of Abbreviations 
 
ACPR  Adequate clinical response 
ADP  Adenosine diphosphate 
AOR  Adjusted odd ratio 
ATP  Adenosine triphosphate  
AL  Artemether plus lumefantrine Y
AMQ   Mefloquine plus artesunate AR
ANOVA  Analysis of variance R
AOR  Adjusted odd ratio LIB
AQ  Amodiaquine N 
AQAS  Artesunate plus amodiaquine A
AQPS   Amodiaquine plus sulphadoxAineD-pyrimethamine 
AQSP  amodiaquine plus sulfa lIenBe-pyrimethamine 
AS   Artesunate  F
AUC0-28d Area undeYr co n
Ocentration time curve from 0 to 28 day 
CI   ConfIidTence interval 
Cl   RCSlearance Cmax  E maximum concentration 
COTI V Co-trimozaxole 
UCNQ  Chloroquine 
CQCP  chloroquine plus chlorpheniramine 
CQKET Chloroquine plus ketotifen 
CQPS  Chloroquine plus sulphadoxine-pyrimethamine 
d  Day 
DCQ   Desethyl chloroquine 
19 
 
 
DEAQ  Desethyl amodiaquine 
df  degree of freedom 
DHA  Dyhydro artemisinin 
DHPS  Dihydropteroate synthase 
DHFR  Dihyrdofolate reductase 
DNA  Deoxyribonucleic acid  
dUMP  deoxyuridine monophosphate RY
dUTP  deoxyuridine triphosphate RA
ETF  Early treatment failure  IB
F  female  L
Fig.  Figure AN-3
gcm   Gramm per centimetre cube D
-3
gdm   Gramm per decimetre cubBe A
GM   Geometric mean F I
GTP  Glutathione r edOuctase 
h  hour Y
Hb  HSaemIo
Tglobin 
HCl  RHydrochloric acid 
HPLCV  EI  High performance liquid chromatography INPT  Intermittent preventive treatment UIS   Internal standard 
ITNs  Insecticide treated nets 
Kg  Kilograms 
L  Litre 
LTF  Late treatment failure 
20 
 
 
M  Molarity 
MDR  Multi drug resistance 
ml  millilitre 
min  minutes 
MQ  Mefloquine 
mV  Millivolt  
n  Sample size RY
NaH2PO4.2H20  Sodium dihydrogen phosphate RA
NaOH  Sodium hydroxide IB
ng/ml  Nano gram per Millilitre  L
o
C  Degree Celsius AN
 OR  Odds ratio D
pABA  Paraaminobenzoic acid BA
PCT   Parasite clearance Ftim eI 
PYR  Pyrimethamin eO alone  
PPV   PapavTerinYe RBM  RSoll BIack Malaria 
SP  R Sulphadoxine-pyrimethamine 
SPPBV  EI Sulphadoxine-pyrimethamine plus probenecid SND   Standard deviation USDX  sulphadoxine alone 
t1/2  Half-life 
tmax  Time at maximum concentration 
V/V/V  Volume/volume/volume 
VS  Versus   
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WHO  World Health Organization 
y   Year 
χ2  Chi square 
 
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CHAPTER ONE 
INTRODUCTION 
Significance of the project 
 Among several tropical diseases that affect humans, malaria poses Yspec ial 
control problems due to increasing population at risk from the disease.  In 2R008, there 
were approximately 232 million malaria cases and 841,000 malaria RdeatAhs, with close 
to 90% of these occurring in sub-Sahara Africa (WHO 2009).I  BNigeria is the largest 
population at risk in Africa.  Being a serious disease th aLt mostly affects children 
under the age of 5 year, it is responsible for 25% Aof iNnfant mortality and 30% of all 
childhood deaths in Nigeria (FMOH 2004). DGlobally, it causes 85% of death in 
children under 5years of age (GlobalI MBal
Aaria, 2008).  It is a significant cause of 
morbidity accounting for about 6F5%  of all clinic attendance in the country (FMH 
2005).  Nigeria is the first  aOmong the top-five countries for malaria death (Global 
Malaria, 2008).  In tThe Yabsence of an effective antimalarial vaccine, chemotherapy 
remains the maiSnstaIy for control of the disease.  However, the malaria parasite has 
developed reRsistance to nearly all the classes of antimalarials including the promising 
artemIVisin
Ein derivatives (Dondorp et al., 2009).  This could be as a result of 
wNidespread and indiscriminate use of antimalarials, which places a strong selective Upressure on malaria parasites to develop high levels of resistance (Olumese 2005; 
WHO 2006).  
 The reports of antimalarial drug resistance in disease endemic countries, 
especially Africa (Sowunmi & Salako 1992; Falade et al., 1997; Wang et al., 1997; 
Sibley 2001; Sowunmi et al., 2004; Talisuna et al., 2004; Pitmang et al., 2005) had 
23 
 
 
made the WHO to recommend Artemisinin based combination therapy (ACTs) for 
the treatment of acute uncomplicated malaria.  Between 2002 and 2005 many African 
countries including Nigeria adopted the use of ACTs (FMH 2004).  Unfortunately, 
ACTs are expensive for poor people living in disease endemic countries and require 
longer dosage regimen which pose challenges in their use.  In addition, the 
emergence and spread of resistance to these front-line antimalarials have necessita ted 
the need to continually field-monitor parasite susceptibility to coRnveYntional 
antimalarial drugs such as  chloroquine (CQ) and sulphadoxinRe-pAyrimethamine 
(SDX) in order to revisit them for the treatment of acute uInBcomplicated malaria 
(Nkoma et al., 2007; Dondorp et al., 2009).  For instan ceL, in Malawi chloroquine 
regained it efficacy for treatment of malaria 12 yeaArs aNfter it was withdrawn from use, 
suggesting that CQ might once again be considDered for treatment of malaria (Laufer 
et al., 2006). BA
 Indeed, it is very importanFt to  Idetermine factors that can be observed prior to 
treatment that can lead to tre aOtment failure especially in children. In this perspective, 
it would clearly help Tif aYll, or, at least, most children at risk of drug treatment failure could be identifSied Isoon after they become ill. This would allow the planning of 
alternative, mRore effective treatment strategies and thus the reduction of malaria-
attriIbuVtab
Ele morbidity and mortality and slowing of the spread of drug resistance.   
 N A fundamental component of the strategy for the control of malaria disease Uis based on prompt and effective treatment.  In vivo tests are much more direct 
measurement of treatment efficacy in a target population.  The assessment of in-vivo 
therapeutic efficacy involves clinical and parasitological outcomes of treatment over 
a certain period following the start of treatment in order to check for the reappearance 
of parasites in the blood.  A significant proportion of treatment failures do not appear 
24 
 
 
until after day 28 in areas of low or high transmission (WHO 2006).  However, it is 
not often clear if patients have adequate drug blood levels at the time of 
recrudescence of infection and this can be determined by monitoring of blood 
concentration after treatment which can also be used to predict treatment outcome 
(White et al., 2008).   Pharmacokinetics can also play a major role in the 
development of drug resistant malaria.  It has been demonstrated t hat 
pharmacokinetics of drugs can be altered during long time use (BousquRet, Y1970). 
Chloroquine and sulphadoxine-pyrimethamine have been common RdruAgs used in the 
treatment of malaria.  The long-time use of CQ and SP might hIaBve caused changes in 
their kinetic disposition which may result in selective dru gL pressure (Gardella et al., 
2008; Hodel et al., 2010).  Thus, the current studAy wNas designed to re-assess drug 
resistance level and determine whether drug Dtreatment failures observed during a 
standard in-vivo test are related to BparAasite factors or whether they are the 
consequence of poor metabolism oFf C QI and SP. 
 Although, chroma toOgraphic techniques have been developed to allow 
accurate and sensitiveT deYtermination of the level of most antimalarial drugs in blood (Karbwang et alS., 19I87; Walker et al., 1983; Walker & Ademowo 1996; Babalola et 
al., 2003; MRinzi et al., 2005; Dua et al., 2007) these techniques are quite expensive, 
timeI cVons
Euming and require the collection and use of venous blood. The sampling 
tNechniques require expertise, are invasive and sampling may not be easy in children Uduring field studies.  These techniques may not be very practicable in analytical 
laboratories in low resource areas where malaria is endemic.  Efforts in this 
project were devoted to the optimization and development of simple and cost 
effective chromatographic techniques for measurement of CQ and sulphadoxine in 
micro blood samples collected on filter paper.  The newly developed method is 
25 
 
 
sensitive and requires less sample which are easy to transport.  This new method of 
analysis will provide a unique opportunity to facilitate SDX blood level 
determination in patients and can also be employed for therapeutic drug monitoring 
during Intermittent Preventive Treatment for malaria control in pregnant (IPTp) 
women or children (IPTc) using sulphadoxine-pyrimethamine.  
 Besides, the determination of antimalarial drug levels has been estimated  in 
general from whole blood, plasma or red cell (White 1992; Salako and RSowYunmi, 
1992; Gbotosho et al., 2009; Obua et al., 2008). However, antimRalarAials have also 
been estimated in saliva, for example, quinine (Salako aInBd Sowunmi, 1992).  
Although, there is much difficulty in measuring artem isLinin drugs in biological 
samples, it is often easier to measure their paArtneNr drugs, e.g. amodiaquine or 
sulphadoxine plus pyrimethamine (Gitau et al.,D 2004; Gbotosho et al., 2009; Obua et 
al., 2008).  An ideal medium from wIBhich
A antimalarial drugs should be measured 
should be non-invasive with resFpec t to sample collection; saliva is one of such 
medium (Salako and Sowun mOi 1992; Wilson et al., 1993). 
 Despite increaTsinYg drug treatment failure, there are no clear guidelines, at least in Nigeria, SaboIut the time to change antimalarial drug treatment if parasites do 
not clear quiRckly from peripheral blood following treatment of uncomplicated acute 
infecItVions
E in African children.  Effort in this thesis was devoted to understanding the 
cNlinical factors that can lead to antimalarial treatment failure and the pharmacokinetic Ubasis of treatment failure in children with acute uncomplicated P. falciparum malaria 
in an area of intense malaria transmission in Nigeria.   
   
Objectives of the research: 
1. To determine the relationship between delay in parasite clearance and 
26 
 
 
antimalarial treatment failure in children with falciparum malaria. 
2. To determine the pharmacokinetic risk factors and the effects of 
pharmacokinetic variability on treatment outcome in children with acute 
uncomplicated falciparum malaria treated with chloroquine. 
3. To develop and evaluate a simple, cost effective and sensitive method for 
quantification of sulphadoxine in small capillary samples of wholeY blo od spotted on filter paper. R
4. To assess the field applicability of the developed method foRr sulAphadoxine: to 
study the pharmacokinetic disposition of sulphadoxine IaBnd evaluate the effect 
of pharmacokinetic variability on therapeutic effica cLy in children.  
5. To evaluate the use of saliva for therapeAutiNc drug monitoring in patients 
treated with amodiaquine-artesunate comDbination. 
 IB
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CHAPTER TWO 
 
 
LTERATURE REVIEW 
 
2.0   Malaria the disease  
Malaria, the most prevalent and most pernicious disease of human, remaYins up 
till now a major endemic parasitic disease and a leading cause of mAorRbidity and 
mortality especially in sub-Sahara Africa (WHO 2006).  In thBe wRorld as a whole, 
malaria has been a major disease of mankind for thousa nLds Iof years.  The global 
morbidity and mortality due to malaria have not signiNficantly changed over the past 
50 years (Greenwood, 2004).  World HealtDh OArganization (WHO) reported an 
increase in malaria clinical cases from 273 Amillions in 1998 to 515 millions cases in 
2002 (Snow et al., 2005).   IB
 F
 O
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TY
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Figure 2.1: Global distribution of Malaria in 2003 
(http://rbm.who.int/wmr2005/html/map1.htm, Adopted from World Malaria Report 
2005). 
28 
 
 
Malaria is a hematoprotozoan parasitic infection transmitted by species of 
anopheles mosquitoes (White 2004).  It is currently endemic in 90 countries of the 
world of which a substantial population is from Africa, South of Sahara (Figure 2.1) 
(Kondrachie & Trigg, 1997; Olumese, 2005).  Malaria accounts for one in five of all 
childhood deaths in Africa (Kager, 2002; RBM, 2006).  The disease causes over 1 
million deaths, 75% of which occur in African children < 5 years.  In Nigeria, mala ria 
accounts for 63% of the diseases reported in health care centres and preRvaleYnce of 
malaria among pregnant women is 48% (FMOH, 2004). Almost USR$ 3A.5 million was 
reported by the Nigerian government for funding of malaria coInBtrol in 2003, with an 
additional US$ 2.3 million from other sources (RBM, 2005 )L. 
Non-immune travellers visiting malaria enAdemNic areas are at risk of malaria 
infection because of lack of immunity which deDvelops after repeated infection (Hviid, 
2005; Stevenson & Zavala, 2006). PreIgnBant
A women especially primigravidae are also 
at risk of malaria infection (WhittFy e t al., 2005; WHO, 2003; Adam et al., 2005) due 
to reduced immunity in preg nOancy and the presence of the plancenta   which offer a 
highly supportive grTowYth environment for the parasite (Serghides et al., 2001).  Malaria in pregSnanIcy leads to maternal anaemia (Miaffo et al., 2004), low birth 
weight and Rpremature delivery, which are associated with an increased risk of 
neonIaVtal 
Edeath and impaired cognitive development. Economically, the impact of 
mNalaria is alarming and contributes to individual, community and country poverty Uthrough lost labour days and expenses incurred for treatment and prevention. The 
disease impairs physical and mental development in children (Kihara et al., 2006). 
2.1   Life Cycle of Plasmodium 
 There are various species of the genus plasmodium responsible for the disease 
29 
 
 
in man which includes P. falciparum, P. vivax, P. malariae and P. ovale (White, 
2004; Crutcher & Hoffman, 2001).  The most important of these is P. falciparum 
because it can rapidly cause fatal infections and it is responsible for the majority of 
malaria related deaths. The life cycle of the malaria parasite is split between a 
vertebrate host and an insect vector (Figure 2.2). The extreme rarity of P. vivax in 
West Africa is apparently due to prevalence of the duffy negative trait in W est 
Africans.  This is an inherited red cell phenotype that lacks the receptor foRr inYvasion 
of the human red cell by the merozoites of P. vivax. RA
When a mosquito infected with the malaria pIarBasite bites human, 
developmental stages of the parasite called sporozoites is  Linjected into the human’s 
bloodstream (A). The sporozoites then travel toA thNe liver (B). Each sporozoite 
undergoes asexual reproduction, in which Aits Dnucleus splits to form two new cells, 
called merozoites.  Merozoites enter tIheB bloodstream and infect red blood cells (C). 
In red blood cells, merozoites Fgro w and divide to produce more merozoites, 
eventually causing the red  Oblood cells to rupture. Some of the newly released 
merozoites infect oIthTer Yred blood cells (D). Some merozoites develop into sexual stages known asS male and female gametocytes (E). When another mosquito bites the 
infected EhumRan, ingesting the gametocytes (F). The gametocytes mature in the 
mosIquVito’s stomach and undergo sexual reproduction, uniting to form a zygote (G). 
TNhe zygote multiplies to form sporozoites, which travel to the mosquito’s salivary Uglands. If this mosquito bites another human, the cycle begins again (H).   
 
30 
 
 
 
 
 
 
 
 
 
  
AR
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BR
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A
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B
 
 O
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Figure  2.2:  PlasmoIdTium spp.  Life Cycle 
 (http://encartaR.msnS.com/media_461541582/Life_Cycle_of_the_Malaria_Parasite.html) E
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31 
 
 
2.2       Malaria Control Methods 
 
 Global Malaria Control and Disease Management 
Malaria control efforts are currently focused on two major interventions 
which include targeting the mosquitoes that transmit malaria by the use of 
insecticide-treated bed net (ITNs) and early diagnosis and treatment of mala ria 
(EDTM). These are two of the pillars of global malaria control campaiRgn Y(WHO 
2000; Olumese, 2005; Barat, 2006; Killeen, 2007).  PreventionA of malaria 
encompasses a variety of measures that may protect against inBfectRI ion or against the 
development of the disease in infected individuals.  Me aLsures that protect against 
infection are directed towards the mosquito vectAor pNreventing the transmission of 
gametocyte.  These include personal (individuDal or household) protection measures 
e.g. protective clothing, repellents, bBednAets or community/population protection 
measure e.g. use of insecticiFdes  Ior environmental management to control 
transmission. Intermittent  Opreventive treatment (IPT) with sulfadoxine-
pyrimethamine (SP) in pYregnant women is the primary prevention strategy that relies 
on the use of mediIcTS ation (White, 2005; Yartey, 2006; Schellenberg et al., 2006; 
Vallely et al.R, 2007) for control of malaria among pregnant women.  
VSeEveral partnerships have been set-up by international organizations for 
eNffecItive control of infectious diseases especially malaria. These organizations Uinclude Malaria Control and Evaluation Partnership in Africa (MACEPA) and Roll 
Back Malaria (RBM). MACEPA’s mission is to demonstrate that scaling up malaria 
prevention and control under national leadership saves lives, reduces illness, and 
increases economic opportunity (Macepa, 2007). The Roll Back Malaria partnership 
is an initiative to improve malaria control in the context of health sector reform. It 
32 
 
 
was initiated in 1998 through a joint partnership of WHO, UNICEF, UNDP and the 
World Bank.  However this joint partnership has now embarked on a Global Malaria 
Control Programme.  This new programmes focused more on reducing the morbidity 
and mortality associated with malaria. Accordingly, the objectives of the global 
malaria control strategy were prioritized as follows: 
 Y 
 Provision of early diagnosis and prompt treatment for the disease; R
 Planning and implementation of selective and sustaiRnabAle preventive                           
measures including vector control IB
 Early detection of and response to malaria epidemi csL, 
 Improved prevention and treatment of malaAria iNn pregnant women. 
 Strengthening of local research capAacitDies to promote regular assessment of countries’ malaria situations, in parItiBcular the ecological, social and economic 
determinants of the disease. F 
Y 
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33 
 
 
 
2.3: Chemotherapy of malaria 
 Chemotherapy remains one of the important practicable tool to control 
falciparum malaria in sub- Sahara Africa where > 90% of the world’s burden of 
malaria mortality and morbidity occurs (Sibley, 2001).  Chemotherapy in malaria as 
in other infectious diseases is based on preventing the growth or survival of infect ing 
agents, by means of drugs without damage to the host.  Effective treRatmYent of 
falciparum malaria depends on a rapid reduction and clearance of pRarasAitaemia.  The 
malaria parasite is however developing resistance rapidly to moIsBt of these drugs. L
 N 
 2.3.1:    Classification of antimalarial drugs DA
 There are many antimalarial drugBs wAith specific effect on various stages of the malaria parasite lifecycle that have b eIen developed and are currently in clinical use. 
The stages of development ofO maFlaria parasite show varying degree of susceptibility 
to antimalarial drugs.  YThe se antimalarial drugs can be categorized according to 
chemical classes orI sTtage of parasite against which they are most effective (Table 
2.1). S
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Table 2.1: Classification of anti-malaria drugs  
 
Class Blood schizontocide Tissue schizontocide 
 4-Aminoquinolines  Chloroquine  
 Amodiaquine  
 Arylaminoalcohols  Quinine  
  Quinidine   
 Y Mefloquine  R
 Phenanthrene-
 Halofanthrine  RAmethanol 
 8-Aminoquinoline   Primaquine IB
 Artemisinin and L
 Dihydroartemisinin   
derivatives N
  Artemether D A  Artesunate A  
 Antimetabolites  Proguanil  IB  Proguanil     PyrimethamFine  
   SufadoxOine  
   SuYlfale ne    IT Dapsone   Antibiotics S  Tetracycline  Tetracycline   R  Doxycycline  Doxycycline 
  E  Minocycline  Minocycline V
NIWHO recommended combination therapy options for Africa 
1U. Artemether plus lumefantrine  
2. Artesunate plus amodiaquine 
3. Artesunate plus sulphadoxine-pyrimethamine 
4. Dihydroartemisinin plus piperaquine 
 
