A Randomized, Open-Label Study to Evaluate Potential Pharmacokinetic Interactions of Orally Administered Artemether-lumefantrine and Amodiaquine in Healthy Adult Subjects
Phase: PHASE1
Conditions: Pharmacokinetic, Healthy, Drug Combination
Sponsor: University of Oxford

 
 
 
 
A Randomized, Open-Label Study to Evaluate Potential Pharmacokinetic 
Interactions of Orally Administered Artemether-lumefantrine and 
Amodiaquine in Healthy Adult Subjects 
 
Short Title: Pharmacokinetic study of artemether-lumefantrine and 
amodiaquine in Healthy Subjects 
 
Protocol version: version 6.0 dated 22 June 2022 
Trial registration number: NCT04080895 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             1 of 43  
A Randomized, Open-Label Study to Evaluate Potential Pharmacokinetic Interactions of 
Orally Administered Artemether-lumefantrine and Amodiaquine in Healthy Adult 
Subjects 
Short Title: Pharmacokinetic study of artemether-lumefantrine and amodiaquine in Healthy 
Subjects 
OxTREC reference Number: 34-19 
Registration number : NCT04080895 
 
Protocol Number:  MAL19003 
EC submission No.: TMEC 19-056 
 
Principal 
Investigator  
Asst. Prof. Borimas Hanboonkunupakarn 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand                               
Phone: 662-354-9168 ext 3160 
Email: Borimas@tropmedres.ac 
Co-Investigator 
Asst. Prof. Podjanee Jittamala  
Mahidol-Oxford Tropical Medicine Research Unit 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand  
Phone: 662-354-8333 # 2404 
Email: 
podjanee@tropmedres.ac  
Co-Investigator 
 
 
Professor Sasithon Pukrittayakamee 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand                             
Phone: 662-354-9100 # 1435 
Email: yon@tropmedres.ac  
 
Co-Investigator 
 
Asst. Prof.  Chayasin Mansanguan 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand                               
Phone : 089 1309481 
Email : 
chayasin.man@mahidol.ac.th 
Co-Investigator 
 
Assoc. Prof. Kitiyod Poovorawan 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand                               
Phone : 662-354-9168 ext 3160 
Email : 
kittiyod.poo@mahidol.ac.th 
Co-Investigator 
Dr James Callery 
Mahidol-Oxford Tropical Medicine Research Unit  
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand 
Phone: 662-203-6333  
Email: James.c@tropmedres.ac 
Co-Investigator  
Professor Nick White  
Mahidol-Oxford Tropical Medicine Research Unit  
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand 
Phone: 662-203-6333 # 6301 
Email: nickw@tropmedres.ac 
Co-Investigator  
Professor Nick Day  
Mahidol-Oxford Tropical Medicine Research Unit 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand 
Phone: 662-203-6333 # 6302 
Email: nickd@tropmedres.ac 
Co-investigator 
 
Professor Joel Tarning 
Mahidol-Oxford Tropical Medicine Research Unit 
Faculty of Tropical Medicine, Mahidol University 
Bangkok, Thailand 
Phone: 662-203-6369 
Email: joel@tropmedres.ac 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             2 of 43  
Sponsoring Institution:  
Oxford University 
Funder: 
 
 
Bill & Melinda Gates Foundation 
Study Site:   
 
 
Hospital for Tropical Diseases,  
Faculty of Tropical Medicine,  
Mahidol University, Thailand 
 
 
 
 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             3 of 43  
Table of Contents 
1. INTRODUCTION .............................................................................................................................. 7 
2. OBJECTIVE(S) .................................................................................................................................. 9 
2.1. Primary Objective ............................................................................................................................ 9 
2.2. Secondary Objectives ....................................................................................................................... 9 
3. ENDPOINT(S) .................................................................................................................................. 10 
3.1. Primary Endpoint ........................................................................................................................... 10 
3.2. Secondary Endpoints ..................................................................................................................... 10 
4. STUDY DESIGN .............................................................................................................................. 10 
5. STUDY POPULATION ................................................................................................................... 11 
5.1. Number of Subjects ........................................................................................................................ 11 
5.2. Inclusion Criteria ........................................................................................................................... 11 
5.3. Exclusion Criteria .......................................................................................................................... 12 
6. STUDY ASSESSMENTS AND PROCEDURES ............................................................................ 13 
6.1 Study Procedures (Appendix 2) ...................................................................................................... 13 
6.1.1 Visit 1 (Screening visit) ............................................................................................................... 13 
6.1.2 Visit 2-4 ....................................................................................................................................... 14 
6.3 Safety Assessments ......................................................................................................................... 19 
6.3.1. Adverse Events ........................................................................................................................... 19 
6.3.2. Vital Signs (BP, RR, BT and HR) .............................................................................................. 19 
6.3.3. ECGs  .......................................................................................................................................... 19 
6.3.4 Holter monitor                                                                                                                                     20   
6.3.5. Clinical Laboratory Assessments ................................................................................................ 20 
6.3.6. Pregnancy .................................................................................................................................... 20 
6.4. Pharmacokinetics ........................................................................................................................... 20 
6.4.1. Pharmacokinetic Samples (Plasma Matrix) ................................................................................ 22 
6.5. Storage of Genetic Samples ........................................................................................................... 22 
7. LIFESTYLE AND/OR DIETARY RESTRICTIONS ...................................................................... 23 
8. PRODUCTS ...................................................................................................................................... 23 
9. CONCOMITANT MEDICATIONS................................................................................................. 23 
9.1. Prohibited Concomitant Medications ............................................................................................. 24 
10. SUBJECT COMPLETION AND WITHDRAWAL ...................................................................... 24 
10.1. Subject Completion ...................................................................................................................... 24 
10.2. Subject withdrawn from the study ............................................................................................... 24 
11. ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS ....................................................... 25 
11.1. Definition of an Adverse Event ................................................................................................... 25 
11.2. Definition of a Serious Adverse Event ........................................................................................ 26 
11.3 Prompt Reporting of Serious Adverse Events .............................................................................. 27 
11.4 Evaluating Adverse Events and Serious Adverse Events ............................................................. 27 
11.4.1. Assessment of Intensity ............................................................................................................ 27 
11.4.2. Assessment of Relatedness ....................................................................................................... 27 
11.4.3. Action Taken ............................................................................................................................. 28 
11.4.4  Outcome .................................................................................................................................... 28 
12. DATA ANALYSIS AND STATISTICAL CONSIDERATIONS ................................................. 28 
12.1 
General Considerations ............................................................................................................. 28 
12.2 
Analysis Sets ............................................................................................................................. 28 
12.3 
Subject Disposition ................................................................................................................... 29 
12.4 
Determination of sample size .................................................................................................... 29 
12.5 
Methods of statistical analysis .................................................................................................. 29 
12.5.1 Pharmacokinetic assessments .................................................................................................... 29 
12.5.2 Safety Analysis .......................................................................................................................... 29 
12.6 Assessment of the relationship between pharmacokinetic parameters and ECG findings............ 30 
12.7. Interim analyses ........................................................................................................................... 30 
13. STUDY CONDUCT CONSIDERATIONS ................................................................................... 30 

Protocol version 6.0 dated 22 June 2022                                                                             4 of 43  
13.1. Regulatory and Ethical Considerations, Including the Informed Consent Process ...................... 30 
13.2. Risks ............................................................................................................................................. 30 
13.3. Benefit .......................................................................................................................................... 30 
13.4. Confidentiality ............................................................................................................................. 31 
13.5. Compensation .............................................................................................................................. 31 
13.6. Data Management ........................................................................................................................ 31 
14. RESEARCH USE OF STORED HUMAN SAMPLES/SPECIMENS........................................... 33 
14.1. Intended Use of the Samples/Specimens ..................................................................................... 33 
14.2. Storage of Samples/Specimens .................................................................................................... 33 
14.3. Storage of Genetic Sample ........................................................................................................... 33 
15. REFERENCES ............................................................................................................................... 33 
Appendix 1 ............................................................................................................................................ 35 
Appendix 2 ............................................................................................................................................ 36 
Appendix 3 ............................................................................................................................................ 37 
Appendix 4 ............................................................................................................................................ 38 
 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             5 of 43  
ABBREVIATIONS  
AE 
 
 
Adverse event  
AL or A/L 
 
Artemether-lumefantrine 
ALP 
 
 
Alkaline phosphatase 
ALT 
 
 
Alanine aminotransferase  
AQ 
 
 
Amodiaquine 
AST 
 
 
Aspartate aminotransferase  
AUC (0-Th)   
Area under the drug concentration curve from time 0 to time T hours  
AUC (0-last)          
Area under the drug concentration curve from time 0 to last time-
point 
AUC (0-∞)          
Area under the drug concentration curve from time 0 to infinity 
BUN  
 