2.3.2: Pharmacology of antimalarial drugs  
35 
 
 
Quinine 
Quinine is a quinoline antimalarial  (Fig. 2.3). It has been the drug of choice for the 
management of severe malaria in most areas of the world (WHO, 2005). Krishna et al 
(2001) reported that malaria parasites still remain sensitive to quinine in Africa 
despite resistance to commonly used quinolines although, in some part of South East 
Asia decrease sensitivity has been detected (RBM 2001). Recently, it was stYron gly 
suggested that parenteral artesunate should replace quinine as the treatmenRt of choice 
for severe falciparum malaria worldwide. Artesunate was well tRolerAated, with no 
serious drug-related adverse effects with significant reductionIs Bin parasite clearance 
time (Roshental., 2008; Dondorp et al., 2010). Quinoline  aLntimalarials are known to 
have similar mechanism of action in malaria chemAothNerapy (Slater, 1993; Francis et 
al., 1997).  They are known to inhibit digeAstioDn of haemoglobin by the parasite and 
thus reduce the supply of amino acidsI nBecessary for parasite viability.  Salako et al. 
(1989) reported a significantly loFwer  clearance and longer half-life in children with 
kwashiorkor than normal ch ilOdren.  Administration of quinine plus nevirapine results 
in significant decreasTes oYf AUC, Cmax and t1/2 in healthy volunteers (Soyinka et al., 
2009).  AdversSe rIeactions are common with quinine therapy, but severe life-
threateninEg tRoxicity is rare.  A characteristic symptom complex known as cinchonism 
is caIuVsed by quinine (Powell & McNamara, 1966). These consist of tinnitus, high 
tNone deafness (Roche et al., 1990), nausea, uneasiness, malaise, and blurred vision. UHypoglycemia is a more commonly encountered problem with quinine treatment. 
Quinine clearance is increased by  
36 
 
 
RY
 
RA
IB  
Figure 2.3: Chemical structure of Quinine 
N 
L
 
 DA
phenobarbitone, rifampicin (PukrittayakBameAe et al., 2003) and smoking, but reduced 
by cimetidine. The pharmacokiFneti cI parameters of quinine are reported to be 
significantly altered during m Oalaria and this alteration is in proportion to the severity 
of the infection. MalariaY infection causes a reduction in volume of distribution and 
clearance leadingS toI a
T prolongation of elimination half-life as well as elevated plasma 
drug concentRrations (Babalola et al., 1998). 
VE
 
UN
I
Mefloquine 
 Mefloquine is a quinoline-methanol compound, which is structurally similar 
to quinine (Fig. 2.4).  It was developed by the Walter Reed Army Institute of 
Research (WRAIR), to combat emerging strains of drug resistant P. falciparum 
(Bryskier et al., 1988).  Structurally, it consists of 2– and piperidyl and 
37 
 
 
trifluoromethyl group in position 2 of the quinoline nucleus.  Mefloquine is effective 
against all malaria species including multidrug resistant P. falciparum. Initially 
mefloquine against chloroquine resistant and sensitive strains of P. falciparum has 
been well reported (Doberstyn et al., 1997; Sowunmi et al., 1990; 1992). However 
significant resistance has developed in South East Asia (WHO 2000a). Oduola et al. 
(1987) reported a reduction in the in-vitro sensitivity of isolates of P. falcipar um 
from West Africa to mefloquine even before the drug was introduced into Rthe rYegion, 
suggesting that parasite with innate resistance to mefloquine may Rbe Apresent in the 
West Africa sub region.  The structure-activity relation is bIaBsed on N-O of 2-  
piperidyl group (Tracy et al., 1996).   L
 The actual mechanism of action of mefloquAineN is still unknown however, it is 
thought to inhibit heme polymerase. MefloquDine forms toxic complexes with free 
heme causing damage to parasite mIeBmbr
Aane and interacts with other plasmodia 
components (Mockenhaupt, 1995F).   Mefloquine is metabolized by CYP3A4. An 
increase in plasma conc enOtration was reported when mefloquine was co-
adminisatered with TketYoconazole, a CYP3A4 inhibitor (Ridtitid et al., 2005). 
Rifampicin whiSch Iinduces CYP3A4 enzyme reduced plasma concentration of 
mefloquine wRhen coadministered (Ridtitid et al., 2000).   Mefloquine is generally 
wellI Vtole
Erated, although nausea, abdominal discomfort, dizziness, vomiting and 
dNiarrhea are the frequent adverse reactions associated with it (Phillips-Howard et al., U1995; Ter Kuile et al., 1995; Sowunmi et al, 1990). There is no established 
biochemical basis behind neurotoxicity of mefloquine but it is found to disrupt 
neuronal calcium homeostasis and induce an endoplasmic reticulum stress response 
at physiologically relevant concentrations effect that may contribute at least in part of 
the neurotoxicity (Geoffrey et al., 2003). It also results in severe neuropsychiatry 
38 
 
 
reactions such as disorientation, seizures, encephalopathy, hallucinations and sleep 
disturbances (nightmares). Recently, mefloquine has been reported to induce 
pneumonitis (Soentjens et al., 2006). 
 
 
AR
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Figure 2.4:  Che TSmicIal structure of Mefloquine
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Halofantrine 
 Halofantrine is a 9-phenanthrene-methanol antimalarial agent (Fig. 2.5). It is a 
schizonticidal drug that probably acts by formation of a complex with 
Ferriprotoporphyrin IX toxic to the parasite. Inhibition of proton pump at the host-
parasite interface has also been hypothesised as an alternate mode of action (Watkins 
et al., 1988; Barriso & Goa 1992).  It is active against all human malarial parasi tes.  
Minor and reversible event including nausea, diarrhea, vomiting and RabdYominal 
discomfort occur but are usually self-limiting.  Pruritus has been reRporAted in 13% of 
Nigerian taking the drug (Sowunmi et al., 1989).  It has no adIvBerse effect on central 
nervous system and is better tolerated than mefloquine  L(TerKuile et al., 1993).  
Halofantrine prolongs Q-Tc interval (Dion et alA., 2N001; Darrel et al., 2001) and 
should not be administered to patients taking drDugs known to prolong the QT interval 
(i.e. Quinine, tricyclic antidepressant, BQuAinidine) or to those who have received 
mefloquine within few days (WhitFe, 1 9I96; Touze et al., 1997).  Oral bioavailability is 
increased up to six-fold if h alOofantrine is taken with fatty meal (Shanks et al., 1992; 
Tracy et al., 1996).   TY
SI  
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Y 
AR
BR
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DA
N
A
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Figure 2.5: Chemical stYruc ture of HalofantrineIT
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Artemisinin and its derivatives 
 The family of trioxane compound derived from the plant artemisia annua 
(qinghaosu) and a series of synthetic derivatives are among the most potent 
antimalarial compounds known (Fig. 2.6).  The trioxane structure contains a peroxide 
group essential for its antimalarial activity (Klayman, 1985; Woerdenbarg et al., 
1994). These naturally occurring sesquiterpene lactones are structurally unrelated  to 
the quinoline antimalarial drugs. Artemisinin derivatives include: ARrtemYisinin, 
deoxyartemisinin, dihydroartemisinin (DHA), artesunate, aRrtemAisinic acid, 
artemisitene, arteether and artemether respectively. Three cIoBmpounds have been 
extensively evaluated: the parent compound artemisinin, t heL water soluble artesunate 
and the oil soluble ether artemether.   AN 4
 Artemisinins act very rapidly reducingD parasitemia by a factor of 10  with 
each cycle (Baird et al., 2005; Baird, 20B05A; Wilairatana et al., 2002). Unfortunately, 
there are reports of reduced susceFpti biIlity of the artemisininsin the Thai/Cambodian 
border (Jambou et al., 2005 ; ODandorp et al., 2009).  Artemisinin can be given orally 
and intramuscularly.  TArtYemisinin, but not other derivatives (Karunajeewa et al., 2004), shows consideraSble Iautoinduction of its own metabolism so that blood concentrations 
after several Rdays of dosing are considerably lower than would be predicted from the 
initiIalV dos
Ee (Ashton et al., 1998).  Oral artesunate and artemether, but not artemisinin, 
aNre hydrolyzed rapidly back to the common metabolite DHA, which is intrinsically Umore active as an antimalarial agent (Li et al., 2007).   Oral artesunate may be 
considered mainly as a prodrug for DHA, as the metabolite is the main contributor to 
overall antimalarial activity (Newton et al., 2000).  
42 
 
 
Y 
AR
LIB
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Artemisinin 
DA
N
 IB
A
OF 
ITY
RS
IV
E
 
 
UANrtemether 
43 
 
 
Y R
BR
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Artesunate DA
N
BA
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Figure 2.6: Chemical struYctu re of Artemisinin derivativesIT
ER
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44 
 
 
2.4: Review of studies on Pharmacokinetic of chloroquine, sulphadoxine-
pyrimethamine and amodiaquine. 
 
Chloroquine: 
Chloroquine, is 7-chloro-4-(4-diethyl amino-1-methylbutyl-amino) quinoline, 
and a tertiary amine (Figure 2.7).  It is the most important of the 4-aminoquinol ine 
compounds for the treatment of malaria.  The 4-aminoquinoline nucleus Yhas a 
th
chlorine atom attached to 7th carbon atom of the nucleus.  The chloriAne aRtom at 7  
position of the quinoline nucleus greatly confers the antimalariaRl activity on the 
molecules (Goodman & Gillman 1996). B
 N 
LI
 
AD
A
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B
Y 
O
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NI Figure 2.7: Chemical structure of Chloroquine U
45 
 
 
 
(i) Mode of action: Chloroquine is a weak base but its accumulation in the parasite 
lysosome is 1000-fold greater than predicted on the basis of a weak base effect  
(Rang et al., 1997). Chloroquine acts on the intraerythrocytic P. falciparum stages 
that are responsible for the clinical manifestation of the disease.  These stages feed on 
the erythrocyte haemoglobin, in acidic vacuoles (lysosomes).  Chloroquine inhib its 
digestion of haemoglobin by the parasite and thus reduces the supply of amRinoY acids 
necessary for parasite viability (Figure 2.4); it is also said to causeR fraAgmentation of 
the parasite RNA and intercalate DNA.  The toxic haem is polyImBerized into insoluble 
non-toxic haemozoin (Sarchez et al., 1997).  Plasmodial  heLme polymerase catalyses 
this polymerization reaction and it is inhibited byA chlNoroquine and other quinolines 
(Slater, 1993; Francis et al., 1997).  A highDer parasite burden is associated with 
increased risk of failure of treatment wiBth CAI Q and mefloquine (Sowunmi et al., 2004 & 2005).   F 
  O
Pharmacokinetics ofT chYloroquine (i) Absorption: S ChIloroquine is rapidly absorbed and widely distributed after oral 
administratioRn in healthy adults (Gustafsson et al., 1983) and children with 
uncoImVpli
Ecated malaria (Adelusi et al., 1982).  In adults with moderately severe 
mNalaria, bioavailability relative to parenteral treatment was 70% compared with 75% Uin healthy subjects (Gustafson, 1983).   In children with uncomplicated malaria given 
an initial oral treatment dose of 10mg base/kg, peak plasma concentration of 
approximately 250µg/L was reached in 2h (Adelusi et al., 1982).  Absorption is very  
 
 
46 
 
 
 
 
Y 
RA
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 OF
Figure 2.8                       Y   M ode of action of Chloroquine 
 IT
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rapid and relatively complete even in very severe infections (White et al., 1987b, 
1988).  The absorption and elimination of chloroquine in children appears to be 
similar to that in adults (WHO 2000). 
(ii) Distribution:  Chloroquine is widely distributed throughout the body after 
absorption. It is extensively bound to tissues particularly liver, spleen, kidney, lun gs, 
connective tissues and tissues containing melanin such as retina resultYing in 
enormous apparent volume of distribution (Walker et al., 1A983R).  The 
pharmacokinetic properties of chloroquine are complex.  The totalR apparent volume 
of distribution is enormous (100-1000 L/kg) because of LexIteBnsive tissue binding 
(Frisk-Holmberg et al., 1984) whereas the volume of Ncen tral compartment is several 
orders of magnitude smaller (0.18 ±0.13L/kg) (LAooareesuwan et al., 1986).  The 
process of distribution from this centrAal Dcompartment determines the blood 
concentration profile during the treat mIeBnt of malaria.  Chloroquine concentration in 
red blood cells is approximatelyF 3-5 times higher than in plasma, and there is 
considerable concentratioYn i n
O granulocytes and platelets (Nosal et al., 1988). Whole 
blood concentrationI iTs 6-10 times higher than plasma concentration (Gustafsson et 
al., 1983) and fuSrther in parasitized red blood cells (Adelusi et al., 1982; Ajayi et al., 
1988).  CEhloRroquine concentration in cerebrospinal fluid are very low, with a mean 
valuIe Vof 2.7% of corresponding whole blood concentration (White, 1988b) and 
UsNlightly concentrated in breast milk with area under the concentration time curve 
milk/plasma ratio of 2.0 - 4.3 (Edstein et al., 1986).  Chloroquine in saliva has a long 
elimination half-life (7-20d) and there is a good correlation between the AUC values 
derived from saliva and plasma (Onyeji and Ogunbona 1996; Ogunbona et al., 1986). 
48 
 
 
(iii) Elimination: Chloroquine is eliminated slowly such that the drug and it 
metabolites can be detected in plasma for 21 - 60 days after single dose of 5mg/kg 
depending on the sensitivity of the assay method.  It is 51% cleared unchanged by the 
kidney (Gustafsson et al., 1983; Frisk-Holmberg et al., 1984). The remainder is 
slowly biotransformed by side chain de-ethylation in the liver leading to the 
formation of the major metabolite, desethyl-chloroquine which is the primary am ine 
that can undergo deamination to form an alcohol - the 4-hydroxyl compoRundY which 
then undergoes oxidation to form the carboxylic acid derivativeRs.  AThe principal 
metabolite, desthyl-chloroquine also exhibits potent antimaIlaBrial activity against 
sensitive isolates of P. falciparum (Oduola et al., 1989 ),L but less active than the 
parent drug and is also eliminated more slowlyA (GNustafsson et al., 1987). The 
terminal elimination half-life is approximatelyD 1-2months but, in terms of curative 
treatment (blood concentration), the IreBal 
Ahalf-life (t1/2) is about 6-7 days (Frisk-
Homberg et al., 1984).  VarFiou s half-lives have been reported after oral 
administration of chlroquine . O This is because the determination of the half-life of a 
drug depends on the TideYntification of the true terminal log linear elimination phase, which is difficulSt toI obtain with chloroquine in view of its continuous redistribution 
from tissuesR to plasma over weeks.  It also depends on the sampling time and 
sensIitVivit
Ey of the assay method.  Drugs that inhibit the actions of liver microsomal 
eNnzymes prolong the half-life of chloroquine (Bowman & Rand, 1980).  U
 (v) Toxicity:  Serious adverse reaction associated with use of chloroquine is rare at 
therapeutic dosage. The common side effect among Nigerians is pruritus (Sowunmi 
et al, 1989).  Some patients may vomit and may complain of blurred vision (Ferreras 
et al., 2007).  It causes disruption of lysosome in living cells (Michihara et al., 2005).  
Cardiovascular abnormalities such as hypotension or cardiac arrhythmia progressing 
49 
 
 
to cardiac arrest and death are often observed after parenteral administration or 
overdose with the drug (Olatunde, 1970; Williams, 1966; Tuboku–Metzger, 1964; 
White, 2007).  For instance, there are reports of sudden death following 
administration of intramuscular chloroquine to children with severe malaria 
(Olatunde, 1970).  Since chloroquine is rapidly absorbed in the gastrointestinal tract, 
the use of parenteral route of administration is not encouraged.  Prolonged treatm ent 
with chloroquine may cause a lichenoid skin eruption in some patieRnts.Y  The 
condition is mild and subsides when the drug is discontinued.  ChloRroqAuine overdose 
(usually self poisoning) is manifested by coma, convulsiIonB, dysrhythmias and 
hypotension. Diazepam is a specific antidote (Riou et  aLl., 1988). Oral activated 
charcoal is administered when an overdose is used.A N
 AD
Sulphadoxine B
F I
Sulfadoxine (SDX) i s Oa weak acid (Fig. 2.9) and 88 - 90% bound to plasma 
protein, mainly albumin Y(Abdi et al., 1995; Mayxay et al., 2001). Body weight and 
age significantlyS inIfl
Tuence the pharmacokinetics of sulphadoxine (Trengue et al., 
2004).  SulfRonamides are structural analogues and competitive antagonists of p-
aminoVbenEzoic acid (Rang et al 1995).  Sulphadoxine inhibit dihydropteroate synthase 
(NDHIPS), a key enzyme in the folate biosynthesis (Rang et al 1995).  Sulphadoxine is Ua partner drug with pyrimethamine for treating malaria. They act through a two steps 
synergistic blockage of plasmodial metabolism. The success of SP depends on this 
synergy: when either component is compromised, the effectiveness is dramatically 
reduced. 
 
50 
 
 
(i) Mode of action: This is the most used of a family of drug combinations which 
antagonize parasite folic acid synthesis. Sulphadoxine act by inhibition of 
dihydropteroate synthase (sulphonamide and sulphones) (Fig 2.10) This leads to a 
decrease level of fully reduced tetrahydrofolate, a necessary co-factor important in 
one-carbon transfer reactions in the purine, pyrimidine and amino acid biosynthetic 
pathway (Ferone, 1977). The lower level of tetrahydrofolate result in decrea sed 
conversion of glycine to serine, reduced methoinine synthesis, and lower thRymYidylate 
levels with a subsequent arrest of DNA replication (Gregson & PlowRe, 2A005). B
 LI
DA
N
A
 IB
 O
F  
 
 TY
Figure 2.9: CheSmicIal structures of Sulphadoxine. 
 
ER
IV
UN
51 
 
 
 
 
Sulphadoxine Pyrimethamine 
 
RY 
Figure 2.10:     Mode of action of sulphadoxine/pyrimethamine A
 BR
Pharmacokinetics of sulphadoxine   LI
(i) Absorption:  Sulphadoxine is well absorbed AoralNly.  In healthy subjects, peak 
plasma concentration is reached in 2-8h (AhAmaDd & Rogers, 1980; Weidekamm et al., 
1982; Dzinjalamala et al., 2005a). AbsIoBrption in uncomplicated malaria is similar to 
that in healthy subjects (WinstanleFy e t al., 1992). O
(ii) Disposition: The apYpare nt volume of distribution of sulfadoxine (0.1-0.2L/kg) 
(WHO, 2000). It isI lTipophilic.  Plasma protein binding of sulphadoxine in healthy 
subjects is RhighS (88%) (Abdi et al.,1995).  Red blood cells concentration of 
sulphadoxEine is less than half of that in plasma (WHO, 2000).  Pregnancy has been 
showInV to have significant effect on disposition of sulphadoxine; it reduces SDX half-
UlNife, lowers the AUC and clearance is significantly greater than in non-pregnant 
women (Green et al., 2007). 
(iii) Elimination:  Sulphadoxine is a slowly eliminated sulfonamide. Unlike other 
sulphonamides, only 5% of sulphadoxine is n-acetylated and eliminated in the urine 
in this form (WHO, 1984). Biotransformation and clearance is approximately 
52 
 
 
0.5mL/kg/h. Elimination half-life of sulphadoxine ranges from 4.8 to 10.6 day 
(Winstanley et al., 1992; Dzinjalamala et al., 2005).  
 (iv) Toxicity:  Sulphadoxine is very well tolerated and severe adverse effects are 
uncommon (WHO, 1985). It has been reported to cause kernicterus in neonates but 
sulphonamide treatment in a lactating woman does not pose a threat to her breast  fed 
neonates unless there is jaundice, prematurity or G-6PD deficiency (WHO, Y2000). 
Life threatening erythema multiforme (Steven Johnson syndromAe) Rand toxic 
epidermal necrolysis has been reported in individuals taking the druRg for prophylaxis 
(Miller et al., 1986). 
 LI
B
 AN
Pyrimethamine AD
Pyrimethamine (PYR) is a  IbBase (Bergqvist et al., 1985)(Fiig. 2.11). 
Pyrimethamine is a competitiOve iFnhibitor of dihydrofolate synthesis (DHFR) (Peter, 1997).  Pyrimethamine aYcts  through a two steps synergistic blockage of plasmodial 
division. The succIesTs of pyrimethamine in treatment of malarial depends on 
sulphadoxine-pySrimethamine synergy: when either component is compromised, the 
effectivenEessR is dramatically reduced. 
 IV
U(Ni) Mode of action: This is the most used of a family of drug combinations which 
antagonize parasite folic acid synthesis. They act by sequential inhibition 
dihydrofolate reductase (pyrimethamine and biguanides) enzymes in the folate 
pathway (Fig 2.6) (Bzik et al., 1987; Triglia and Cowman, 1994; Triglia et al., 1999; 
Cowman, 1997).  This leads to a decrease level of fully reduced tetrahydrofolate, a 
necessary co-factor important in one-carbon transfer reactions in the purine, 
53 
 
 
pyrimidine and amino acid biosynthetic pathway (Ferone, 1977). The lower level of 
tetrahydrofolate result in decreased conversion of glycine to serine, reduced 
methoinine synthesis, and lower thymidylate levels with a subsequent arrest of DNA 
replication (Gregson & Plowe, 2005). 
 