Blood urea nitrogen  
BW 
 
 
Body weight 
BP 
 
 
Blood pressure  
Clcr   
 
Creatinine clearance  
Cmax   
 
Maximum observed drug concentration  
CK  
 
 
Creatine kinase  
CQ 
 
 
Chloroquine 
CRF   
 
Case report form 
ECG  
Electrocardiogram  
 
EDTA  
 
Ethylenediaminetetraacetic acid 
FBS 
 
 
Fasting blood sugar 
GCP   
 
Good clinical practice  
HBsAg  
 
Hepatitis B surface antigen 
HIV   
 
Human immunodeficiency virus  
HR  
 
 
Heart rate  
IC50  
 
Half maximal inhibitory concentration 
EC  
 
 
Ethics committee  
IRB 
 
 
Institutional review board 
kg  
 
 
Kilograms  
MetHb                         Methemoglobin 
mg  
Milligram  
 
mL  
 
 
Milliliter  
mmHg  
 
Millimeters of mercury  
MORU 
 
Mahidol-Oxford Tropical Medicine Research Unit 

Protocol version 6.0 dated 22 June 2022                                                                             6 of 43  
MQ 
 
 
Mefloquine 
PGx   
 
Pharmacogenetic  
PK 
 
 
Pharmacokinetics  
Pfcrt 
 
 
Plasmodium falciparum chloroquine resistance transporter  
Pfmdr1 
 
Plasmodium falciparum multidrug resistance protein-1 
PO 
 
 
Per os (by mouth or orally) 
RBC   
 
Red blood cell  
SAE   
 
Serious adverse event  
Scr  
 
 
Serum creatinine  
TG 
 
 
Triglyceride 
Tmax   
 
Time to maximum observed drug concentration  
t½  
 
 
Terminal elimination half-life  
ULN   
 
Upper limit of normal  
WBC   
 
White blood cell  
Wt 
 
 
Weight  
Vd  
 
 
Volume of distribution based on the terminal phase  
λz  
 
 
Terminal elimination rate constant   

Protocol version 6.0 dated 22 June 2022                                                                             7 of 43  
1. INTRODUCTION 
Artemisinin combination therapies (ACTs) have been a major driving force behind 
substantial reductions in global malaria morbidity and mortality over recent years. However, 
further gains are threatened by the recent emergence of artemisinin resistance in Southeast 
Asia (1), a region that has been the epicentre for the evolution and spread of resistance to 
every important class of antimalarials.  
ACT is the simultaneous use of two or more blood schizontocidal drugs with independent 
modes of action. The rationale behind ACT is that the chance of parasites simultaneously 
developing resistance to two drugs with different modes of action is much lower than the 
chance of parasites developing resistance to single drugs. Rapid clearance of parasitemia and 
resolution of symptoms is dependent on artemisinin while the complete clearance of the 
remaining parasites is dependent on the partner medicine (2). With the emergence of 
artemisinin resistance, partner drugs have been left exposed to a much larger biomass of 
parasites and unsurprisingly partner drug resistance has followed (3). 
Treatment failures after artemisinin combination therapies are becoming more widespread in 
Southeast Asia. Failures rates of around 30% for dihydroartemisinin-piperaquine and 
mefloquine-artesunate have been documented in western Cambodia (4) and western Thailand 
border areas (5) respectively. Failure of first line ACTs will damage current control and 
elimination efforts and accelerate the emergence and spread of resistance. A major concern is 
that artemisinin and partner drug resistance may spread across a wider geographic area, as 
chloroquine resistance did in the 1960s and 1970s, moving from Southeast Asia to the Indian 
subcontinent and Africa. Furthermore, while the resistance phenotype is currently limited to 
the ring stage of the asexual cycle, there is a chance that the resistance itself could ‘deepen’ 
by extending beyond this stage, causing more problems in the management of malaria . 
There is an urgent need to evaluate alternative treatments where standard courses of ACTs 
are failing, and to develop combinations of drugs which will not fall rapidly to resistance and 
can be deployed immediately. As new drugs are at least five years away, we propose to 
combine artemisinin derivatives with two existing slowly-eliminated partner drugs; triple 
artemisinin combination therapy (TACT).  
The principle that multiple drugs with independent mechanisms of action prevent the 
emergence of drug resistance is proven in a range of human diseases. In HIV and tuberculosis 
for example, the occurrence and spread of drug resistance can be prevented by use of a 
combination of three or more antiretroviral or antimycobacterial therapies respectively, but 
until now this was not thought necessary in malaria. Interestingly, there is a fortuitous inverse 
correlation between susceptibility to two important antimalarials, amodiaquine and 
lumefantrine, which will be exploited in the TACTs. 
Choosing partner drug combinations 
In general the ‘malaria world’ can be divided into different areas, based on current resistance 
patterns and current antimalarial drug use. 
The decision on which two partner drugs to combine in any new regimen must take into 
account pharmacokinetic and pharmacodynamic interactions, locally existing resistance 
profiles to artemisinin derivatives and the individual potential partner drugs, and the current 
antimalarial treatment policy. 
The global spread of chloroquine (CQ) resistance was also associated with cross-resistance to 
amodiaquine. However amodiaquine continued to be used in some areas of Southeast Asia, 
such as Myanmar, owing to its low cost. Relatively few trials of the ACT artesunate-