 
RY
 
BR
A
 LIN
DA
 IB
A
F  
Figure 2.11: Chemical stru ctOures of Pyrimethamine 
 Y
PharmacokinetiScs oIf
T Pyrimethamine 
(i) AbsorEptioRn:  Pyrimethamine is well absorbed orally.  In healthy subjects, peak 
plasImVa concentration is reached in 2-8h (Ahmad & Rogers, 1980; Weidekamm et al., 
U1N982; Dzinjalamala et al., 2005a). Absorption in uncomplicated malaria is similar to that in healthy subjects (Winstanley et al., 1992). 
(ii) Disposition: The apparent volume of distribution of pyrimethamine is 2-3 L/kg 
(Weidekamm et al., 1987). This is considerably larger than volume of the sulfadoxine 
(0.1-0.2L/kg) (WHO, 2000). Both drugs are lipophilics.  Plasma protein binding of 
pyrimethamine in healthy subjects is high (93%) (Abdi et al.,1995).   
54 
 
 
(iii) Elimination: Biotransformation and clearance is approximately 0.5mL/kg/h. 
Elimination half-life of pyrimethamine  is 3.3 to 4.8 day (Winstanley et al., 1992; 
Dzinjalamala et al., 2005).  Pyrimethamine is transformed to several unidentified 
metabolites and is cleared predominantly by hepatic biotransformation.  
(iv) Toxicity:  Pyrimethamine is very well tolerated and adverse effects are r are 
(WHO, 1985). Prolong use of pyrimethamine may provoke folate deficieYncy in 
vulnerable subjects (pregnant or malnourished patients. No record of caArdioRtoxicity of 
pyrimethamine has been recorded either in animal or human exRperiment (White, 
2007). LIB 
 N
Amodiaquine DA
 Amodiaquine is a 4-aminiquIiBnoli
Ane similar to chloroquine (Fig 2.11).  
Amodiaquine was synthesized durFing  the 2nd world war and has a similar action with 
chlroquine since both are str ucOtural analog (Brykier et al., 1988).  It is made up of the 
same quinoline nucleTus Ywith lateral para-hydroxylphenyl group linked to a methyl-
aminodiethyl chSain.I  Amodiaquine is as effective as chloroquine and also used 
against chlorRoquine-resistant strains of P. falciparum (Van Dillen et al., 1999; Olliaro 
et alI.,V 199
E6).    
 
UNMode of action of amodiaquine 
Amodiaquine is a schizonticidal antimalarial drug. The mechanism of action of 
amodiaquine is not completely certain. Like other quinoline derivatives, it is thought 
to inhibit heme polymerase activity. This results in accumulation of free heme, which 
is toxic to the parasites and leads to the parasite death.  
55 
 
 
 
Amodiaquine Y 
 R
BR
A
LI
N 
AD
A
B  
N-desethyl amodiaquine  I
  O
F
Figure 2.12:  Chemical Ystructure of amodiaquine and N-desethyl amodiaquine 
PharmacokinetiScs oIf
T amodiaquine 
(i) AbsorEptioRn:  Amodiaquine is rapidly absorbed, widely distributed and extensively 
metaIbVolized to a pharmacologically active metabolite, desethyl amodiaquine, 
UfNollowing oral administration and it seems that this metabolite is responsible for the antimalarial activity of the compound (Winstanley et al., 1985). The main metabolite 
of AQ is N-desethylamodiaquine (DEAQ) with other minor metabolites being 2-
hydroxyl-DEAQ and N-bisdesethylAQ (bis-DEAQ) (Churchill et al., 1985, 1986; 
Mount et al., 1986). The primary route of AQ metabolism to DEAQ is via 
polymorphic CYP2C8 enzyme (Li et al., 2002; Adejei et al., 2008). The absorption 
56 
 
 
and elimination of amodiaquine in children appears to be similar to that in adults 
(WHO 2000). 
 (ii)  Distribution:  Amodiaquine is concentrated in the red blood cells, the whole 
blood to plasma concentration ratio being 3.1. The AUC0-24h for DEAQ in whole 
blood is significantly higher than that in plasma (Winstanley et al., 1987). The to tal 
apparent volume of distribution of DEAQ is small compared to chloroquine (Y87.9 – 
243.1L/kg) (Stepniewska et al., 2009; Orrell et al., 2008). Both amoAdiaRquine and 
DEAQ were found to be highly bound to plasma protein with a meRan bound fraction 
of 92 and 85% respectively (Li et al., 2003).   LIB
(iii) Elimination:  The formation of DEAQ is rapid, itNs el imination is very slow with 
a terminal half-life of over 100 h (Winstanley et alA., 1987; Laurent et al., 1993; Adjei 
et al., 2008; Orrell et al., 2008). Excretion oAf DDEAQ is slow and is detectable in urine 
5 months after dosing (Wintsanley et  aIl.B, 1987).   
(iv) Toxicity: Amodiaquine at usuFal doses has similar adverse effect to chloroquine 
but high prevalence of aYgran u
Olocytosis and hepatitis (Hatton et al., 1986) after long-
term use.  Hepatitis IhTas been observed to occur from as early as 3 weeks (exposure to 
3 weekly dosesS) to as late as 10 months of prophylaxis. Patients with severe 
amodiaquEineR induced hepatitis may remain jaundiced for 3-6 months. Severe 
neutIroVpenia may occur if amodiaquine is used in anti-inflammatory doses for 
UrNheumatoid arthritis.  It seems to be an unstable molecule and undergoes auto-
oxidation in aqueous solution to yield a quinoneimine, which may be implicated in 
the drug’s toxicity (Maggs et al., 1987).  However, the adverse reactions appear to be 
idiosyncratic and have not been described when amodiaquine is used in malaria 
therapy (Olliaro et al., 1996).   
 
57 
 
 
2.5: Antimalarial Drug Resistance 
 The emergence and spread of drug resistant malaria globally has become one 
of the most important problems in malaria control in recent years (Bradley, 1996; 
Olliaro et al., 2004). The parasites that caused the disease have developed resistance 
to all antimalarial including artemisinin derivatives (WHO 2006; Jambou et al., 2005; 
Dondorp et al., 2009).  Chloroquine resistance is now common in every region wh ere 
P. falciparum occurs (Edward & Biagini, 2006).  Chloroquine was formerlRy reYplaced 
by sulphadoxine-pyrimethamine until when this combination succRumAbed in South-
east Asia, South America and most recent, Africa (WHO 2I0B06).  The impact of 
resistant malaria is considerable and compounds the seri oLusness of malaria related 
morbidity and mortality (Price et al, 2001).  It is tAhe Nmost critical factor in reducing 
the useful life span of a drug and undermining the drug policy.  
AD
 Antimalarial drug resistance isI Bdefined as the ability of a parasite strain to 
survive and/ or multiply despite thFe a dministration and absorption of a drug given in 
doses equal to or higher than  Othose usually recommended but within the tolerance of 
the subject; and the IdTrugY in question must gain access to the parasite or the infected red blood cell foSr the duration of time necessary for its normal action (WHO, 2001). 
The mainE mRechanism underlying the development of resistance includes, naturally 
occuIrVring genetic mutations in the malaria parasite. These mutations result in a 
dNecline in drug sensitivity depending on the class of antimalarial drug (Olliaro, 2004). UInadequate treatment (e.g. sub-therapeutics dose, sub-optimal drug) of a high biomass 
infection will not kill the mutant parasites and is the main selective pressure for 
resistance. Resistant parasites are then transmitted to other individuals by mosquitoes. 
In addition, drugs with long half-lives are more likely to select for resistance because 
low drug concentrations linger on and are only able to kill sensitive parasites (Nzila 
58 
 
 
et al., 2000).  
 The need to extend the clinical use of chloroquine has become important for 
several reasons including the socioeconomic situation in endemic areas of Africa and 
South America.  The availability, tolerability and it low cost which had been the main 
cause to retain it in malaria control efforts.  In Nigeria, chloroquine and 
sulphadoxine-pyrimethamine were antimalarial drugs of choice prior to chaYnge  of 
treatment policy to artemisinin based combination therapy (ACTs) in 20R04 (FMH, 
2004).  In few malaria endemic areas such as Malawi, the efficacyR ofA CQ has been 
renewed through withdrawal of CQ from circulation to reduceI dBrug pressure (Laufer 
et al., 2006).  Chloroquine has also been combined wit hL artesunate yielding high 
efficacy (Fehintola and Balogun 2010).   SulphadAoxiNne-pyrimethamine is currently 
employed as Intermittent Preventive TreaAtmeDnt in children (IPTc) and pregnant 
women (IPTp).  It is essential toI Bstudy the effect of drug resistance on 
pharmacokinetics of CQ or SP duFrin g acute infections in children.  With respect to 
drug level estimation, the  Odetermination of antimalarial drug levels has been 
estimated in generalI fTromY whole blood, plasma or red cell (White., 1992; Gbotosho et al., 2009; Obua eSt al., 2008).  
 HEoweRver, antimalarials have also been estimated in saliva, for example, 
quinIinVe (Salako and Sowunmi 1992; Wilson et al., 1993, Babalola et al 1996).  
UANlthough, there is much difficulty in measuring artemisinin drugs in biological 
samples, it is often easier to measure their partner drugs, e.g. amodiaquine or 
sulphadoxine plus pyrimethamine (Gitau et al., 2004; Gbotosho et al., 2009; Obua et 
al., 2008).  An ideal medium from which antimalarial drugs should be measured 
should be non-invasive with respect to sample collection; saliva is one such medium 
(Salako and Sowunmi 1992; Wilson et al., 1993).  This will provide useful 
59 
 
 
information that may guide drug combination strategies and drug therapeutic 
monitoring in order to revisit existing but abandoned drugs as a result of drug 
resistance to all antimalarial drugs (Dondorp et al., 2009).  
 
RY
RA
 LI
B
DA
N
BA
F I
Y 
O
IT
RS
IV
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60 
 
 
 
 
CHAPTER THREE 
 
MATERIALS AND METHODS 
  
Study 1: Studies on risk factors contributing to delay in parasite clearaRnceY in 
children with acute uncomplicated falciparum malaria. A
Drug resistance in Plasmodium falciparum is commoBn iRn many endemic 
countries but there is no clear recommendation on whe n Lto Ichange therapy when 
there is delay in parasite clearance after initiation of thNerapy in African children.  This 
study reports the relationship between delay iDn paArasite clearance and anti-malarial 
treatment failure in children with fBalciAparum malaria in an area of intense transmission in south-western Nigeria ,I where resistance in Plasmodium falciparum to 
CQ and SP has increase steadiOly oFver the past ten years. 
 Y 
3.0:  Patients andI dTrug treatment.  
The sRtudSies were conducted between April 2002 and July 2010 in patients 
presenVtinEg at the Malaria Research Laboratories Clinic, University College Hospital, INbadIan.  Ibadan is an hyper-endemic area for malaria in southwestern Nigeria (Salako Uet al., 1990).  Ethical clearance was provided by University of Ibadan/University 
College Hospital ethics committee and Oyo State Ministry of Health, Secretariat 
Ibadan.  During the period, a series of antimalarial drug efficacy studies were 
conducted to evaluate the efficacy and safety of different treatment regimens (Table 
3.1).  The study was conducted with the assistance of a physician (Professor A. 
Sowunmi). 
61 
 
 
Briefly, children with symptoms compatible with acute uncomplicated 
falciparum malaria who fulfilled the following criteria were enlisted in the study: age 
≤12 years, pure P. falciparum parasitaemia greater than 2000 asexual forms/µl blood, 
negative urine tests for antimalarial drugs (Dill-Glazko and lignin tests), absence of 
concomitant illness, no evidence of severe malaria (WHO, 2000) and written 
informed consent given by parents or guardians.  Clinical and parasitologi cal 
evaluation was done at enrollment (day 0) and on days 1-7, 14, 21 and Rday Y28. In 
patients who received ACTs, follow-up was for 42 d.  Clinical evaluRatioAn consisted of 
a general clinical examination including measurement of weIigBht, core temperature 
and physical examination.  L
 AN
3.1: Sample collection. D
Prior to treatment, 100µl of caIpBilla
Ary blood sample was obtained from each 
patient and spotted on filter paperF (1 10mm Whatman Filter paper) for determination 
of chloroquine or sulphadox inOe levels.  During follow-up, on day 1, 2, 3, 4, 5, 6, 7, 
14, 21 and 28 thick bTlooYd films were prepared and 100µl of capillary blood samples were obtained anSd sIpotted on filter paper.  The filter paper samples were stored in 
desiccated rReseal able plastic bags at room temperature for measurement of 
chlo EIroVquine and sulphadoxine concentrations.  
UN
62 
 
 
Table 3.1:  Antimalarial drugs and their treatment regimens with time of study 
administered to children with acute uncomplicated falciparum malaria. 
Treatment Drugs*                                               Regimens† No of Years 
Group patients of study 
Monotherapy AQ 30 mg/kg of amodiaquine base over 3 days, that is, 10 mg/kg 573 2005/6 
  daily   
 AS Artesunate given as 28 mg/kg over 7 days, that is, 4 mg/kg 120 2006 
  daily   
 CQ 30 mg/kg of chloroquine base over 3 days, that is, 10 mg/kg 388 2002-4 
  daily   
 MQ Mefloquine given as 25mg/kg at presentation 176  2007/8 
    Y  
Artemisinin AQAS Artesunate given as 4 mg/kg daily for 3 d  plus amodiaquine 142 2004/5/
Combination  given as in AQ above R 10 
Therapy AMQ Mefloquine given as 25mg/kg at presentation plus artesunAate 174 2006 
  as given in AQAS above R   
 AL Artemether (20mg) plus lumefantrine (120mg) given thus: 5- 90 2007/8 
  14kg received 1 tab., 15-24kg received 2 taIbB., 25-34kg   
  received 3 tab., > 34kg received4 tab. at pres enLtation, 8 h later     and at 24, 36, 48 and 60 h after first dose   
    
Non- AQSP Amodiaquine given as in AQ abAoveN plus sulphadoxine- 69 2002-4 Artemisinin  pyrimethamine given as 25 mg/kg of the sulphadoxine   
Combination  component at presentation   
Therapy AQSFP Amodiaquine given as inA ADQ above plus sulfalene- 91 2006  pyrimethamine given as 25 mg/kg of the sulfalene component   
 at presentation B   
COT Co-trimoxazole giFven  Ias 20 mg of the sulphamethoxazole 104 2003  component twice daily   
CQCP 30 mg/kg of  cOhloroquine base over 3 days, that is, 10 mg/kg 315 2003  daily plus chlorpheniramine 8mg start and 4 mg 8 hourly for 5    d.   
CQKET 30 mg/kYg of CQ base over 3 days, i.e., 10 mg/kg daily plus 70 2004 
 ketoItTifen 25mg/kg start followed by 0.125 mg/kg 8 hourly for    S4 d.   CQSPR 30 mg/kg of chloroquine base over 3 days, that is, 10 mg/kg 107 2007/8  daily plus sulphadoxine-pyrimethamine given as 25 mg/kg of E    the sulphadoxine component at presentation   VSP  Sulphadoxine-pyrimethamine given as 25 mg/kg of the I 291 2003  sulphadoxine component at presentation    N  Sulphadoxine-pyrimethamine given as in SP above plus   U SPPB probenecid at 20-25mg/kg in two divided doses daily for 3 day 42 2003 † All drugs were administered orally.  AQ, amodiaquine; AQAS, amodiaquine plus artesunate; AQSP, amodiaquine plus sulphadoxine-pyrimethamine; AQSFP, amodiaquine-sulfalene-pyrimethamine; AMQ, mefloquine plus artesunate; AL, 
artemether plus lumefantrine; AS, artesunate; COT, co-trimoxazole; CQ, chloroquine; CQCP, chloroquine plus 
chlorpheniramine; CQKET, chloroquine plus ketotifen; CQSP, chloroquine plus sulphadoxine- pyrimethamine; MQ, 
mefloquine; SP, Sulphadoxine-pyrimethamine; SPPB, sulphadoxine-pyrimethamine plus probenecid; 
63 
 
 
 3.2: Assessment of parasitaemia. 
Thick and thin blood films prepared from a finger prick were Giemsa-stained 
and were examined by light microscopy under an oil-immersion objective, at X100 
magnification and re-examined by an independent microscopist.  Parasitaemia in 
thick films was estimated by counting asexual parasites relative to 1000 leukocytes, 
or 500 asexual forms, whichever occurred first.  From this figure, the parasite dens ity 
was calculated assuming a leukocyte count of 6000/L of blood.  GametocyteYs were 
also counted in thick blood films against 1000 leukocytes assuminAg aRn average 
leukocyte count of 6000/L of blood (Shaper & Lewis, 1971; EzBeiloR, 1971; Sowunmi 
et al., 1995).    LI
 
AN
3.3: Evaluation of response to drug treatment. D
Response to drug treatment wIasB as
Asessed using World Health Organization 
(WHO) criteria (WHO, 1973 or 20F03 ) as shown in Table 3. 2.  In those with sensitive 
or RI response, parasite clea raOnce time (PCT) was defined as the time elapsing from 
drug administration uTntilY there was no patent parasitaemia for at least 72 h.  Delay in parasite clearancSe wIas defined as a parasite clearance time > 2 d, and was based on 
the asexual liRfe cycle of 48 h in the infected erythrocyte (White, 1997). E
 V
UN
I
64 
 
 
Table 3.2: Evaluation of clinical response of children to treatment with 
antimalarial drugs 
Treatment Response Description 
S (sensitive) clearance of parasitaemia without recurrence 
RI (mild resistance)  parasitaemia disappears but reappears within 7 to 14 
days; 
RII (moderate resistance)  decrease of parasitaemia but no complete clearance  
from peripheral blood; RY
RIII (severe resistance)  no pronounced decrease or increase inR parAasitaemia at 48 hours after treatment B
                                                                       LI
OR N 
 ACPR (adequate clinical and if there was no reappAearance of parasites or fever by 
parasitological response) day 14 D
ETF ( Early treatment failure) if dangerI sBign
As or severe malaria developed on days 
1, 2F and  3 in the presence of parasitaemia, or Oparasitaemia on day 2 higher than day 0 irrespective 
Y of temperature, or parasitaemia on day 3 with fever T o≥37.5 C, or day 3 parasitaemia ≥25% of the count on 
SI day 0 
LCF (Late cliniRcal failure)  if danger signs or severe malaria developed after day E 3 in the presence of parasitaemia or presence of 
IV oparasitaemia and fever (≥37.5 C) on any day between N day 4 and day 14; ULPF (Late parasitological if there was parasitaemia on day 14 and axillary 
o
failure)  temperature less than 37.5 C without meeting any of 
the previous conditions 
65 
 
 
Study 2: Pharmacokinetic determinants of response to treatment with 
chloroquine in children with acute uncomplicated Plasmodium falciparum 
malaria. 
Resistance to antimalarial has become an important issue in infectious 
diseases research. The usefulness of the common antimalarials, such as chloroquine 
and sulphadxoine-pyrimethamine is fading out as a result of resistance to the drugs. 
Apart from the issue that the parasites are resistant to these drugs, the way indivYidu als 
respond to drug therapy varies considerably within a population. RThere are 
interindividual differences in absorption, distribution, metabolism aRnd Aelimination of 
drug.  
LIB
Monitoring of efficacy and evaluation of thNe p harmacokinetic parameters 
requires routine collection of whole blood for drugA analysis.  The use of whole blood 
collected through finger prick and adsorbAed Don filter paper has reduced risk of 
infection (Green et al., 2002) and se mIi-Bskilled field workers can collect the samples 
after minimal training with little Fdiscomfort or risk to the patient or volunteer.  In 
addition, blood collectioYn o n
O filter paper reduces the need to provide facilities for 
separating and storIinTg blood samples in the field.  However, collection of field 
samples for phaSrmacokinetic studies in children has particular challenges since 
frequent EsamRpling over a follow up period is required.  Children tend to be less 
williInVg to have repeated finger pricks.  When such situations arise, it may be difficult 
UtNo obtain the correct blood volumes especially using capillary tubes.  Different 
methods for measuring drug levels on filter paper have been developed (Minzi et al., 
2003; Gitau et al., 2004; Hoegberg et al., 2005). 
The pharmacokinetic determinants of antimalarial treatment failure are 
important in chemotherapy. This will help in prompt treatment of drug failure 
especially in children and pregnant women who are the vulnerable groups.  It is 
66 
 
 
important to recommend correct optimal dosage regimen where it is insufficient 
especially in children and pregnant women in who low drug levels have been clearly 
documented (Barnes et al., 2008; Gbotosho et al 2009). Sub-therapeutic  
concentrations and variability in pharmacokinetic disposition to antimalarial drugs 
contribute to poorer responses  to treatment and increase the spread of antimalarial 
drug resistance.  Suboptimal drug concentrations which could result from inadequ ate 
drug absorption, an unusually large apparent volume of distribution or duRe toY rapid 
clearance of the drug.  Response to treatment is ensured if aRntimAalarial drugs 
concentrations produce maximum effect until all malaria parasiIteBs are eliminated.  
 The use of Pharmacokinetic parameters to pred iLct treatment outcome is 
important and this has not been utilised in antimaAlarNial drug clinical trials to asses 
progression of responses to drug resistAancDe.  Routine measurement of drug 
concentrations of a slowly eliminatedI aBntimalarial drug such as CQ can be used to 
determine the minimum therapeFutic  concentration during treatment. For a drug 
eliminated slowly, the area  uOnder blood or plasma concentration time curve (AUC) 
could be a useful phIaTrmYacokinetic predictor of treatment outcome in uncomplicated malaria becauseS it captures both the drug concentration and duration of exposure 
(White etE alR., 2008).  The AUC comprises both the absorption and the elimination 
phasIeVs of a drug and provides a measure of parasite exposure to the anitmalarial 
dNrugs.     U
3.4: Study population 
From the delay in parasite clearance study, it was shown that children who 
were treated with chloroquine and sulphadoxine-pyrimethamine had highest 
proportions of delay in parasite clearance 70.8% (CQ) and 63.9 % (SP).  A cohort of 
children was randomly selected during enrolment for identification of 
67 
 
 
pharmacokinetic determinants of responses to chloroquine and assessment of 
pharmacokinetic disposition of sulphadoxine.   
In this study, children, aged between 6 months – 12years were treated with 
standard doses of chloroquine (30 mg/kg of chloroquine base over 3 days, that is, 10 
mg/kg daily). Children had microscopically confirmed infection with pure P. 
falciparum at parasiteamia greater than or equal to 2000 asexual parasites perY mi cro 
liter of blood before enrollment.  Prior to treatment, 100µl of capillary bloRod sample 
was obtained from each patient and spotted on filter paper (110mmR WAhatman filter 
paper) for determination of chloroquine or sulphadoxine level.I  BDuring follow-up, on 
day 1, 2, 3, 4, 5, 6, 7, 14, 21 and 28 thick blood films w eLre prepared and 100µl of 
capillary blood samples were obtained and spottedA onN filter paper.  The filter paper 
samples were stored in desiccated resealabAle Dplastic bags at room temperature for 
measurement of chloroquine and sulphIadBoxine concentrations. 
 F 
3.5: Drug analysis  O
3.5.1: Reagent for aInTalyYsis. Chloroquine, DeSsethyl chloroquine and Papaverine reference standard were obtained 
from WEalteRr Reed Army Institute, USA; Diethyl ether, Sodium hydroxide, 
HydIroVchloric acid and perchloric acid were obtained from BDH chemical England; 
ANcetonitrile, Methanol and Water were HPLC grade obtained from LiChrosolv®, E. UMerck, Germany. All other reagents were analytical grade. 
3.5.2: Preparation of Stock and Working solutions. 
1. Sodium hydroxide (NaOH, 5M) working solution was prepared by weighing 
3 
40g of NaOH pellets and dissolving in 200cm of distilled water in a 
volumetric flask. 
68 
 