Protocol version 6.0 dated 22 June 2022                                                                             8 of 43  
amodiaquine have been conducted. The largest showed borderline efficacy in Myanmar with 
recrudescence rates of about 10%. Studies in Vietnam, as well as India and Africa, have 
reported cure rates above 95% (6-10). 
Transfection data provide the most reliable means of understanding the molecular basis of 
amodiaquine resistance. Fidock and colleagues introduced two major patterns of mutations 
(Dd2 and 7G8) into wild-type pfcrt-encoding parasites and were able to confirm that these 
mutations were causative factors in CQ resistance (11). Notably there was a clear inverse 
correlation between the IC50 values for CQ and mefloquine (MQ) as well as for amodiaquine 
and MQ. In other words, resistance to chloroquine/amodiaquine caused by specific pfcrt-
mutations was associated with mefloquine hypersensitivity. 
A second locus, pfmdr1, also appears to be involved in amodiaquine resistance, with the N86Y 
mutation associated with resistance in vitro and increased clinical failures with amodiaquine 
monotherapy (12-14). A recent paper by Venkatesan et al. suggests that recurrent infection 
after artemether-lumefantrine and artesunate-amodiaquine is associated with specific 
mutations in the pfmdr1 gene, but that the two partner drugs select alternative and drug 
resistance opposing alleles (15).  
In theory, the concurrent use of lumefantrine combined with amodiaquine exploits these 
opposing selection pressure effects. Potentially, this will prevent or delay emergence of high-
level resistance to both drugs at the same time. This would be expected to prolong or 
maintain the overall efficacy of both partner drug components (16).  
To assess the safety and tolerability and pharmacological interactions of the combination of 
artemether-lumefantrine and amodiaquine we propose an open label sequential trial of 
artemether-lumefantrine combined with amodiaquine.  
As we propose to combine artemether-lumefantrine, and amodiaquine, it is necessary that the 
potential interactions of these drugs be characterized. These drugs are metabolized by 
cytochrome P450 (abbreviated CYP) enzymes which potentially causes pharmacokinetic 
alteration, resulting in clinically significant drug-drug interactions that can cause unanticipated 
adverse reactions or therapeutic failures because of the suboptimal exposure of the parasite.  
 
Artemether-lumefantrine data 
Following administration of artemether-lumefantrine to both healthy volunteers and patients 
with malaria, artemether is absorbed with peak plasma concentrations reached about 2 hours 
after dosing. Absorption of lumefantrine, a highly lipophilic compound, starts after a lag-time 
of up to 2 hours, with peak plasma concentrations about 6 to 8 hours after administration. Food 
enhances the absorption of both artemether and lumefantrine. In healthy volunteers, the relative 
bioavailability of artemether was increased between 2- to 3-fold, and that of lumefantrine 16-
fold when artemether-lumefantrine tablets were taken after a high-fat meal compared to under 
fasted conditions. Artemether and lumefantrine are both highly bound to human serum proteins 
in vitro (95.4% and 99.7%, respectively). In human liver microsomes and recombinant 
CYP450 enzymes, the metabolism of artemether was catalyzed predominantly by CYP3A4/5.  
Artemether is cleared from plasma with an elimination half-life of about 2 hours. Lumefantrine 
is eliminated more slowly, with an elimination half-life of 3 to 6 days in healthy volunteers and 
in patients with falciparum malaria. Demographic characteristics such as sex and weight appear 
to have no clinically relevant effects on the pharmacokinetics of artemether and lumefantrine 
(17). 
 