 
2. The working solution of Sodium dihydrogen phosphate (NaH2PO4.2H2O, 
0.02M) was made by weighing 3.1202g of the compound and dissolving in 
3
1000cm  of distilled water. 
3. Stock solution of chloroquine, desethyl chloroquine and papaverine (internal 
standard) were prepared by weighing 5mg of each of the drugs and dissolving 
in 5ml of 0.1M HCl. The working solution (100µg/ml) was prepared fr om 
1mg/ml stock solution by making 1:10 dilution of 1mg/ml. OneR mYilliliter 
(1ml) of 1mg/ml stock was made up to 10ml with 0.1M HCRl inA a volumetric 
flask or centrifuge tube. IB
4. Preparation of 0.1M HCl was made from conc enLtrated hydrochloric acid 
(HCl).  The concentrated acid used hadA peNrcentage purity of 35% with 
specific gravity of 1.18g/ml.  The mDolarity of the concentrated HCl was 
calculated as follows; BA
% purity of acid =35%F    I molar mass of HCl =36.5 
 
Concentration  Oof HCl in 1.18g/ml of the acid content = % purity x 
specific gIrTaviYty = (35x1.18)/100g/ml = 0.413g/ml 
 i.eS. 413 g of HCl in 1000ml (A) 
ERWeight equivalent to molar mass of any compound in 1000ml of distilled 
wIVater = 1M of the compound. 
UN If 36.5g of HCl (was dissolved in 1000ml solvent) = 1M HCl (B) 
 Therefore if A=B 
then, 413.0g (in 1000ml solvent) = (413g x 1M)/36.5g = 11.315M (C1).  
The molarity of the concentrated acid was 11.315M (C1).  To prepare 1000ml 
(V2) of 0.1M HCl (C2).  The volume (V1) of concentrated acid required can be 
69 
 
 
obtained from the formula, C1V1 = C2V2, where C1 is initial concentration of the 
acid, C2 is final concentration, V1 volume of initial concentrated acid require and 
V2 is final volume respectively. 
 V1= (C2V2)/C1      = (0.1M X 1000ml)/11.315M  
 = (100ml)/11.315 = 8.837ml of concentrated acid.  
Therefore, 0.1M was prepared by adding 8.837ml of concentrated acid to dYistilled 
water in volumetric flask and making up to 1000ml with distilled water.
RA
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3.5.3: Analysis of chloroquine from filter paper samples.  LN
(a) Instrumentation. A
Chromatographic separations were carried AoutD on a Cecil Adept, High performance 
liquid chromatography unit. The unit  cIoBnsists of Cecil 4100 solvent delivery system 
with a Rheodyne valve injectioFn system and a Cecil 4200 variable wavelength 
ultraviolet detector operatin g Oat 254nm with a Cecil 4900 chromatographic system 
manager.  The sepaIrTatioYn was carried out on a Waters C18 Reversed-Phase 10m 
Bondapak coluSmn of 3.9mm X 300mm dimension stainless steel maintained at 
room temEperRature. 
 IV
U(Nb)  Mobile Phase Composition. 
The mobile phase used for the analysis consisted of 0.02M phosphate buffer: 
methanol: acetonitrile (58:27:15 V/V/V) adjusted to pH 2.5 with perchloric acid. The 
mobile phase was delivered to the system at a flow rate 1ml/min. 
 
70 
 
 
(c) Extraction of chloroquine whole blood filter paper samples. 
Whole blood concentrations of chloroquine were measured from filter paper blood 
spots using a modified HPLC analysis (Ogunbona et al, 1986).  Briefly, filter paper 
sample was cut into pieces and the pieces were transferred into clean extraction tubes.  
The pieces of filter paper were soaked in 200µl of 0.1M HCl for 30 minutes.  Two 
hundred nanogram (40µl of 5µg/ml) of papaverine as internal standard was add ed.  
Two hundred microlitres (200µl) of 5M NaOH was added to the content ofR eacYh tube 
to basify the sample.  A fixed volume (2ml) of diethyl ether was RalsoA added as the 
extraction solvent.  The content was vortexed for sixty secondIsB and then centrifuged 
for 10 minutes at 2000g.  After centrifugation the organic  sLupernatant of the mixture 
was transferred into a clean centrifuge tube. One hAundNred microlitre (100µl) of 0.1M 
HCl was added to the organic supernatant andD vortexed for 2 minutes.  The mixture 
was thereafter centrifuged for ten mi AInButes at 2000g. The organic supernatant was removed carefully and discarded. FTw enty microlitre (20µl) of the aqueous phase was 
injected onto the column for  aOnalysis. 
 TY
(d) Calibration CSurvIes. 
 CEalibRration curve is a plot of detector response (peak/ratio) with change in 
concIeVntration of known sample. It is a general method for determining the 
UcNoncentration of a substance in an unknown sample (blood) by comparing the 
unknown to a set of standard samples of known concentrations. Calibration curves 
based on peak-height ratios were prepared by spiking drug free blood sample with 
working solutions of chloroquine and desethyl-chloroquine to yield final 
concentration of 0 – 2000ng/ml for chloroquine and desethyl-chloroquine on filter 
paper.  Chloroquine metabolite, desethyl-chloroquine was not considered because 
71 
 
 
desethyl-chloroquine standard powder was not available. The peak-height ratio for 
both the drug and the internal standard were recorded. Concentration of drug in the 
blood samples was extrapolated from the calibration curve as shown in Figure 4.4. 
 
(e) Precision and percentage recovery of the analytical method.  
Filter paper samples (drug free) were spiked with known concenRtratiYon of chloroquine (between 0 - 2000ng/ml of chloroquine).  A 40µl (of 5µg/Aml) aliquot of 
internal standard was added to the cut filter paper in extractioBn tRube. The samples 
were prepared (n=5), taken through the extraction procedu rLe aIs described previously 
and injected onto the column. Another set of standaNrd chloroquine solutions were 
prepared to yield actual concentration in the spiDkedA filter papers.  An aliquot (20µl) of 
the solution was injected directly into the cAolumn. The peak height of the extracted 
standard samples and the solutions in jeIcBted onto the system were compared to obtain 
the percentage recovery of theO meFthod, intra-assay (within day) and inter-assay (day-
to-day) coefficient of vaYria tion (C.V.).   Limit of detection of the drug was also 
determined. IT
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Study 3:  Development of High Performance Liquid Chromatography analytical 
method for measurement of sulphadoxine concentrations in filter paper sample. 
 Sulphadoxine-pyrimethamine ( SP) analysis from filter paper has been a very 
difficult analysis because of the nature of chemical composition for sulphadoxine and 
pyrimethamine.  Sulphadoxine is both an acid and a weak base while pyrimethamine 
is a weak base (Green et al., 2002).  This method was developed in order to Yredu ce 
cost of analysis and the burden of sample storage especially during fRiled trials.  
Although this method analysed both sulphadoxine-pyrimethamine (RSP) Abut only SDX 
was considered.  It has been reported that there is a correlIaBtion between plasma 
concentration of SDX and PYR (Bergvist et al., 1 LN987 ), however knowledge of sulphadoxine concentrations provides some inforAmation of the magnitude of the 
accompanied pyrimethamine concentrationA asD well. The ratio of combination of 
sulphadoxine-pyrimethamine is 2I0:B1 (500mg of sulphadoxine:25mg of 
pyrimethamine) in a tablet. 
OF
 
 Y 
3.6: Analysis of sulpIhTadoxine  from filter paper samples. 
a.  InstrumenRtatiSon. 
The IsVame
E HPLC unit as in CQ analysis was used for sulphadoxine analysis but the 
cNolumn used was different. The eluent was monitored using a UV detector operated Uat 240nm with a Cecil 4900 chromatographic system manager.  The separation was 
carried out on a Beckman Coulter ODS 5µm column of 4.6mm X 15cm dimension 
stainless steel maintained at room temperature. 
 
 
73 
 
 
b. Preparation of Stock and Working solutions. 
Stock solution of sulphadoxine, pyrimethamine and sulisoxazole (internal standard) 
were prepared by weighing 5mg of each of the drugs and dissolving in 5ml of 70% 
ethanol prepared with 0.1M HCl solution.  Working solution (100µg/ml) was 
prepared from 1mg/ml stock solution by making 1:10 dilution of 1mg/ml.  One 
milliliter (1ml) of 1mg/ml stock was made up to 10ml with 0.1MHCl in a voluYme tric 
flask or centrifuge tube. 
AR R
c. Mobile phase composition. IB
The mobile phase used for the analysis consisted of  1L% triethylamine solution 
containing 0.05M phosphate buffer: methanol: acetAonitNrile (70:17:13 V/V/V) adjusted 
to pH 3.4 with phosphoric acid.  The mobiAle pDhase was delivered to the system at a 
flow rate of 0.9ml/min. 
F I
B
 
 d. Measurement of sulphado xOine from whole blood on filter paper samples. 
Sulphadoxine (SDXI) TexYtraction was performed by cutting filter paper samples into pieces and transSferring the pieces into clean extraction tubes. The pieces of filter 
paper weEre sRoaked in 200µl of 0.1MHCl for 30 minutes and twenty microlitre (20µl) 
of 5I0V0µg/ml sulisoxazole as internal standard was added.  Two millitres (2ml) of 
aNcetonitrile was also added as an extracting solvent. The content was vortexed for Utwo minutes and then centrifuged for 10 minutes at 2000g. After centrifugation the 
acetonitrile supernatant of the mixture was transferred into a clean centrifuge tube 
O
and dried under a stream of nitrogen gas in a water bath at 50 C. One hundred 
microlitres (100µl) of 0.1M HCl was added to reconstitute the dried extracted sample, 
vortexed for 2 minutes and centrifuged for ten minutes at 2000g.  Twenty microlitres 
74 
 
 
(20µl) of the aqueous phases was injected onto the column for analysis. The retention 
time for sulphadoxine and sulisoxazole were determined. 
 
 e. Calibration curve for sulphadoxine. 
The calibration curve for sulphadoxine was prepared by spiking drug free blo od 
sample with working solutions of SDX to yield final concentration of 0 – 60µgY/ml for 
sulphadoxine.   Using an adjustable pipette, 100µl of drug-spiked donoAr wRhole blood 
o
was adsorbed onto the filter paper.  The samples were placed in an Rincubator at 37 C 
and allowed to dry overnight.  The spiked filter paper bLlooIdB sample were taken 
through the normal assay procedure as discussed abovNe, a nd calibration graphs were 
constructed using the peak-height ratio of SDAX to internal standard against 
concentration. AD
 B
F I
 f.   Precision and Recovery.  O
To assess precision, cToefYficient of variation (CV) was determined for intra- and inter 
assay variabilityS.  TIhis was achieved by carrying out repeated analysis of drug-free 
whole bloodR spiked with different concentrations of sulphadoxine (n>5 for each 
concIeVntra
Etion) on each of six days. Calibration curves were prepared from the 
mNeasurement of peak height ratios of the SDX and internal standard. Extraction Urecovery was determined for each concentration by comparing peak heights ratio of 
the extracted known standards with the directly injected standard concentrations.  
Commonly used antimalarial drugs including chloroquine, mefloquine, quinine, 
sulphamethoxazole and trimethoprim were studied for interference by spiking the 
drugs in the blank whole blood. The drugs were extracted according to the method 
75 
 
 
described above. The presence of peaks was monitored after injection of 20µl of the 
reconstituted sample. 
Y R
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Study 4: Pharmacokinetic disposition of sulphadoxine in children with acute 
uncomplicated malaria treated with standard dose of sulphadoxine-
pyrimethamine. 
 Sulphadoxine-pyrimethamine is currently recommended by WHO as a partner 
drug with artesunate in the chemotherapy of malaria and it is also being used alone 
for intermittent preventive treatment for malaria in pregnancy and in infants Y(WH O 
2006; WHO 2007; May et al., 2008). However, information on pharmacoRkinetics of 
SDX-pyrimethamine in children with acute uncomplicated malaria Ris liAmited (Barnes 
et al., 2006; Dzinjalamala et al 2005; Obua et al 2008; IHBellgren et al 1990; 
Winstanley et al., 1992 and Aubouy et al 2003).  ENffor ts
L in this study were thus 
devoted to studying the pharmacokinetic dispositioAn of SDX and evaluating the effect 
of pharmacokinetic variability on therapeutiAc efDficacy in children between the ages of 
6 months and 12 years with acute uIncBomplicated falciparum malaria treated with 
standard dose of SP.  F 
 Seventy-four paYtien t
Os treated with standard dose of sulphadoxine-
pyrimethamine (25 ImTg/kg of the sulphadoxine component) were randomly selected 
from cohort of cShildren who had highest proportion of delay in parasite clearance. 
The simpEle cRost-effective high performance chromatographic analytical method that 
was IdVeveloped for the analysis of sulphadoxine in capillary whole blood collected on 
UfNilter paper in study 3 was used for the analysis of these samples.  Samples were 
collected before treatment (day 0), 1, 2, 3, 4, 5, 6, 7, 14, 21 and day 28 post treatment 
respectively. Reagent and chromatographic conditions are as stated in study 2 and 
study 3. 
77 
 
 
 
Study 5: Evaluation of the use of saliva for therapeutic drug monitoring in 
children with uncomplicated Plasmodium falciparum malaria treated with 
amodiaquine-artesuante. 
 Saliva offers an easily accessible and sometimes useful body fluid  for 
therapeutic drug monitoring and for pharmacokinetic and pharmacodynamic sYtudies. 
Saliva is limited to the unbound fraction of the drug. Thus, saliva levelsA maRy be more 
reflective of drug concentrations at the site of action thanR are total drug 
concentrations in plasma or whole blood. It has beenL sIhBown that the saliva 
concentrations of particular drug can be used to predicNt how  much of those drugs is in 
the blood (Fatah and Cohn, 2003).   A
 
BA
D
3.7: Patients enrolled in the studFy a nId sample collection. 
 Patients were random Oly selected from cohort of children who received oral 
doses of artesunate-amTodYiaquine (4mg/kg artesunate plus 30mg/kg amodiaquine base 
over 3 days).  SSeveIn children were selected from this group of children. Venous 
blood (5ml) Rwas collected at 0hr before treatment, 4hr post treatment and thereafter 
on dIaVy 7,
E 14, 21, 28 and 35.  Blood samples were collected into heparinised tube and 
wNere immediately centrifuged at 2000g for 10 minutes to separate the plasma and red U Oblood cells, which were stored at -20 C until analysed. Saliva samples were taken at 
the same times that venepuntures were done.  The samples were collected after 
rinsing the mouth with water and without any special stimulus to increase saliva flow.  
The saliva (3-5ml) was immediately centrifuged and the clear fluid was removed and 
O
stored at -20 C until analysed.  
78 
 
 
3.8: Analysis of amodiaquine or desethyl amodiaquine from plasma or saliva 
samples. 
(a) Instrumentation. 
Chromatographic separations were carried out on the same HPLC unit as in CQ study 
(study 2).  
 Y 
(b). Preparation of Stock and Working solutions. R
Stock solution of amodiaquine, desethyl amodiaquine and quRinidAine (internal 
standard) were prepared by weighing 5mg of each of the drugsI aBnd dissolving in 5ml 
of 70% ethanol prepared with distilled water.  WorNking  
Lsolution (100µg/ml) was 
prepared from 1mg/ml stock solution by makingA 1:10 dilution of 1mg/ml.  One 
milliliter (1ml) of 1mg/ml stock was madAe uDp to 10ml with distilled water in a 
volumetric flask or centrifuge tube. 
 IB F
(c)  Mobile Phase Compositi oOn. 
The mobile phase IcoTmpYrised 0.02M potassium diphosphate buffer, methanol and acetonitrile in thSe ratio 75:23:2 (V/V/V) adjusted to pH 2.65 with phosphoric acid.  
The separEatioRn was carried out on a Hypersil Reversed-Phase 5µ column of 4.6 mm x 
250 ImVm dimension stainless steel maintained at room temperature.  The UV detector 
wNas set at 254nm.  The mobile phase was delivered to the system at a flow rate U1ml/min.   
 
( d ) Extraction of amodiaquine/desethyl amodiaquine from plasma or saliva sample. 
Amodiaquine and desethylamodiaquine in plasma and saliva was assayed by a 
specific high performance liquid chromatography (HPLC) with ultraviolet detector 
79 
 
 
using a modification of a method described by Gitua and others (2004).  Briefly, 0.5 
ml of plasma or saliva was transferred into clean extraction tubes.  Two hundred and 
fifty nanogramm (5µl of 50µg/ml) of quinidine was added as internal standard (IS).  
A fixed volume (5 ml) of diethylether was also added as the extraction solvent.  The 
content was vortexed for sixty seconds and then centrifuged for 10 minutes at 2000g.  
After centrifugation the organic supernatant of the mixture was transferred into  a 
O
clean centrifuge tube and dried in a fume cupboard at 40 C.  The reRsiduYe was 
reconstituted in sixty-five microlitre (65µl) of mobile phase anRd fiAfty microlitre 
(50µl) injected onto the column. 
LIB N 
(e) Measurement of saliva pH.   
DA
The pH of saliva was measured immediAately after collection using a pH meter 
(Mettler-Toledo GmbH, Sonnenbergs trIaBsse, Switzerland) and recorded immediately.   
  O
F
(f) Calibration CurvesT. I Y
Stock solutionsS of amodiaquine, desethyl amodiaquine (1mg/ml) and internal 
standard EquinRidine (1mg/ml), was prepared in 70% methanol.  Drug free plasma and 
salivIaV was spiked with the working solution containing both AQ and DEAQ to yield 
fNinal concentrations ranging from 600 – 100ng/ml of AQ and DEAQ.  The samples Uwere assayed as described above and this was used for calibration curve. 
Concentration of drug in the plasma or saliva samples was extrapolated from a 
calibration curve. 
(g) Precision and percentage recovery of the analytical method. 
Drug free plasma or saliva samples were spiked with known concentration of AQ or 
80 
 
 
DEAQ (between 100 -800ng/ml) and 5µl of 50µg/ml aliquot of internal standard was 
added.   The samples (n=5), were taken through the extraction procedures as 
described previously and injected onto the column. Another set of standard AQ and 
DEAQ solutions were prepared to yield actual concentration in the spiked plasma or 
saliva sample.  An aliquot (20µl) of the solution was injected directly onto the 
column. The peak height of the extracted standard samples and the solutions inYjec ted onto the system were compared to obtain the percentage recovery of thRe method, 
intra-assay (within day) and inter-assay (day-to-day) coefficient of vRariaAtion (C.V.).    
 
LIB
3.9: Statistical analysis for all studies. N 
Data were analysed using version 6 ofD theA Epi-Info software (Anon., 1994), 
and the statistical program SPSS for WAindows version 10.01 (Anon., 1999).  
Proportions were compared by calc uIlaBting 2 with Yates' correction.  Normally 
distributed, continuous data  Ower
Fe compared by Student's t-tests and analysis of 
variance (ANOVA).  DatYa not conforming to a normal distribution were compared by 
the Mann-Whitney IUT-test and the Kruskal-Wallis test (or by Wilcoxon rank sum 
test).  The vRalueSs presented are generally means and standard deviations (s.d.) or 
standaVrd Eerror of mean (s.e.m.). 
NI In order to determine the risk factors for delay in parasite clearance and Upharmacokinetic factors associated with CQ treatment failure, the relationship 
between each of the clinical or parasitological data at enrolment and pharmacokinetic 
parameters following treatment was investigated by univariate analysis.  A multiple 
logistic regression model was used to test the association between response to drug 
treatment (sensitive or resistant) and the factors that were significant in the univariate 
81 
 
 
O
analysis: age, presence of fever (temperature ≥ 37.5 C) at presentation, PC > 2 d (yes 
or no at presentation or during follow up): presence of fever, asexual parasitaemia at 
presentation or during follow-up, a history of vomiting, and drug treatment.  All these 
factors were used in multiple logistic regression analysis models to test the 
association with delay in parasite clearance. 
GraphPad Prism (version 3.02) and Microsoft Excel statistical softwar e’s 
were used for collation of all the drug concentrations obtained from each pRatieYnt and 
used for test of linearity of the standard curves.  PharmacokineticR parAameters were 
obtained using Turbo Ken software (designed by the DIepBartment of Clinical 
Pharmacology, University of Southampton, United Kingd oLm). P values < 0.05 were 
considered significant for all statistical analysis exAcepNt in multivariate analysis of t1/2 
where P < 0.3 was ‘falsed’ for the analysiDs.  In comparison of disposition of 
amodiaquine and desethylamodiaquiIneB in
A plasma and saliva in children with 
uncomplicated Plasmodium falciFpar um malaria, plasma and saliva concentration-
time curve was plotted for  eOach subject. Oral clearance was calculated from the 
equation CL= D/AUCT, DY is the dose given. The apparent volume of distribution (Vd) was calculated frSomI the equation Vd=CL/β.  
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CHAPTER FOUR 
 
 
RESULTS 
 
Study 1:  Risk factors contributing to delay in parasite clearance in 
uncomplicated falciparum malaria in children. 
  
4.0: Study Population Y
Between April 2002 and July 2010, 2,752 children (1,342 femaAles Rand 1,410 
males) were enrolled into the antimalarial drug studies.  ABll cRhildren who were 
recruited into these studies had primary infections  wLithI P. falciparum.  There 
were 1,716 under five-year olds.  The geoNmetric mean parasitaemia at 
enrolment was 34,044 asexual parasites/µDl oAf blood (95% CI 30,400 – 33,664) 
as shown in Table 4.1.   
  IB
A
4.1: Drug treatment andO delFay in parasite clearance 
Overall, delay in pYara site clearance occurred in 1,237 of the 2,752 children 
(45%) (FigureI 4T.1 and Table 4.2).  The highest proportions of children showing 
delay iRn paSrasite clearance were found in those treated with chloroquine (CQ), 
sulpEhadoxine-pyrimethamine (SP), chloroquine plus chlorpheniramine (CQCP), 
IVN co-trimoxazole (COT), amodiaquine (AQ), chloroquine plus ketotifen U (CQKET), chloroquine plus sulphadoxine-pyrimethamine (CQSP), 
amodiaquine plus sulphadoxine-pyrimethamine (AQSP), sulphadoxine-
pyrimethamine plus probenecid (SPPB) and mefloquine (MQ).  The 
proportions of children with delay in parasite clearance were significantly lower 
in those treated with AS, AQAS, AL and AMQ when compared with the latter 
2
group above (χ  = 447.91 df = 8, P < 0.0000001).  There was no significant 
83 
 
 
difference in the proportions of children with delay in clearance in those treated 
2
with AS (3 of 120), AQAS (14 of 142), AL (6 of 90) and AMQ (10 of 174) (χ  
= 6.1, df =3, P = 0.10). Infection in 291 of the 2,752 (10.6%) children failed to 
respond to treatment (Table 4.3). 
 
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Table 4.1:  Baseline clinical and parasitological parameters of 2752 children with 
acute uncomplicated falciparum malaria enrolled in the study. 
 
 Variables    Mean ± SD (range) 95% CI 
Age (year)    6.1 ± 3.0 (0.5-12) 6.0-6.2 
     No. < 5 years   1,716 (63%) 
Weight (kg)    17.3 ± 6.4 (5-47) 17.0-17.6 
O  
Axillary temperature ( C)  Y
(n = 2428)    38.3 ± 1.2 (34-42) 38.2-38R.3 
o
    No. with > 40 C   210  A
Haematocrit (%) (n = 994)  30.5 ± 4.8 (10-51) 3R0.1-30.7 
    No. with < 30%   380 IB
Parasitaemia (/µl)  GM   34,044   L        30,400-33,664 
       Range     2009-1,19N4,285 
       No with > 100,000 (/µl)  638 (2A3.2%) 
       No. with > 250,000 (/µl)  18D7 (6.8%) 
Gametocytaemia (/µl) GM  BA27 (6 - 4188) 
Duration of illness (d)  F  I 3.0 ± 1.4 (1-14) 2.9-3.0 Duration of vomiting (Od)  1.3 ± 1.4 (1-9)  1.2-1.3 ____________________Y___ _______________  _________________ GM, geometriTc mean; parasitaemia= asexual parasites/µl of blood 
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Table 4.2: Proportions of children with Delay in Parasite Clearance 
following treatment with standard doses of selected antimalarial drugs. 
Drug Proportion with delay in PC (%) 
Total with delay in PC  45 (1237) 
Chloroquine (CQ) 70.8 
Sulphadoxine-pyrimethamine (SP) 63.9  
Chloroquine  + chlorpheniramine (CQCP) 60.0 RY
Co-trimoxazole (COT) 58.6 A
Amodiaquine (AQ) 52.0 BR
Chloroquine + Ketotifen (CQKET) 51.4 LI
Chloroquine + Sulphadoxine- 42N.0  
pyrimethamine(CQSP) A
Amodiaquine + sulfalene-pyrimethamineA D 37.6 
(AQSFP) IB
Sulphadoxine-pyrimethamine F + 30.9 
probenecid (SPPB)  O
Mefloquine (MQ) Y 22.7 
Artesunate + amIoTdiaquine (AQAS) 9.9 
ArtesunRate +S lumefantrine (AL) 6.6 
VArteEsunate + Mefloquine (AMQ) 5.7 
NIArtesunate (AS) ( monotherapy) 2.5 U† All drugs were administered orally.  AQ, amodiaquine; AQAS, amodiaquine plus artesunate; AQSP, amodiaquine plus sulphadoxine-pyrimethamine; AQSFP, amodiaquine-sulfalene-pyrimethamine; 
AMQ, mefloquine plus artesunate; AL, artemether plus lumefantrine; AS, artesunate; COT, co-
trimoxazole; CQ, chloroquine; CQCP, chloroquine plus chlorpheniramine; CQKET, chloroquine plus 
ketotifen; CQSP, chloroquine plus sulphadoxine- pyrimethamine; MQ, mefloquine; SP, Sulphadoxine-
pyrimethamine; SPPB, sulphadoxine-pyrimethamine plus probenecid. 
86 
 
 
 
 
 
Table 4.3:  Proportion of children with falciparum malaria who failed to 
respond to treatment with standard doses of antimalarial drugs. 
  
Day                Total failure  Failure rate (%) Y
        7                     57          2.1 AR
       14                    136          5.3 R
       21                     291    L    I 1B0.6 2
χ  for trend = 158, p < 0.000001; Infection in 291N of  the 2,752 (10.6%) children 
failed to respond to treatment. 
DA
 IB
A
 O
F
ITYS
ER
NI
V
U
87 
 
 
 
 
 
 
 
 
 
 
 
Δ Parasite clearance > 2 d who 
proceded to failure. Y 
Δ No. with parasite clearanRce > 2 d   
Δ Total enrolled 
BR
A
N 
LI
DA
BA
OF
 I
 
SI
TY
 
 
 R
Figure 4.1:  NEumbers of children with delay in parasite clearance (PC > 2 d) following 
treatment wIiVth antimalarial drugs. 
UN
88 
 
 
4.2:  Risk factors for delay in parasite clearance at enrolment 
The following were found to be independent risk factors for delay in parasite 
clearance at enrolment (Table 4.4):  age ≤ 2 years (Adjusted odds ratio [AOR] = 2.13, 
95% confidence interval [CI]1.44-3.15, P < 0.0001), presence of fever (AOR = 1.33, 
95% CI = 1.04 –1.69, P = 0.019)  and parasitaemia >50,000  asexual parasite/µl of 
blood (AOR = 2.21, 95% CI = 1.70 - 2.75, P < 0.0001). A history of vomiting w as 
associated with an increased risk of delay in clearance (crude odds ratio = R1.34Y, 95% 
CI = 1.07 -1.58, P = 0.009). A
 IB
R
4.3:  Risk factors for delay in parasite clearance following  iLnitiation of treatment 
o
Following treatment, a body temperature > 38 C aAnd Nparasitaemia > 20,000 asexual 
parasite/µl of blood a day after treatment begDan, were independent risk factors for 
delay in clearance (Table 4.5).  Non aBrtemAisinin monotherapy was associated with 
delay in clearance.  I
  O
F
4.4:  Delay in parasitTe clYearance and treatment failure Figure 4.2 showSs thIe proportions of children treated with various antimalarials who 
had delay in Rparasite clearance and who subsequently failed treatment.  Overall, 291 
childIrVen f
Eailed treatment.  Of these, 211 (72%) had delay in parasite clearance.  The 
lNatter represent 17% of the total children (1237) with delay in parasite clearance.  In Usummary 13 of 20, 7 of 10, 119 of 146, 14 of 15, 5 of 19 and 50 of 60 children treated 
with AQ, COT, CQ, CQCP, MQ, and SP respectively were those who failed 
treatment and previously had delay in parasite clearance. 
 
 
89 
 
 
Table 4.4:  Predictors of delay in parasite clearance at presentation in children with 
acute falciparum malaria treated with antimalarial drugs. 
 
Variables Number PC Crude OR P Adjusted OR (95% P 
enrolled >2 d (95% CI) Value CI) Value 
Age (years)       
   >2 2446 1076 1  1  
   ≤2 267 140 1.40 (1.09 –1.80) 0.008 2.13(1.44 –3.15) <0.000
Gender       1  
   Female 1342 607 1   Y  
   Male 1240 557 0.98 (0.84 –1.15) 0.870 - R - 
      A  
Fever*       
   Absent 686 252 1  1 R  
   Present 1743 842 1.6 (1.34 -1.93) <0.0001 B1.33 (1.04-1.69) 0.019 
     I   
Duration of      L   
illness (d)     N   
   < 3 2085 926 1    
   > 3 521 246 1.12 (0.90 – 1.3D0) A0.225 - -  
Haematocrit (%)    A    
  ≥ 30 614 182 1 B    
  <30 380 114 1.01  (0I.76 – 1.34) 0.904 - -   
Parasitemia (/µl    O 
F    
blood)       
   < 50,000 1607 634 1  1  
   > 50,000 1145 IT60Y3 1.70 (1.46 –1.99) <0.0001 2.21 (1.77 – 2.75) < 0.0001 
       
Gametocytemia  S      
   Absent E2R086 896 1       Present V 232 90 0.84 (0.63 – 1.10) 0.224 - -        
VomitingN I         No 1005 519 1  1  
  Yes U 665 387 1.34 (1.07-1.58) 0.009 1.21 (0.90-1.51) 0.089        
Hepatomegaly       
   Absent 471 237 1    
   Present 798 430 1.54 (0.91 – 2.44) 0.219 - - 
       
 
o
CI, confidence interval; OR, odd ratio; PC, parasite clearance, *Body temperature > 37.5 C 
90 
 
 
 
Table 4.5:  Predictors of delay in parasite clearance on day 1 after treatment in 
children with acute falciparum malaria. 
       
Variables Number PC Crude OR P Adjusted OR P 
enrolled >2 d (95% CI) Value (95% CI) Value 
Axillary       
O
temperature ( C)        
   <38.0 2326 1012 1  1 Y  
   ≥38.0 228 139 2.02 (1.53 -2.67) <0.0001 1.80 (1.3R0-2.50) < 0.001 
       
Parasitaemia (/µl      A  
blood)     B R  ≤ 20,000 1328 463 1  1  
>20,000 683 467 5.25 (4.20 – 6.48) <0. 0L001I 5.13 (4.14 – 6.35) < 0.001        
Drug treatment *       
CQ 388 275 1 AN1 1   AQ 573 298 0.44 (0.33 – 0D.58) <0.0001 0.79 (0.63 – 0.98) < 0.031 AQAS 142 14 0.05 (0.03A – 0.08) <0.0001 0.24 (0.10 – 0.57) < 0.0001 AQSP 69 26 0.24 (0.14 – 0.42) <0.0001 0.44 (0.27 – 0.73) 0.002 
AQSF 91 34 0.25 (0.15 – 0.39) <0.0001 0.43 (0.29 – 0.68) < 0.0001 
AMQ 174 10 0.03 
F I
(0B.01 – 0.05) <0.0001 0.04 (0.02 – 0.09) < 0.0001 
AL 90 6 0.03  (0.01 – 0.07) <0.0001 0.05 (0.02 – 0.12) < 0.0001 
AS 120 3 O0.01 (0.00 – 0.03) <0.0001 0.02 (0.00 – 0.06) < 0.0001 COT 104 61  0.58 (0.37 – 0.91)    0.017 1.03 (0.68 – 1.36) 0.877 CQCP 315 189Y 0.61 (0.45 - 0.84)    0.002 1.09 (0.83 – 1.42) 0.512 CQKET 70 IT36 0.43 (0.25 – 0.73)   0.001 0.77 (0.47 – 1.25) 0.298 CQSP 107 46 0.31 (0.19 – 0.48) <0.0001 0.54 (0.36 – 0.82) 0.004 MQ 176 S 40 0.12 (0.08 – 0.18) <0.0001     0  . 2  1   (  0  . 1 5 - 0.31) < 0.0001 SP 29R1 186 0.72 (0.52 – 1.00) 0.055 - - SPPB E42 13 0.18 (0.09 - 0.37)      <0.0001 0.33 (0.17 -0.98)  0.001    
 CI, confidence interval; OR, odd ratio; PC, parasite clearance, * Values of OR represent chances of delay in parasite clearance.  
† All drugs were IadVministered orally.  AQ, amodiaquine; AQAS, amodiaquine plus artesunate; AQSP, amodiaquine plus 
sulphadoxine-pNyrimethamine; AQSFP, amodiaquine-sulfalene-pyrimethamine; AMQ, mefloquine plus artesunate; AL, artemetherU plus lumefantrine; AS, artesunate; COT, co-trimoxazole; CQ, chloroquine; CQCP, chloroquine plus chlorpheniramine; CQKET, chloroquine plus ketotifen; CQSP, chloroquine plus sulphadoxine- pyrimethamine; MQ, mefloquine; SP, Sulphadoxine-pyrimethamine; SPPB, sulphadoxine-pyrimethamine plus probenecid; 
 
 
91 
 
 
 
 
 
 
 Δ Delay in PC Δ Total failure 
RY
 
A
LIB
R
AN
 
AD
F I
B
 O
ITYS
VE
R
I   
FigUureN 4.2: The proportions of children treated with various antimalarials who had delay in parasite clearance (PC) and subsequently failed treatment. (AQ, amodiaquine; AQAS, amodiaquine plus artesunate; AQSP, amodiaquine plus sulphadoxine-pyrimethamine; AQSFP, 
amodiaquine-sulfalene-pyrimethamine; AMQ, mefloquine plus artesunate; AL, artemether plus 
lumefantrine; AS, artesunate; COT, co-trimoxazole; CQ, chloroquine; CQCP, chloroquine plus 
chlorpheniramine; CQKET, chloroquine plus ketotifen; CQSP, chloroquine plus sulphadoxine- 
pyrimethamine; MQ, mefloquine; SP, Sulphadoxine-pyrimethamine; SPPB, sulphadoxine-pyrimethamine 
plus probenecid) 
  
92 
 
 
 
Study 2:  Pharmacokinetic determinants of response to treatment with 
chloroquine in children with acute uncomplicated Plasmodium falciparum 
malaria. 
Chloroquine was well resolved in the analysis using this method. There was a 
2
good linearity (r  = 0.9992; Figure 4.3 and Appendix Table 4.a) in the standard cu rve 
obtained for the analysis of chloroquine (CQ). In addition, the percentage rRecovYery of 
CQ as well as the precision and accuracy of the analytical method for CAQ are shown 
in Table 4.6 and 4.7.  Figure 4.4 shows the chromatogramBs Rfor CQ, desethyl 
chloroquine (DCQ) or papaverine (PPV), internal standar dL obItained during analysis. 
There was no interference with other drugs (e.g. paracNetamol, amodiaquine and other 
commonly used drugs) with the peaks obtaineDd (FAigure 4.4). The limit of detection 
for chloroquine was 5ng/ml.  The retention Atime for DCQ, CQ and the PPV were 5.5 
minutes, 6.5 minutes and 10.5 minute
F 
sI rBespectively.  
 O
TY
RS
I
VE
NIU
93 
 
 
 
10.0
7.5
5.0  
RY
2.5
RA
0.0 IB
0 500 1000 1500  L2000 2500
Concentration (AngN/ml).  
 AD
Figure 4.3: Standard curve of chloroq uIiBne for extrapolation of unknown 2 concentrations from whole blood sFpotted on filter paper.  (r = 0.9992)
 O
TY
RS
I
E
IV
UN
94 
 
Peak height ratio
 
 
 
Table 4.6:   Percentage recovery of chloroquine from whole blood spotted on 
filter paper using the analytical method for chloroquine. 
 
 
 
  Concentration % Recovery n 
 (ng/mL)    (S.D) 
     Y 
 Whole blood 200 85.65.9 5 
  400 87.52.5 4 R
  800 89.61.9 4 A
  R
 B
S.D= Standard deviation 
N 
LI
DA
BA
OF
 I
 
SI
TY
R
IV
E
UN
95 
 
 
 
 
 
Table 4.7:  Precision of analytical method for extraction of chloroquine from 
whole blood spotted on filter paper. 
 
 
  Concentration C.V (%) n 
(ng/mL) 
     
Intra-assay Chloroquine Y
 100 2.9 5 
 200 1.9 5 R
 1000 2.5 4 A
    R
Inter-assay  Chloroquine B
 100 3.2  L5I  200 3.1 6  1000 2.8 4 
 C.V= coefficient variation AN
BA
D
OF
 I
ITY
 
ER
S
NI
V
U
96 
 
 
A 
IS 
 
AR
Y
 
 BB R
N 
LI
IS 
DA
BACQ  IDCQ 
 O
F
SI
TY
VE
R
 
NI Time (min)  UFigure 4.4: Chromatograms showing chloroquine (CQ) and desethyl chloroquine 
(DCQ) from whole blood sample collected from a patient  and spotted on filter paper 
on day 0 (A) and day 3 (B). 
 
 
 
97 
 
 
4.5:  Study Population 
The characteristics of the 74 children who were randomly selected into the 
study are shown in Table 4.8.  Thirty eight (51.4%) children were aged < 5 years. The 
geometric mean parasite density was 52,467 (95% confidence interval, 38,194 – 
71,944 asexual parasite/µl of blood).  Seventy eight percent (58/74) of the children 
were febrile at enrolment.   
 Y 
4.6:  Pharmacokinetics parameters of all patients enrolled in the study R
All the patients or guardians gave a negative history of chloRroqAuine ingestion 
in the preceding two week but analysis of the pre-treatment IbBlood sample showed 
substantial chloroquine levels in 43 (58.1%) of the 7N4 ch i
Lldren enrolled. The mean 
CQ concentration was 916.86±89.79ng/ml (range,A 25.6 – 2643.0ng/ml) (Table 4.8).  
The children were therefore divided into 4 AgrouDps for the analysis of the data; all the 
74 children that were enrolled into tIhBe study; 48 children with delay in parasite 
clearance, 43 children with pre-dFosin g detectable chloroquine concentration in their 
blood and 31 children with n oO chloroquine in the pre-treatment blood sample.  
Drug conceInTtratYion before drug administration on day 2 (48h) was 1740.81±137.69nSg/ml and the decline of chloroquine concentration after day 2 was 
mono expEonRential with a terminal half-life (t1/2) of 306.3±20.99h (95% CI, 264.4 - 
348.I1Vh).  Table 4.9 is the summary of pharmacokinetic parameters of all patients 
iNrrespective of pre-dosing chloroquine blood concentration at enrolment.   The AUC0-U28d and CL were 920.6±69.6ng.h/ml and 633.6±60.2ml/h respectively.  
 