Protocol version 6.0 dated 22 June 2022                                                                             9 of 43  
Amodiaquine data 
Amodiaquine by oral administration is absorbed rapidly, and then extensively metabolized to 
N-desethylamodiaquine (active metabolite) and 2-hydroxy-desethyl-amodiaquine by hepatic 
first-pass effect. Cmax of amodiaquine in plasma, whole blood and packed cells is 32±3ng/ml, 
60±10ng/ml and 42±6ng/ml respectively; Tmax of amodiaquine is 0.5±0.03h, 0.5±0.1h and 
0.5±0.1h respectively; AUC0-8 of amodiaquine in plasma, whole blood and packed cells is 
99±19ng·ml-1h, 
148±25ng·ml-1h 
and 
167±72ng·ml-1h 
respectively. 
Cmax 
of 
desethylamodiaquine in plasma, whole blood and packed cells is 181±26ng/ml (19 fold higher 
than those of the parent drug), 561±70ng/ml and 561±143ng/ml respectively; Tmax for 
desethylamodiaquine is 3.4±0.8h, 2.2±0.5h and 3.6±1.1h respectively; and AUC0-24 for 
desethylamodiaquine in plasma, whole blood and packed cells is 2304±37ng·ml-1h, 
6811±752ng·ml-1h and 5713±1269ng·ml-1h respectively (whole blood AUC was higher than 
plasma AUC, P<0.001). The trial of 14C-labelled amodiaquine administered orally to Wistar 
rats showed that amodiaquine can be distributed widely in body with the main organs of 
accumulation being kidney, liver, bone marrow and spleen. Amodiaquine is not observed in 
plasma 8 hours after administration, whereas the concentration of desethylamodiaquine in 
plasma at 96h is 29±8ml-1. The half-life of amodiaquine in plasma is 5.2±1.7h, and elimination 
half-life (βt1/2) of desethylamodiaquine in plasma is 9-31 days. The bioavailability of 
amodiaquine is decreased due to first-pass hepatic metabolism. The plasma protein binding 
rate of amodiaquine and desethylamodiaquine is greater than 90%. The Tmax of amodiaquine 
taken orally by patients with falciparum malaria is 1.75h, distinctly longer than the Tmax of 
healthy volunteers (18). 
 
Potential interactions through hepatic metabolism 
All study drugs are metabolized in the liver. Artemether and lumefantrine are primarily 
metabolized via CYP3A4 (19). Artemether metabolism through CYP3A4 produces an active 
metabolite, dihydroartemisinin (DHA) that contributes substantially to its antimalarial activity. 
Artemether is also a CYP3A4 inducer, while lumefantrine inhibits CYP2D6 (20).  The 
clearance of amodiaquine and metabolism to its main metabolite, N-desethylamodiaquine, is 
catalyzed primarily by CYP2C8 (21).  As some of these drugs share the same enzymes or have 
effects on the enzymes for other drug metabolism, the co-administration may result in clinically 
significant drug-drug interactions that can cause unanticipated adverse reactions or therapeutic 
failures. To evaluate whether such effects take place, we propose to co-administer these drugs 
in this healthy volunteer study. 
 
 
2. OBJECTIVE(S)  
2.1. Primary Objective 
 To characterize the potential pharmacokinetic interactions of artemether -
lumefantrine and amodiaquine in healthy adult subjects. 
2.2. Secondary Objectives 
 To characterize the pharmacokinetic properties of artemether-lumefantrine and 
amodiaquine when given alone and in combination.  
 To evaluate the safety and tolerability of co-administered artemether-lumefantrine and 
amodiaquine. 

Protocol version 6.0 dated 22 June 2022                                                                             10 of 43  
 To investigate pharmacogenetic polymorphisms affecting drug levels of artemether-
lumefantrine and amodiaquine and their metabolites.  
2.3. Exploratory Objective 
 To evaluate independent renal markers against standard blood urea nitrogen/ 
creatinine levels as markers of renal toxicity. 
 