98 
 
 
Table 4.8:  Baseline clinical and demographic characteristics of seventy-four 
children with acute uncomplicated malaria treated with standard doses of 
chloroquine. 
 
 
Parameters Mean±SD 95% CI 
Number enrolled 74       - 
Age (y)  5.2 ± 2.9 4.5 - 5.9  
Range Y0.5 - 12.0       - R
No with age < 5 y 38 (51.4%)       - A
Gender ( M:F) 38:36   B     -R 
Weight (kg)  16.8 ± 6.5 LI15.2 - 18.3 
Range  
5.0 - 42.0 N       -  
O
Axillary Temperature ( C) 38.4 ± 1.1A 38.1 – 38.6 
 Range 36A.3 -D 40.5       - 
No. with Fever   IB58 (78.4%)       - 
Haematocrit (%) F  30.6±5.9 29.2 – 32.1 
Range  O 20.0 – 46.0       - 
No. with PCV < T30%Y 27 (36.5%)       - 
ParasitaemSia/µIl (GM) 52,467 38,194 – 71,944 
Range R 2,156 - 404,666  -  
VCQE concentration (ng/ml) on  916.8±89.7* 735.6 – 1098.0 
NI     day 0 (n=43) U  Range 25.6 – 2643.0 
O
Fever, temperature ≥ 37.5 C;  * Mean±s.e.m; CI, confidence interval; GM, geometric mean 
99 
 
 
 
 
 
Table 4.9:  Pharmacokinetic parameters of chloroquine determined in children with falciparum 
malaria and treated standard doses of chloroquine. 
 
Parameters Mean ±s.e.m Y 
t1/2 (h) 306.3 ± 20.9 R
95% CI 
264.4 – R348.A1 Range 
97I.2B – 958.3 
AUC0-28d (ng.h/ml)   L920.6 ± 69.6 
95% CI 
AN 781.9 – 1059.3 Range D 185.8 – 3509.2 
CL (mL/h) BA 633.6 ± 60.2 95% CI  I 513.6 – 753.5 
Range F
 O 69.0 – 2579.0 Cl= clearance, AUC0-28d =Y area under concentration-time curve from day 0 -28,    t 
1/2= half-life IT
ER
S
IV
UN
100 
 
 
4.7:  Comparison of blood chloroquine concentrations and pharmacokinetic 
parameters in children with early or delay in parasite clearance. 
Figure 4.5 illustrates whole blood chloroquine concentrations versus time in 
those children with early parasite clearance or delay in parasite clearance.  In these 
patients day 1 chloroquine concentrations were 1169.06±167.21 and 
1958.23±213.80ng/ml, respectively.  The difference between mean day 1 chlorYoqu ine concentration was not significant (P = 0.061) but higher in children witRh delay in 
parasite clearance.  There was no correlation between delay Rin Aparasite and 
chloroquine concentration (P = 0.084, r = 0.237).  Table 4.1I0 Bis a summary of the 
pharmacokinetic parameters of CQ in the children with  eLarly parasite clearance or 
delay in parasite clearance.  There was no differAentN between the pharmacokinetic 
parameters of chloroquine in children who hadD delay in parasite clearance compared 
to those whose parasites cleared earIlyB fr
Aom the peripheral blood. Although the 
chloroquine half-life in childreFn w ith delay in parasite clearance was higher 
compared to those that clea reOd parasite early (321.3±27.1 versus 219.6±25.3h, P = 
0.065).  
ITY 
ER
S
IV
UN
101 
 
 
 
 
 
 
 
 
2500  
 Early PC Y
2000  DelaAy PRC
1500 R
1000  LI
B
N
500 DA
0 A
0 5 10 B15 20 25 30
F I  Time (days)  
Figure 4.5: Mean wholeY bl o
Ood concentration-time curves of chloroquine following 
oral administration ofT 25mg/kg of chloroquine base over 3 days in children infected with PlasmodiumS faIlciparum malaria with early or delay in parasite clearance (PC).  
 
VE
R
NIU
102 
 
Chloroquine concentration
(ng/ml)
 
 
 
 
 
Table 4.10:  Pharmacokinetic parameters of chloroquine in children with acute 
uncomplicated malaria who had delay in parasite clearance after treatment with 
standard doses of chloroquine  
 RY
Parameters Children with Children withR  AP value 
PC  ≤ 2d PC > 2d IB
 n=13 n= 48  L
t1/2 (h) 219.69  ± 25.35 3N21.35  ± 27.13 0.065 
Range 
108.0 – 379.9D A97.2 – 958.3 
AUC0-28d (ng/ml.h)  728.58B  ± A122.99 1011.92  ± 95.17 0.150 Range 
F198 .7I  – 1577.5 185.8 – 3509.1 CL (mL/h) 616.84  ± 92.37 612.15  ± 75.65 0.976 
Range  OY 215.0 – 1505.1 69.0 – 1952.2 
   Mean±ISTEM,  *significant value 
ER
S
IV
UN
103 
 
 
 
4.8:  Comparison of pharmacokinetic parameters in children with or without 
pretreatment chloroquine concentration. 
Figure 4.6 illustrates whole blood chloroquine concentrations versus time in 
those children with or without CQ in their blood at enrolment.  In these patients day 1 
chloroquine concentrations were 1741.87±209.16 and 1640.35±226.95ng/ ml, 
respectively.  The difference between mean day 1 chloroquine concentratioRn wYas not 
significant (P = 0.749).  Day 2 concentrations were also similar: 1703.9A8±172.92 and 
1796.90±231.34ng/ml, respectively.  The difference betweenB daRy 2 chloroquine 
concentration was not significant (P = 0.715).  Ta blLe I4.11 summarizes the 
pharmacokinetic parameters of children with and wNithout detectable chloroquine 
concentration pre-dosing.  Despite presence ofD CQA in their blood pre-treatment there 
was no difference in the pharmacokinBeticA parameters between patients with pre-treatment CQ levels and those withou t Idetectable pre-dosing chloroquine. 
 OF  
ITY
ER
S
IV
UN
104 
 
 
 
 
 
 
 
 
  
2500 RY
CQ absent
2000 ACQ presBentR
1500  LI
1000 AND
500
BA I0
0 5 10 F 15 20 25 30
 O Time (days)  
 TY
Figure 4.6: MeaSn wIhole blood concentration-time curves of chloroquine in children 
infected withR Plasmodium falciparum malaria treated with oral doses of 25mg/kg 
chloroquiEne base over 3 days with or without chloroquine in their blood at enrolment.  
IV  
UN
105 
 
Chloroquine concentration (ng/ml)
 
 
 
 
 
 
Table 4.11:  Pharmacokinetic parameters of chloroquine in children with an d 
without pretreatment chloroquine levels following treatment with stRandYard 
doses of chloroquine (n=74). 
RA
Parameters Children without Children with IBP value 
chloroquine on chloroquine oLn 
day 0 day 0 N 
 n=31 n=43 A
t1/2 (h) 276.30  ± 23.94 A32D7.90  ± 31.57 0.228 
Range 
97.2 – 666.4  IB 108.0 – 958.3 
AUC0-28d (ng/ml.h)  877.64  ± 1F25.86 951.60  ± 79.25 0.648 
Range 
1Y85.8  
O– 3509.1 227.9 – 2640.0 
CL (mL/h) IT666.77  ± 103.53 610.41  ± 73.09 0.604 Range S 143.1 – 2579.0 69.0 – 1952.2 
Pre-dosing R CQ  916.86  ± 89.79  
concIeVntr
Eation 
             -       - 
(ng/ml) 
 25.6 – 2643.0 
URNange    Mean±s.e.m,  *significant value 
106 
 
 
4.9:  Comparison of clinical parameters and treatment outcome in children with 
or without pretreatment chloroquine concentration.   
 
Table 14.12 shows, the clinical characteristics and response to treatment in children 
with or without pretreatment CQ.  Of the 43 or 31 children with or without CQ before 
treatment, 23 (53.5%) or 16 (51.6%) responded to treatment, respectively.  In 
children with or without pretreatment CQ, 11 (25.6%), 6 (13.9%) and 3 (7.0%Y) o r 9 
(29.0%), 4 (13.0%) and 2 (6.4%) had RI, RII and RIII responses, reRspectively.  
Response to treatment in children with or without pretreatment CQR werAe thus similar 
2
(χ =0.01, P=0.9389).   B
 N 
LI
 A
4.10:  Comparison of blood chloroquine concDentrations in children with sensitive 
and resistant responses to chloroquineB. A I
Table 4.13 is a summary of the wFho le blood CQ concentrations following treatment 
in the children with sensiti veO or resistant responses to chloroquine.  Mean whole 
blood CQ concentraItiTon Yon day 3 was significantly higher in children with sensitive response compaSred with children with resistant responses (1856.7±204.7 vs 
1276.3±1E38.R2ng/ml, P = 0.011). 
IV
UN
107 
 
 
 
Table 4.12:   Comparison of clinical parameters and treatment outcome in 
children with acute uncomplicated malaria who had or without pretreatment 
chloroquine concentration (n=74). 
 
Parameters Children without Children with P value 
chloroquine on day 0 chloroquine on day 0   Y 
 n= 31 n= 43 R
Age (y) 5.0  ± 2.6 5.3  ± 3.1 0.A64 
Range R
1.0 – 12.0 0.5 – 12.0 IB
Sex (M:F) 17:14 21:22  L  
Weight (Kg)  15.9 ± 5.2 17.3  A± 7.N2 0.38 
7.5 – 30.0 6.D0 – 42.0 
FCT (d) 1.8 ± 1.1 BAI 1.9 ± 1.0 0.56 1 – 5 F 1 – 5 
PCT (d) 3.3 ± 1. 1 O 3.5 ± 1.3 0.56 
2.0 –Y 7.0 2.0 – 6.0 
Parasitaemia/µl TS I50,839 53,987 0.65 (GMPD) R 3,125 – 313,538 2,156 – 404,666 Range 
RespIoVnse
E    
SNensitive 16 (51.6%) 23 (53.5%) 0.77 URI 9 (29.0%) 11 (25.6%) 
RII 4 (13.0%) 6 (13.9%) 
RIII 2 (6.4%) 3 (7.0%) 
  
GMPD, geometric mean parasite density, Mean±s.d
108 
 
 
Table 4.13:  Whole blood chloroquine concentrations in children with 
uncomplicated falciparum malaria with sensitive or resistant response after 
administration of 25mg/kg chloroquine base (n=74). 
*Mean 
Time Whole blood chloroquine concentration (ng/ml) 
whole 
(days) 
blood 
 Sensitive  n=39 Resistant  n=35 P value chlor oqu
0  496.47 ± 97.33* 573.22 ± 114.33 0.609 iYne 
0.0 – 2643.0** 0.0 – 2119.0 Rconcentr
ation 
1 1814.96 ±  219.88 1560.11 ± 212.00 0.687A 
±s.e.m,  
375.7 – 5192.0 74.6 – 4680.8 BR ** 
2  1873.61 ± 223.98 1592.08 ± 148.76 I 0.312 Range; 
500.7 – 5378.1 732.9 – 4166.0  L RI= 
3 1856.65 ± 204.70 1276.32 ± 1A38N.22 0.011 parasitaeD mia 472.5 – 5776.3 135.0 – 3086.2 disappea
7 1427.26 ± 254.75 910A.20 ± 89.14 0.087 ra but 
43.7 – 5920.3 IB103.6 – 1892.4 reappear
14 1214.36 ±170.27  1131.82 ± 244.01 0.789 s within 
335.8 – 2319.6  O
F 254.5 – 1625.6 7 to 14 
days; 
21 1090.23 ± Y259.37 757.24 ± 147.57 0.308 RII= 77.7 – 2T994.4 248.5 – 1625.9 decrease 
28 86S9.94I ±165.21 609.50 ± 120.68 0.328 of R34.6 – 2096.3 151.6 – 1067.0 parasitae
OutcomeE    mia but no 
SensIitVive 39  complet
RNI - 20 (57.1%) e URII - 10 (28.6%) clearanc
RIII - 5 (14.3%) e from 
peripher
al blood; RIII= no pronounced decrease or increase in parasitaemia at 48hr after treatment; S= 
sensitive.   
109 
 
 
4.11:  Comparison of chloroquine pharmacokinetic parameters in children 
whose infection responded or failed to respond to treatment. 
 
Table 4.14 is a summary of the pharmacokinetic parameters of the children with 
sensitive or resistant responses to chloroquine.  The AUC0-28d of chloroquine was 
significantly higher in children with sensitive response compared with those with 
resistant responses (1052.1 ± 116.3 vs 774.1± 62.9 ng/ml.h, P = 0.01).   The AUYC0 -28d 
values in children with sensitive response was approximately 1.5 fold Rthose with 
resistance response (Figure 4.7 and 4.8).  RA
 
 LI
B
 
4.12:  Pharmacokinetic risk factors associated Nwith chloroquine treatment 
failure 
 DA
Table 4.15 shows the univariate and mBultAivariate analysis of pharmacokinetic risk 
factors for chloroquine treatmenFt fa ilIure.  Day 3 CQ concentration ≤ 1750ng/ml 
{Crude odd ratio (COR), 4. 0O8; 95% CI, 1.13 – 14.64; P = 0.027} and AUC0-28d ≤ 
950ng/ml.h (COR, 2.8T9; Y95% CI, 1.05 –7.93, P = 0.037) were significantly associated with a risk of treSatmIent failure.  
 R
HowIeVver
E, if a P value less than 0.3 was taken (‘falsed’) to indicate significant 
dNifference in a univariate analysis, a terminal elimination half-life less than 220h U{Adjusted odds ratio (AOR), 0.28; 95% CI, 0.08  – 0.98, P = 0.047}, and an AUC 0-
28d  less than 950ng/ml.h (AOR, 4.12; 95% CI,1.09 – 15.52, P = 0.036} were 
independent predictors of chloroquine treatment failure (Table 4.16). 
 
 
110 
 
 
 
Table 4.14:  Pharmacokinetic parameters of chloroquine determined in children 
with acute uncomplicated falciparum malaria who had sensitive or resistant 
responses after treatment with standard doses of chloroquine (n=74). 
  
Parameters Sensitive n=39 Resistant n=35 P value  
t1/2 (h) 293.8 ± 28.8 320.2 – 31.0 0.97 Y
95% CI 235.5 – 352.0 257.2 – 383.2 R
Range 97.2 – 958.3 114.0 – 875.0 A
   BR
AUC0-28d (ng/ml.h)  1052.1 ± 116.3 774.1 ± 62.9L I 0.01* 
95% CI 816.6 – 1285.0 646.33 – 9 01.8 
Range 185.8 – 3509.2 202
 D A
.9 –N 1890.8 
CL (mL/h) 647.9  84.5 A 617.1 ± 86.7 0.46 
95% CI  476.9 – 818 .9I B 440.7 – 793.6 Range 69.01 – 1F952.2 107.2 – 2579.0 
Mean±s.e.m, * significan tO
SI
TY
R
IV
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111 
 
 
 
 
 
 
 
 
  
2500 Y
All patients R
RespondedA to treatment
2000 Fail
LIB
ed Rtreatment
N 1500
DA
1000 A
 IB
500  O
F
0 SI
TY
0 5 10 15 20 25 30
ER Time (days)
 V
FNiguIre 4.7: Mean concentration-time curve of chloroquine for all patients, whose Uinfection responded or failed to respond to treatment after oral standard doses 
chloroquine base given over 3 days. 
 
112 
 
Chloroquine concentration (ng/ml)
 
 
 Table 4.15: Pharmacokinetic predictors of treatment failure in children with 
acute uncomplicated malaria treated with standard doses of choroquine. 
     No  of  children Univariate analysis Logistic  regression 
analysis  
 Examined Failing Crude odds ratio p value Adjusted odds P 
treatment (95% confidence ratio (95% CRI) YValue by day 14 interval) 
CQ      A  
concentration 
     R  
(ng/mL) on 
day 3      IB  
>1750 18 4 1   L1  
≤1750 39 21 4.08 (1.13 – 14.64) 0.0N27 3.25  (0.86 – 12.23) 0.081 
t1/2 (h)     A   
>220 42 22 1 D    
≤ 220 32 13 0.62 (0.24 – 1A.57) 0.300 - - 
AUC0-28d       
(ng/ml.h) B
   I    
>950 F 26 8 1  1  
≤ 950 
48 27  O2.89  (1.05 –7.93) 0.0307 3.25  (0.93 – 11.27) 0.063 
ITYS
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113 
 
 
Table 4.16: Pharmacokinetic risk factors of treatment failure in children with 
uncomplicated malaria treated with standard doses of chloroquine (P value 0.3 
is considered). 
     No  of  children Univariate analysis Logistic  regression 
analysis 
 Examined Failing Crude odds p Adjusted odds P 
treatment ratio (95% value ratio (95% CI) Value 
by day 14 confidence 
interval)  
CQ      Y 
concentration 
     R  
(ng/mL) on 
day 3      A  
>1750 18 4 1  1 R  
≤1750 39 21 4.08 (1.13 – 14.64) 0.027 3.057I  B(0.77 -  12.13) 0.112 
t1/2 (h)      
N 
 L  
>220 42 22 1  1  
@
≤ 220 32 13 0.62 (0.24 – 1.57) 0.3A00  0.28  (0.08  – 0.98) 0.047* 
AUC0-28d       
(ng/ml.h) D
   A    >950 
26 8 1 IB  1  ≤ 950 48 27 2.89F  (1. 05 –7.93) 0.030 4.12  (1.09  – 15.52) 0.036* @false to MultiOple regression; * significant 
ITY
 
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114 
 
 
 
 
 
 
. 
 CQ present  
 
 CQ absent AR
Y
 
BR
 LI
DA
N
A
IB   
    TimFe (d ays) 
Figure 4.8: Decline of chloroqOuine concentration in children with or without 
chloroquine in their pre-tYreat ment whole blood samples following oral administration 
of 25mg/kg of chlorIoqTuine base over 3 days. S
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115 
 
Chloroquine concentration (ng/ml) 
 
 
  
Study 3: Development of a simple cost effective high performance liquid 
chromatography assay of sulphadoxine in whole blood spotted on filter paper 
for field studies. 
 
4.13:  Chromatography techniques  
Standard curves from sulphadoxine spiked blood added to filter paper wereY linear 
over the concentration ranged studied.  Linear regression analysis yieldAed cRorrelation 
coefficient r > 0.99 (n = 6, Figure 4.9).  Sulphadoxine peak waBs wRell resolved from 
the internal standard (sulizoxasole) at the calibration  rLangIes of 0 – 60 µg/ml 
(Appendix Table 4.b).  The retention times (tR) of sulpNhadoxine and internal standard 
(IS) were 6.3 and 7.2min respectively (Figure D4.10A).  The separation chromatograms 
of sulphadoxine and the internal standaArd from spiked whole blood samples 
corresponded with those of blood sa mIpBles obtained from a patient at time 0 before 
drug administration and day O3 afFter an oral standard single dose of sulphadoxine-
pyrimethamine (SP) (25Ymg /kg body weight of sulphadoxine and 1.25mg/kg body 
weight of pyrimethaImTine (Figure 4.10 A, B and C respectively).    S
ER
NI
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U
116 
 
 
3
2
Y R
1
BR
A
N 
LI
0
0 10 20 30 4D0 A 50 60 70Sulphadoxine concAentration (µg/ml)B
F I  
Figure 4.9: Sulphadoxine  Ostandard curve for extrapolation of sulphadoxine 2
concentrations from unknYown blood sample spotted on filter paper (r =0.99518).  IT
ER
S
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117 
 
Peak height ratio
Chromatogram - Cgm006 30-11-05 cali .20.0ug ( \siju\ )
 
1125
 
984
A 
844
2 
1 
703
Y 
562 R
00:00 02:24 04:48 07:12 09:36 12:00
Chromatogram - Cgm015 11-01-06 mc125d0 ( \siju\ ) Time [mm:ss]  A
640 R
B 
2 B
600
N 
LI
559
A
518 D
478 A
00:00 02:24 04:48 07:12 09:36 12:00
Time [mm:ss]  B  
Chromatogram - Cgm010 06-01-06 mc135d1 ( \siju\ )
F I713
C O
630  
ITY 2 546
RS 1 
462
VE
N37I8 00:00 02:24 04:48 07:12 09:36 12:00U Time [mm:ss]    Figure 4.10: Chromatograms showing peaks after extraction of sulphadoxine (1) and 
internal standard (2) from drug free whole blood spiked with 60µg/ml sulphadoxine 
(A), day 0  (B) and day 3 (C) of sample collected from a patient who was 
administered with a standard dose of sulphadoxine/pyrimethamine. 
118 
 
Signal [mV] Signal [mV] Signal [mV]
 
  
4.14:  Recovery, calibration curves and reproducibility 
The extraction recoveries for 25µg/ml, 60µg/ml and 100µg/ml of sulphadoxine were 
82.66±4.0 (n=6), 81.02±3.24 (n=5) and 85.60±1.9 (n=5) per cent respectively (Table 
4.17).  The intra-day recovery deviation at 60µg/ml and 100µg/ml of SDX were 3. 7% 
and 4.6% respectively (n=5) (Table 4.18). The intra-day precision was <5.0Y% and 
inter-day accuracy ranged from 4.1 to 5.3% respectively.  The limit oAf deRtection of 
sulphadoxine defined as a concentration giving a peak four times theR baseline noise in 
all biological fluids was 120ng/mL at 0.05 absorbance units full Bscale (aufs).  
N 
LI
A
BA
D
F IO
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119 
 
 
 
Table 4.17:    Percentage recovery of sulphadoxine from whole blood 
samples collected on filter paper. 
 
 Concentration % Recovery N  
(µg/mL)    S.D Y
Whole 25 82.64.0 6 R
blood A
 60 81.03.2 5B R 100 85.61.9 LI5 
SD= standard deviation 
 AN
 
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120 
 
 
 
Table 4.18:   Precision of analytical method for sulphadoxine determination 
using whole blood spotted on filter paper. 
 
 Concentration C.V (%) n 
 (µg/mL) 
Intra-Assay Sulphadoxine    
 60 3.73.0 5 Y
 100 4.62.5 5 AR    R
Inter-Assay  Sulphadoxine   IB
 10 6.02.0  LN 5 
 100 4.12.0A 6 
CV= coefficient of variation D
BA
OF
 I
 
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121 
 
 
4.15:  Interference 
There was no interference from endogenous compounds in the biological sample.  
There was no interference with the peaks of sulizoxasole (IS) and other commonly 
used antimalarial, analgesic and anti-infective drugs. 
  
4.16:  Clinical application RY
The concentration-time curve in whole blood of a patient who was admAinistered with 
a standard single oral dose of sulphadoxine-pyrimethamine RI B(SP) (25mg/kg body 
weight of sulphadoxine and 1.25mg/kg body weight of py rLimethamine i.e 500mg of 
sulphadoxine and 25mg of pyrimethamine) is shNown in Figure 4.11.  The 
pharmacokinetic parameters of SDX in the indiDvidAual are shown in Table 4.19.  Peak 
blood concentration of 212.02µg/ml BwasA reached after day 1.  The calculated 
elimination half-life was 3.50d wFhil e Ithe areas under the concentration time curve 
(AUC0-28d) were 884.84µg/m lO.d.  The profile show the applicability of the method for 
measuring SDX in whoYle blood dried on filter. The pharmacokinetic result agrees 
with previous reSportIs
T (Dzinjalamala et al., 2005).   The method was not sensitive to 
detect pyrimRethamine, although pyrimethamine peak was detected and separated 
duringV thEe standard calibration preparations. This insensitivity may be due to the fact 
tNhat Iamount of pyrimethamine in tablet was small (1:20 of sulphadoxine) and only a Usmall volume of blood used for the analysis. 
 
122 
 
 
  
 
 
250
RY
 
200
BR
A
150  LIN
100
DA
50
 IB
A
0 OF0
ITY
 10 20 30
Time (days)
Figure 4.11:  CoSncentration–time curve for sulphadoxine in whole blood following 
single oraEl doRse of sulphadoxine-pyrimethamine (25mg/kg body weight of 
sulpIhaVdoxine and 1.25mg/kg body weight of pyrimethamine). 
UN
123 
 
Sulphadoxine concentration (g/ml)
 
 
Table 4.19:  Demographic and pharmacokinetic parameters of a patient who 
received a standard dose of sulphadoxine-pyrimethamine. 
 
Parameters Mean 
Age (year) 6.0 Y  
Weight (Kg) 16.0 R
 A
t 1/2 (d) 3.50 
 BR
Tmax (d) 1.00 
  LI
Cmax (µg/ml) 212.02 N
 A
AUC0-28d (µg/ml.d) 88D4.84 
 
Cl (ml/d) A
  IB
524.50 
 
AUC F0-28, area under concentratioOn time –curve from time 0 to day 28, Cmax, peak blood 
concentration; Tmax, time tYo p eak blood concentration; t1/2 elimination half-life; Cl, clearance.T
RS
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124 
 
 
Study 4: Pharmacokinetic disposition of sulphadoxine in children with 
uncomplicated P. falciparum malaria treated with standard dose of 
sulphadoxine-pyrimethamine. 
 
4.17:  Study population 
A total of 74 patients with acute uncomplicated malaria were randomly selected i nto 
the study. The clinical and demographic parameters of the children areR shoYwn in 
Table 4.20. The geometric mean parasite density was 48,953 (RangRe, 2A011- 461,333  
asexual parasite/µl of blood). The mean age of the children waIsB 5.07±3.02 years and 
their mean weight was 16.55±7.7kg.   L
AN
 
AD
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B
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125 
 
 
Table 4.20: Baseline clinical and demographic characteristics of children with 
acute uncomplicated falciparum malaria treated with standard dose of 
sulphadoxine-pyrimethamine (n=74). 
 
Parameters Mean±SD 95% CI 
Number enrolled 74       - Y 
Age (y)  5.2 ± 3.0 4.5 - 5.9 R
Range 0.5 - 12.0       R- A
No with age < 5year 38 (51.4%) IB      - 
Gender ( M:F) 35:39   L       - 
Weight (kg)  16.9 ± 7.A6 N 15.1 - 18.7 
Range 4.0 –D 38.0       -  
O
Axillary Temperature ( C) B3A8.2 ± 1.0 37.2 – 38.4 
 Range F I 36.1 - 40.5       - 
No. with fever   O 57 (77.0%)       - 
Haematocrit (%T) Y  30.7±4.0 29.1 – 32.3 
Range SI 25.0 – 38.0       - 
No. wiRth PCV < 30% 27 (36.5%)       - 
IVPar
Easitaemia/µl (GM) 48,953  
N Range 2011- 461,333  -  U SDX Concentration (µg/ml) on 8.9 ± 3.5* (n=13)  
day 0  
 Range 2.6 – 168.3 
O
Fever, temperature >37.5 C; * Mean±s.e.m; CI, confidence interval; GM, geometric mean 
126 
 
 
4.18:  Pharmacokinetic parameters of sulphadoxine in children with 
uncomplicated falciparum malaria after administration of standard dose of 
sulphadoxine-pyrimethamine. 
 Thirteen patients (17.6%) had pretreatment levels of SDX and their data were 
excluded from the final analysis. The mean SDX concentration was 8.9±3.5µg/ml 
(range, 2.6 – 168.3µg/ml) (Table 4.20). The patients were therefore divided Yinto  4 
groups for the analysis of the data; the 74 children that were enrolled intoR the study; 
45 with delay in parasite clearance; 13 with detectable preR-treAatment SDX 
concentration and 61 children without SDX before treatment.  
LIB
 N 
Table 4.21 is the summary of the pharmacokDineAtic parameters of all the patients 
irrespective of pre-treatment SDX blood coAncentration at enrolment.  The mean tmax 
and elimination half-life of SDX in ca pIiBllary whole blood were 2.1±0.1 and 5.5±0.3d. 
The peak whole blood conceOntraFtion (Cmax) ranged from 25.5 to 745.69µg/ml with 
mean of 214.9±16.9µg/mYl.   The AUC0-28d varied between 91.19 and 3768.62µg/ml.d 
(mean 1252.9±106.1IµTg/ml.d).  The clearance was 437.1±39.9ml/d (ranged, 86.2 – 
1555.3). RS
 
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127 
 
 
Table 4.21: Pharmacokinetic parameters of sulphadoxine in seventy-four 
children treated with standard dose of sulphadoxine-pyrimethamine. 
 
Parameters Mean ±s.e.m 
Cmax (µg/ml) 214.9 ± 16.9  
Range 25.5 – 745.69 Y
Tmax (d) 2.1 ± 0.1 AR
Range 1 – 5 R
t1/2 (d) 5.5 ± 0.3I B
Range 
1.1 –  1L7.0 
AUC0-28d (µg/ml.d)  A125N2.9 ± 106.1 
Range 
AD 91.1 – 3768.6 
CL (mL/d) IB 437.1 ± 39.9 Range F 86.2 – 1555.5 
Day 3 SDX concentration   (µOg/ml) 184.9 ± 20.0 
Range 
TY 19.7 – 856.9 
Day 7 SDX concentIration (µg/ml) 85.0 ± 11.2 
Range RS 3.0 – 368.9 
DayI 1V4 S
EDX concentration (µg/ml) 43.9 ± 6.6 
RNange 3.7 – 151.6 U 
128 
 
 
 4.19: Comparison of blood sulphadoxine concentrations and pharmacokinetic 
parameters in children with early parasite clearance or delay in parasite 
clearance. 
 
Figure 4.12 illustrates whole blood sulphadoxine concentrations versus time in th ose 
children with early parasite clearance or delay in parasite clearance.  In these pYatients 
day one sulphadoxine concentrations were 127.0±19.5 or 215.2A±25R.5 µg/ml, 
respectively.  The difference between mean day 1 SDX cRoncentration was 
significantly (P = 0.049) higher in children with delay in LparIasBite clearance.  Table 
4.22 is a summary of the pharmacokinetic parameterNs of  SDX in the children with 
early parasite clearance or delay in parasite cleaArance.  There was no difference 
between the pharmacokinetic parameters ofA suDlphadoxine in children who had delay 
in parasite clearance compared to th oIsBe that their parasites cleared early from the 
peripheral blood.  Afterwards, thFe concentrations of SDX in children who cleared 
parasite early were higheYr, a lt
Ohough not statistically significant. The mean maximum 
concentration of SDIXT (Cmax) obtained in children who cleared parasite early was high 
but not significanSt. 
 ER
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129 
 
 
 
 
 
 
 
300
Early PC Y
Dealy PC AR
200
LIB
R
N 
100 A
BA
D
0 I
0 3 6 9 F 12 15 18 21 24OTime (days)
ITY
  
Figure 4.12: Mean whole blood concentration-time curves of sulphadoxine in 
children infeRctedS with Plasmodium falciparum malaria with early or delay in parasite 
clearance. 
VE
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130 
 
Sulphadoxine concentration (µg/ml)
 
 
Table 4.22:  Comparison of pharmacokinetic disposition of sulphadoxine in 
children with and without delay in parasite clearance (n=65) 
Parameters Children with PC  Children with  P value 
≤ 2d PC > 2d  
 (n=20) (n= 45) RY
tmax (d) 1.95  ± 0.23 2.09  ± 0.15 0A.611 
Range R
1.0 – 4.0 1.0 – 4.0 IB
Cmax (µg/ml) 218.49  ± 38.65 194.1N0  ±  
L18.64 0.522 
Range 
137.5 – 299.4 D1A56.4 – 231.7 
t1/2 (d) 5.01  ± 0.53 BA 5.59  ± 0.55 0.524 Range 
1.4 – 1F1.0  I 1.16 – 17.0 
AUC0-28d (µg/ml.d)  1 1O02.45  ± 187.78 1011.92  ± 95.17 0.150 
Range 
ITY91.1  – 2568.9 185.8 – 3509.1 
CL (mL/d)R S 616.84  ± 92.37 612.15  ± 75.65 0.976 
Range 
IV
E 215.0 – 1505.1 69.0 – 1952.2 
   Mean±s.e.m,  *significant value 
UN
131 
 
 
4.20:  Comparison of disposition of sulphadoxine in children with and without 
detectable blood levels of sulphadoxine at enrolment before treatment. 
 
Figure 4.13 illustrates whole blood sulphadoxine concentrations versus time in those 
children with or without SDX in their blood at enrolment.  The mean concentration of 
SDX at presentation in 13 patients who had detectable SDX level before treYatm ent 
was 8.90±3.5µg/ml (range 2.99 - 168.3µg/ml). In children with or withoRut SDX in 
their blood at enrolment, day 1 SDX concentrations were R18A8.94±22.2 or 
274.04±71.4µg/ml (P=0.225) respectively.  Day 2 concentratiIoBns were also similar; 
211.57±18.1 or 261.69±45.4µg/ml (P=0.343) betweenN chi ld
Lren with or without SDX 
in their blood at enrolment.  Comparison of dispAosition of SDX in children with 
pretreatment SDX levels and those without ASDXD showed a similar disposition profile; 
Cmax (P=0.325), Tmax (p=0.128), Cl (PI=B0.377), day 3 concentration (P=0.343), day 7 
concentration (P=0.249), and daFy  14 SDX concentration (P=0.245). Table 4.23 
summarizes the pharmacoki neOtic parameters of children with and without detectable 
sulphadoxine concentTratiYon pre-treatment.  However, a trend towards a shorter mean 
elimination half-SlifeI (3.91±0.40d versus 5.85.69±0.44d, P=0.063) and a higher AUC0-
28d (1660E.33R±172.59 versus 1161.62±121.39 µg/ml.d, P=0.069) was observed in 
patieInVts with pre-treatment SDX levels.  
 
UN
132 
 
 
Table 4.23: Pharmacokinetic parameters of sulphadoxine in children with and 
without sulphadoxine in pretreatment whole blood samples. 
Parameters Children without Children with P values 
sulphadoxine on sulphadoxine on 
day 0  (n=61) day 0 (n=13) 
t1/2 (d) 5.85±0.44 3.91±0.40 0.063 
Range 1.16-17.01 2.31-7.04 
Cmax (µg/mL) 207.75±19.31 254.10±27.24 0.325 
 
Range Y25.5-856.9 112.6-413.6 R
t 0.128 max (d) 2.19±0.14 1.66±0.28 A
Range 1.0-5.0 0.0-3.0 R
-1
AUC0-28d (µgmL .d)  1161.62±121.39 1660.33±17I2.B59 0.069 
Range 91.1-3768.6 221.6-2 5L40.9 
Cl (mL/d) 454.26±47.19 36N3.16±59.03 0.377 
Range 86.2-1555.3 DA96.0-936.58 Geometric mean 47081.70 59293.28 0.925 
(parasite/µL blood)  A
Range 
2011 -4 6IB
 
1333 6973-33684 
Day 0  Conc. (µg/ml) - 
OF
8.90±3.54 - 
Range  2.62-168.32 
Day 3 Conc. (µgT/mlY) 175.81±22.59 225.17±42.67 
0.343 
Range I 19.71-856.98 60.45-636.54 
Day 7  ConcS.(µg/ml) 78.86±13.07 112.74±47.44 0.249 
Range 
ER 19.71-856.98 66.00-205.32  
IRVesponse 
   
ACPR 50 (82.0%) 6 (46.2%) 
N ETF 2 (3.2%) 2 (15.4%) U LCF 9 (14.8%) 5 (38.4%) 
 Values are in Mean±s.e.m 
133 
 
 
 
 
 
 
 
 
Y 350
300 SDX abseRnt A
R
250 SDXI pBresent L200 N
150
DA
100
50  IB
A
0 F
0 5  O 10 15 20
Time (days)
ITY  Figure 4.13: MeSan whole blood sulphadoxine concentration-time curves in children 
with PlasEmoRdium falciparum malaria with or without sulphadoxine in their blood at 
enroIlmVent. 
 
UN 
 
 
 
 
134 
 
Sulphadoxine concentration (µg/ml)
 
4.21: Pharmacokinetics of sulphadoxine in 61 children without pretreatment 
sulphadoxine at enrolment. 
The characteristics and demographic parameters of the 61 children without 
pretreatment SDX are shown in Table 4.24.  Thirty one (51.6%) children were aged < 
5 years. The geometric mean parasite density was 472,467 (range 2011 - 461,333 
asexual parasite/µl).  Infection in 50 (81.9%) of the patients exhibited adequ ate 
clinical response (ACPR), whereas 3.3% (2/61) of the patient exhibRitedY early 
treatment failure (ETF) and 14.8% (9/61) had late parasitological faiRlureA (LPF). B
 LI
DA
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A
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Table 4.24:  Clinical and laboratory data at enrolment of children with acute, 
uncomplicated falciparum malaria treated with standard dose of sulphadoxine-
pyrimethamine without pre-treatment sulphadoxine levels. 
 
Parameters    Sulphadoxine 
     
Number enrolled   61 Y
Gender (no. of males: no of females)  31:30 R
Age (yr) 5.16±3.0 A
Range  0.5-12.0 BR
Weight (kg) 16.04±6.9 LI
Range     4.0-38.0  
O
Axillary Temperature ( C)  38.16±1.1 N
Range    36.1D-40A.5 
Geometric Mean (Asexual 4A7,081 
parasite per µl of blood) B 
Range F I 2,011-461,333  
Cure rate on day 14 (% ) O  ACPR  Y 81.9 (50/61)  ETF  IT 3.3 (2/61) LPF S 14.8 (9/61) 
Values are inR Mean±Standard deviation,   
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136 
 
 
4.22:  Comparison of blood sulphadoxine concentrations in children with 
sensitive and resistant responses to sulphadoxine-pyrimethamine. 
 
Table 4.25 is a summary of the whole blood SDX concentrations following treatment 
in children whose infection were sensitive or resistant to sulphadoxine-
pyrimethamine. There was no difference in whole blood sulphadoxine concenYtrat ion 
in children whose infection were sensitive or resistant to treatment.  FRigure 4.14 
shows the mean concentration-time curves for the disposition of RSDAX in children 
whose infections were sensitive or resistant to SP treatmeInBt.  There was inter-
individual variation in the achieved sulphadoxine concNentr at
Lion in the children.    
 
DA
4.23:  Comparison of disposition of sulAphadoxine in children whose infection 
responded or failed to respo nIdB to standard dose of sulphadoxine-
pyrimethamine. F
 O
Table 4.26 is a summarYy of the pharmacokinetic parameters of the children whose 
infection were s TSensIitive or resistant to sulphadoxine-pyrimethamine.  The t1/2 of sulphadoxineR in children whose infections were sensitive or resistant to SP was 
5.98±V0.5 Eor 5.26±1.0d respectively.  The Cmax of SDX ranged from 42.1 to 
7N45.I69µg/mL and 25.5 to 565.12µg/mL in patients whose infections were sensitive Uand resistant to sulphadoxine-pyrimethamine.  There was no difference between the 
mean peak sulphadoxine concentration (Cmax) in patients who failed to respond to 
treatment (163.09±30.3µg/mL) and those whose infection were sensitive 
(217.78±22.5µg/mL) to treatment.  
137 
 
 
Table 4.25: Whole blood sulphadoxine concentration in children with 
uncomplicated falciparum malaria with sensitive or resistant response after oral 
administration of a standard dose of sulphadoxine-pyrimethamine (n=61). 
*
Time Whole blood sulphadoxine concentration (µg/ml) Mean±
(days) s em 
 Sensitive  (n=50) Resistant  (n=11) P value RYwhole 
0  0.0 0.0 - A blood 
1 191.03 ±  23.43* 171.65 ± 81.15 RIB0.791 SDX 
22.3 – 565.1** 62.1 – 413.1  L concen
2  215.60 ± 20.37 192.72 ± 42
82.3 – 421.3 49.5 – 2D70.A
.73N 0.644 tration,  
9 ** 
3 179.06 ± 25.83 B157A.53 ± 38.23 0.737 Range 
19.7 – 856.9 F I39.2 – 347.9 
7 84.24 ± 14.92  O 51.95 ± 23.32 0.365 
3.0 – 368.9Y 3.7 – 155.5 
14 50.21 I±TS  8.85 14.59 ± 5.95 0.151 R4.7 – 151.6 3.7  – 24.1 
21 VE 35.09 ± 11.17 8.40 ± 6.25 0.310 
NI 2.6 – 81.8 2.16 – 14.66 U
138 
 
 
Table 4.26: Pharmacokinetic parameters of sulphadoxine in children without 
pre-treatment sulphadoxine with uncomplicated falciparum malaria who had 
sensitive or resistant responses to oral administration of sulphadoxine-
pyrimethamine (n=61). 
 
Parameters Sensitive (n=50) Resistant (n=11) P value   
Cmax (µg/ml) 217.78 ± 22.5 163.09 ± 30.35 0.277 RY
Range 42.1 – 745.6 25.5 – 347.9 A
T Rmax (d) 2.10 ± 0.14 2.63 ± 0.41 IB 0.148 
Range 1.0-5.0 1.0 – 5.0  L
t1/2 (d) 5.98 ± 0.50 5.2A6 ± N1.01 0.540 Range 1.1 – 17.0 D1.2 – 13.3 
AUC0-28d (µg/ml.d) 1196.18 ± 131.B83 A 1013.93 ± 314.00 0.561 
Range 91.1 – 36F79 .1I 125.4 – 3768.6 
CL (mL/d) 437 .O45  49.16 531.61 ± 140.81 0.450 
Range Y86.2 – 1356.1 98.3 – 1555.3 
Parasite dSensIit
Ty 47260 46311 0.830 
(GM) R   
RangeV E 2011 – 461,333 2574 – 231,333 
NIU
139 
 
 
 
 
 
 
 
300 Responded to treatmenRt YFailed treatment
RA
200 B
N 
LI
A
100 AD
F I
B
0 O
0 5  10 15 20
ITY Time (days)S  
Figure 4.14.: MEeanR whole blood concentration-time profile of sulphadoxine in children 
whose infecItVion responded and whose infection failed to respond to treatment after oral 
adminisNtration of standard dose of sulphadoxine-pyrimethamine (25mg/kg S-1.25mg/kg P). U
140 
 
Concentration of Sulphadoxine
(µg/ml)
 
4.24: Relationship between age and sulphadoxine pharmacokinetic parameters 
in children treated with standard oral dose of sulphadoxine-pyrimethamine. 
 
Figure 4.15 shows the mean concentration-time profile of sulphadoxine in children < 
5 years and ≥ 5years who took oral dose of sulphadoxine-pyrimethamine for  the 
treatment of uncomplicated malaria during infection. The mean SDX concenYtration 
on day 1 (126.9±30.5 versus 235.9±27.9µg/ml, P=0.015), day 3 (98.A1±1R5.9 versus 
245.1±35.7µg/ml, P=0.001) and day 7 (54.1±10.5 versus 122.5±27R.7µg/ml, P=0.01) 
were significantly higher in children older than 5 years coLmpIarBed to those less than 
5years of age (Table 4.27). N 
 DA
Table 4.28 describes the effect of ageB onA disposition of sulphadoxine in children 
infected with Plasmodium falciparFum . I A significant difference in disposition of SDX 
was observed between child reOn younger and older than 5 years of age.  The Cmax of 
SDX in ≥5 years age groYup (295.7 ± 28.7 µg/ml) was significantly (p<0.001) higher 
than age group Sof cIh
Tildren <5years (125.4 ± 15.2µg/ml).  The time (tmax) taken to 
reach this peRak concentration were similar (2.0 ± 0.18d versus 2.4± 0.2d, p=0.160).  
The eVxteEnt of exposure (AUC0-28d) of SDX obtained in children ≥5 years of age 
(N156I2.9 ± 202.3µg/ml.d) was significantly higher (p<0.001) compared to the age Ugroup less than 5 years (812.0 ± 112.7µg/ml.d) respectively. The values of Cmax and 
AUC in older children were twice as high as those in younger children.  In contrast, 
the mean clearance of SDX was significantly lower in older children (334.3 ± 45.8 
versus 574.1±76.8 ml/d, P=0.010).  However, response to treatment in the two age 
141 
 
 
groups was not significantly different (P=0.352) though a higher proportion of 
children below 5 years failed treatment with SP.   
 
RY
 
RA
 LI
B
AN
BA
D
F I
 O
SI
TY
VE
R
UN
I
142 
 
 
Table 4.27: Whole blood sulphadoxine concentration in children with 
uncomplicated falciparum malaria with sensitive or resistant responses after 
administration of standard oral dose sulphadoxine-pyrimethamine (n=61). 
 
  Whole blood sulphadoxine concentration (µg/ml) 
Time (days) <5 years  (n= 31) ≥ 5 years  (n=30) P value Y 
0  0.0 0.0 - R
1 126.99 ±  13.09* 235.93 ± 27.92 0.R015A 
36.1 – 413.1** 98.7 – 565.1 IB
2  182.57 ± 40.75 225.36 ± 20.39  L 0.344 
49.1 – 312.1 103.2 – 421A.3 N
3 98.10 ± 15.94 245.19 D± 35.79 0.001 
22.1 – 347.9 
 IB
191A.7 – 856.9 
7 54.17 ± 10.59 F 122.54 ± 27.70 0.010 
3.0 – 217.7  O 17.3 – 368.9 
14 38.64T ± Y9.01 57.61 ± 15.52 0.268 
S3.7I – 115.3 12.3  – 151.6 
21 R 28.28 ± 13.62 30.67 ± 11.69 0.930 
VE 14.6 – 41.9 10.2– 81.6 
NI *Mean±sem whole blood SDX concentration,  ** Range U
143 
 
 
Table 4.28:  Relationship between age and pharmacokinetic parameters of 
sulphadoxine in children with uncomplicated falciparum malaria treated with 
standard sulphadoxine-pyrimethamine (n=61). 
 
Parameters <5 year ≥5 Year P value 
 
 (n=31) (n=30)  
Cmax (µg/ml) 125.45 ± 15.22 295.72 ± 28.72 0.0Y00 
Range 25.5 – 347.9 98.0 – 745.6 R
Tmax (d) 2.00 ± 0.18 2.40 ± 0.21 A 0.160 
Range 1.0-4.0 1.0 – 5.0B Rt1/2 (d) 5.61 ± 0.58 6.10 ± 0I.70 0.593 
Range 1.2 – 13.8 1.1  –L 17.0 
AUC0-28d (µg/ml.d) 812.09 ± 112.77 AN1562.93 ± 202.3 0.001* Range 91.1 – 2194.5 222.9 – 3768.6 
Cl (mL/d) 574.14  7A6.84D 334.39 ± 45.88 0.010* 
Range 86.2 – 1B555.3 86.6 – 867.9 
Parasite density 3854 0I 57133 0.891 
(GM)   F  
Range OY 2011 – 461333 2574 – 231,333  IT    Parasite S reduction 2.29 ± 0.98 3.32 ± 0.62 0.001* ratio (PRR)  
REespRonse    
IVACPR 24 (77.4%) 26 (86.7%)  N ETF 1 (3.2%) 1 (3.3%)  U LPF 6 (19.4%) 3 (10.0%) 0.352 
144 
 
 
 
 
 
 
 
300 Y 
 children >A 5 RyearschildreRn < 5 years200 B
 LI
100 N
DA
0 A
0.0 2.5 5.0 B 7.5 10.0 12.5 15.0
OF
 ITime (days)
Figure 4.15: Mean conYcen tration-time curve of sulphadoxine in capillary whole blood of children yoIuTnger than 5 years or older than 5 years after oral administration 
of standard dose Sof sulphadoxine-pyrimethamine. 
VE
R
UN
I
145 
 
Concentration of sulphadoxine
(g/ml)
 
Study 5: Evaluation of the use of saliva for therapeutic drug monitoring in 
children with uncomplicated Plasmodium falciparum malaria treated with 
amodiaquine-artesuante. 
 
Amodiaquine (AQ) and desethyl amodiaquine (DEAQ) were well resolved in the 
analysis using the modified method of Gitau and others (Gitau et al., 2004).Y Th ere 2was a good linearity (r  = 0.9907 plasma; Figure 4.16 and appendix TabRle 4.c; and 
2
saliva, r =0.9838, Figure 4.18 and appendix Table 4.d) in the standaRrd cAurve obtained 
for desethyl amodiaquine in plasma and saliva. In additioInB, the recoveries for 
amodiaquine and desethylamodiaquine over the concentra tiLon range of 100 ng/ml to 
600 ng/ml were between 69.7 and 79.4% for AplaNsma and saliva, respectively.  
Coefficient of variation within samples wereD 5.7% - 2.0%, and between samples 
7.2% - 3.5%, for concentrations of I1B00 
Ang/ml to 800 ng/ml, respectively.  The 
compound eluted from the systemF in  the order of quinidine, desethylamodiaquine and 
amodiaquine.  Figure 4.18  sOhows the chromatograms for IS, DEAQ and AQ in 
plasma and saliva. ThereY was no interference with other drugs (e.g. paracetamol and 
other commonlyS useId
T drug such as antimalarial, analgesic and anti-infective drugs) 
with the peaRks obtained (Figure 4.18). The lower limit of detection for desethyl 
amoIdViaqu
Eine was 15ng/ml at 0.05 absorbance units’ full scale (aufs). The retention 
tNime for IS, DEAQ and AQ were 5.5, 9.5 and 11.5 minutes respectively.  U
146 
 
 
 
 
 
 
 
0.75
  
 Y
0.50
 RA
R
 
0.25
  LI
B
 N
0.00 A
 0 250 500 D 750 1000
ConcentrationA (ng/ml) 
 F I
B
Figure 4.16: Standard curve  oOf desethyl amodiaquine for extrapolation of unknown 
2
concentrations of DEATQY in plasma (r  = 0.9907).
RS
I
IV
E
UN
147 
 
Peak height ratio
 
 
 
 
 
 
2.5
  
2.0
 RY
 1.5
BR
A
 1.0 I
 0.5  L
    
0.0 N
0 250 500 750 A 1000
 
Concentration (ng/Aml)D B
Figure 4.17: Standard curve of dFese thIyl amodiaquine for extrapolation of unknown 2
concentrations of desethyl am Oodiaquine in saliva (r  = 0.9838). 
SI
TY
ER
IV
UN
148 
 
Peak height ratio
 
 
 
 
 
 
IS   
A IS B 
AR
Y
BR AQ 
DEAQ  LI DEAQ 
AQ N
AD
A
IB
Time (min.)  F  Time (min.) 
 
Figure 4.18: Chromatograms  shOowing amodiaquine (AQ) and desethylamodiaquine 
(DEAQ) in plasma (A) IaTnd Ysaliva (B) of a patient at 4 h after the first dose of oral 
artesunate-amodiaquiSne. Quinidine is the internal standard (IS) 
 
VE
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UN
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149 
 
 
4.25:  Patients enrolled in the study 
The characteristics of seven children who were randomly selected from the group of 
children treated with artesunate-amodiaquine combination (30mg/kg of amodiaquine 
base over 3 days that is 10mg/kg daily) are shown in Table 4.29.  The seven children 
enrolled in the study aged between 6-13 years.  The mean age was 10.8 ± 2.8 years 
and mean weight was 26.2 ± 5.9kg. The geometric mean parasite densitYy w as 43797/µl of blood.  The pH of saliva generally ranged between 6.8 andR 7.8 in all 
children. RA
LIB
AN
 
D
 IB
A
 O
F
SI
TY
VE
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UN
I
150 
 
 
 
 
 
Table 4.29:  Baseline clinical and demographic characteristics of seven children 
with acute uncomplicated falciparum malaria enrolled in the study. 
 Y 
Parameters Mean±SD R
Age (y) 10.8±2.8 A
Range 7 – 13 R
Weight (kg) 26.2±5.9 LIBRange 16 – 33  
Gender (F:M) 4:3 N
O
Axillary Temperature ( C) 38.2±0.65 A
Range 36.9 – 38.6D 
% PCV 32I.6B±4 
A
Range 2 9 – 39 
Geometric Mean of parasiteF 43797 
density /µl of blood  O  
Range Y 19,009 – 150,000 
SD= standardI Tdeviation, PVC= pack cell volume, 
 
ER
S
V
UN
I
151 
 
 
4.26: Pharmacokinetic parameters of Desethyl amodiaquine in children with 
uncomplicated falciparum malaria after administration of standard doses of 
artesunate-amodiaquine 
Two of the seven children who were enrolled had AQ and DEAQ in their blood pre-
dosing and were excluded from the analysis.  Therefore data from 5 children out of 
the 7 who were enrolled were used for the pharmacokinetic analysis. Amodiaqu ine 
was rapidly converted to desethylamodiaquine, which appeared in the pRlasmYa and 
saliva within 40 minutes in all subjects. All children had detRectaAble levels of 
amodiaquine in plasma and saliva up to 40 h. The plasma anId Bsaliva concentration-
time curve for desethylamodiaquine for the five children  aLre shown in Figure 4.19.  
The concentration on day 7 was 210.3ng/ml in AplaNsma and 131.8ng/ml in saliva 
(Table 4.30).  The decline phases of the desethyDlamodiaquine in saliva concentration-
time curves were approximately paralleBl toA that in plasma (Figure 4.19).  The saliva 
concentration was approximately aF qu aIrter and two-fifth that in plasma. 
The pharmacokinetic param eOters derived from the oral plasma concentration-time 
curve are summarizeTd iYn Table 4.31.  The saliva pharmacokinetic parameters are summarized in TSablIe 4.32.  The plasma half-life (t1/2) was 156.7±20.6h (s.e.m).  The 
elimination Rhalf-life from saliva was also similar at 139.1±8.3h. There was no 
signIifVican
Et difference between plasma and saliva half-life (P = 0.16) (Table 4.33).  
TNhe mean AUC were 96003.6 ± 12344.6 ng/ml.h (s.e.m) for plasma and 74004.2 ± U9514.3 ng/ml.h for the saliva and were similar (P = 0.196) (Table 4.33).  Comparison 
of other pharmacokinetic parameters in the 5 patients gave a mean volume of 
distribution (Vd) of 74.1 ± 16.2 L/kg (s.e.m) for plasma and 91.5±21.8 L/kg for DAQ 
in saliva.  The plasma oral clearance calculated from the expression CLp=fD/AUC, 
152 
 
 
was found to be 325.4±48.8ml/h/kg (s.e.m).  This, too, was not significantly different 
from the clearance of 443.7±85.1ml/h/kg obtained from saliva data. 
Y 
AR
BR
N 
LI
DAA
F I
B
 O
ITY
RS
IV
E
UN
153 
 
 
Table 4.30:  Saliva-plasma ratio of desethyl amodiaquine concentrations in five 
children with falciparum malaria treated with standard oral doses of 
artesunate-amodiaquine. 
 
 Desethyl amodiaquine concentration (ng/ml)  
Time Plasma (n=5) Saliva (n=5) Saliva/plasma P value  
(days) ratio Y
0 0 0 ND NAD R
7 210.3±31.59* 131.8±19.5 0.626 R0.068 
121.9 – 314.9** 75.5 – 176.1 IB
14 193.9±47.1 141.0±24.0 0.727  L 0.358 
98.7 – 293.3 92.5 – 183.4 AN
28 111.3±48.1 41.4±24.0 D 0.119 0.237 
63.1 – 159.4 9.0 – 8B8.5 A
35 27.6±5.4 6F.4± 3I.1 0.233 0.064 
19.2 – 37.7  O3.3 – 9.6 *Mean±sem, ** Range 
ITY
RS
IV
E
UN
154 
 
 
 
 
 
  
 
 
 Y 
Plasma 
 Saliva 
R
2.25 A
 
LIB
R
1. 25
N  
0. 25 DA
 0 100 200 300 400 A500 600 700 800 900
T
  IimBe (h)
 OF
Figure 4.19: PlasmYa  and Saliva log concentration-time profile of 
desethylamodiaquinIe T(DEAQ) in children infected with P. falciparum malaria who 
were treRatedS with oral doses of artesunate-amodiaquine.E
IV
UN
155 
 
DEAQ concentration (ng/ml)
 
 
Table 4.31:  Pharmacokinetic disposition of desethyl-amodiaquine in plasma 
after standard oral doses of artesunate-amodiaquine (30mg/kg amodiaquine) in 
five children with uncomplicated malaria. 
 
 
  
Subject Weight AUC0-35d YExtrapolated 
(Kg) -1 -1 -1 -1(n=5) t1/2 (h) Vd (Lkg ) Cl (mlh kg ) (ng.ml h) R AUC (%) 
1 27 132.9 87.2 454.8 71370.0 A 2.6 
2 29 233.3 131.2 389.7 708I5B9.4
R 11.0 
3 20 128.8 43.5 234.0  L124593.5 2.7 
4 29 121.5 62.4 355.9 AN 87015.4 0.2 5 33 167.2 46.5 192.D8 126179.8 10.7 
Mean ±sem 27.6±2.1 156.7±20.6 74.1±16.2 B3A25.4±48.8 96003.6±12344.6 5.4±2.2 
s.e.m= Standard error of mean 
OF
 I
 
  
TY
RS
I
IV
E
UN
156 
 
 
Table 4.32:  Pharmacokinetic disposition of desethylamodiaquine in saliva after 
standard oral doses of artesunate-amodiaquine (30mg/kg amodiaquine) in five 
children with uncomplicated malaria. 
 
Subject Weight AUC0-35d Extrapolated 
(Kg) -1 -1 -1 -1(n=5) t1/2 (h) Vd (Lkg ) Cl (mlh Kg ) (ngml h) A UC (%) 
1 27 133.6 66.7 346.0 94390.0 RY2.0 
2 29 135.8 119.2 608.1 50376.9 A 1.2 
3 20 114.5 52.3 316.7 907B43.9R 4.1 
4 29 165.7 164.2 686.6  L42I804.1 5.2 
5 33 146.1 55.0 260.9 N 85843.5 17.8 
Mean±sem 27.6±2.1 139.1±8.3 91.4±21.8 443.D7±8A5.1 72831.7±10864.5 6.1±3.0 
s.e.m= Standard error of mean BA
 F I  O
ITY
 
ER
S
IV
UN
157 
 
 
Table 4.33: Comparison of pharmacokinetic disposition of desethyl amodiaquine 
in plasma and saliva after standard oral doses of artesunate-amodiaquine 
(30mg/kg amodiaquine) in children with uncomplicated malaria (n=5). 
 
Parameters Plasma n=5 Saliva n=5 P value 
t1/2 (h)* 156.7 ± 20.6 139.1 ±8.3 0.453 
95% CI 99.3 – 214.2 115.8 – 162.4  
Range** 121.5 – 233.3 114.5 – 165.7 Y
    AR
-1
AUC0-35d (ngml .h)  96003.6±12344.661 72831.7±10864B.54R0.196 
95% CI 729.3 – 130277.9 2666.8–1 02L99I6.6 
Range 87015.4 – 126179.9 42804N.1 – 94390.0 
 D A  
-1 -1
CL (mLh Kg ) 325.4±48.8 A 443.7±85.1 0.302 
95% CI  189.4-461.1 B 207.8-680.2 
Range 192O.8 –F
 I
 454.8 260.9 – 680.1 
 Y    
-1
Vd (LKg ) IT 74.1  16.2 91.5 ± 21.8 0.542 
95% CI  RS 29.1 – 119.2 30.8 – 152.1 
RangVe E 43.5 – 131.2 52.3 – 164.2 
*NMeIan±sem, ** Range U 
158 
 
 
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RS
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Appendices N 
AD
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TY
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177 
 
 
 
. 
 
 
 
Table 4.a:  Standard curve table for extrapolation of unknown concentratiYon o f 
chloroquine in whole blood on filter paper. 
AR R
 
 LI
B
Concentration CQ peak Papaverine (PPV) NRatio of CQ/PPV 
Height (mV) peak Height (mV) peak Height 
(ng/mL) DA
0 0 6.1 A 0 
100 2.8 6.4  IB 0.4375 
200 6.5 OF6.7 0.9701 400 12.4 Y 6.8 1.8235 
800 21I.2T 6.2 3.4193 
1000 RS26.1 5.8 4.5000 2000 E 48.3 5.3 9.1320 
2
r = 0.V99158 
UN
I
178 
 
 
 
Table 4.b: Standard curve table for extrapolation of unknown concentration of 
sulphadoxine in whole blood on filter paper 
Concentration Sulphadoxine Sulisoxazole Ratio of 
(IS)Peak Height SDX/SXZ 
(µg/mL) Peak Height (mV) 
(mV) Peak Heights  
0 0 110.9 0 AR
Y
10 45.9 107.0 0.B428R9 I
20 123.6 171.7 
N 
L0.7198 
40 146.8 145.3 A 1.0103 
50 220.0 1A47.D0 1.4959 
60 219.9  IB106.9 2.0579 
2
r = 0.99158  F
Y 
O   
 T
 
RS
I
VE
UN
I
179 
 
 
Table 4.c: Standard curve table for extrapolation of unknown concentration of 
desethyl amodiaquine in plasma. 
 
Concentration IS (QND) DAQ  Peak heights 
(ng/ml) peak height peak height DEAQ/QND 
ratio 
 
0 31.6 - 0.00 Y 
100 36.7 3.2 0.08 R
200 38.7 4.9 0.12 RA
400 41.7 13.0 0.31 IB
600 36.0 18.3 0 .5L0 
800 37.1 22.5 N0.60 
2
r = 0.9907   DA  
 
 IB
A
F
Y 
O
T
RS
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E
NI
V
U
180 
 
 
Table 4.d: Standard curve table for extrapolation of unknown concentration of 
desethyl amodiaquine in saliva. 
 
Concentration IS (QND) peak DEAQ peak  Peak height 
(ng/ml) height height DEAQ/QND 
ratio 
0 30.9 - 0.00  
100 32.4 13.6 0.41 Y
200 24.4 14.6 0.59 AR
400 28.8 29.4 1.02 R
600 27.9 42.8 1.5L3 IB
800 28.7 60.0 N2 .09 
2
r = 0.9838   A  
 D
 BA I
  
 
 
  O
F
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 T
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ER
IV
UN
181 
 
 
 
 
RY
 
BR
A
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DA
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BA
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O
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182