3. ENDPOINT(S) 
3.1. Primary Endpoint 
 Area under the concentration-time curve (AUC0-∞ and AUC0-last) and maximum 
concentration (Cmax) of artemether, lumefantrine and amodiaquine and their 
metabolites when given alone and in combination.  
3.2. Secondary Endpoints 
 Elimination clearance (CL/F), terminal elimination half-life (t1/2) and apparent 
volume of distribution (Vd) of artemether, lumefantrine and amodiaquine and their 
metabolites when given alone and in combination.  
 Safety and tolerability parameters, including adverse events, electrocardiographic 
changes, vital signs and biochemical assessments. 
 Pharmacogenetic polymorphisms in the case of unusual metabolizer. 
3.3. Exploratory Endpoint 
 Independent renal marker blood levels and standard blood urea nitrogen/creatinine 
levels.  
 
4. STUDY DESIGN 
This is an open-label pharmacokinetic study in 16 healthy Thai subjects. Subjects will be 
admitted in the inpatient ward and will be randomized to group A or group B to receive 3 
drug regimens described in the following table 1.  Every subject will have 1 screening and 
3 admissions in the hospital.  
Table 1: Study Schedule 
Visit 1 
Screening 
Within 14 days 
 
Group A (n = 8) 
Group B ( n = 8) 
Visit 2 
Regimen 1  
Artemether-lumefantrine  
on Day 0, 1, and 2 
Regimen 2 
Amodiaquine  
on Day 0, 1 and 2 
Washout period:  > 6  weeks (after day 3) 
Visit 3 
Regimen 2  
Amodiaquine  
Regimen 1 
Artemether-lumefantrine  

Protocol version 6.0 dated 22 June 2022                                                                             11 of 43  
on Day 0, 1 and 2 
on Day 0, 1 and 2 
Washout period: > 6  weeks (after day 3) 
Visit 4 
Regimen 3 
Artemether-lumefantrine + Amodiaquine on Day 0, 1 and 2 
The total duration for each subject's participation in the study is approximately 6 - 8 months 
depend on washout period. Study duration is approximately 12 months. 
5. STUDY POPULATION 
5.1. Number of Subjects 
This study will enroll 16 healthy subjects both male and female, aged 18-60 years, at the 
pharmacokinetic unit Faculty of Tropical Medicine, Mahidol University. Subjects will be 
healthy HIV-1, hepatitis B and C uninfected individuals who comprehend the purpose of the 
study and have provided written consent. All subjects will undergo screening assessments 
(visit 1). Screening assessments (visit 1) may be carried out over more than one day, 
provided that all required assessments are completed within the 14 days prior to visit 2. If 
the interval between screening (visit 1) and day -1 visit 2 is three days or less, the clinical 
laboratory screening test result and serum pregnancy test result can be used for enrolment 
evaluation on day -1 visit 2. In such cases, these tests would not need to be repeated at day-
1 visit 2. 
 
5.2. Inclusion Criteria 
In order to be eligible to enter the protocol, subjects must meet the following criteria: 
1. Healthy as judged by a responsible physician with no abnormality identified on a 
medical evaluation including medical history and physical examination.  
2. Male or female non-smoker aged between 18 years to 60 years. 
3. A female is eligible to enter and participate in this study if she is:  
 
of non-childbearing potential including pre-menopausal females with 
documented (medical report verification) hysterectomy or double 
oophorectomy   
 
or postmenopausal defined as 12 months of spontaneous amenorrhea or 6 
months of spontaneous amenorrhea with serum follicle stimulating hormone 
levels >40 mIU/mL or 6 weeks postsurgical bilateral oophorectomy with or 
without hysterectomy 
 
or of childbearing potential, has a negative serum pregnancy test at screening 
and prior to start the study drug in each period, and agrees to abstain from 
sexual intercourse or use effective contraceptive methods (e.g., intrauterine 
device, hormonal contraceptive drug, tubal ligation or female barrier method 
with spermicide) during the study until completion of the follow-up procedures 
4. A male is eligible to enter and participate in this study if he: agrees to abstain from 
sexual intercourse with females of childbearing potential or lactating females; or is 
willing to use a condom/spermicide, during the study until completion of the 
follow-up procedures. 

Protocol version 6.0 dated 22 June 2022                                                                             12 of 43  
5. Normal electrocardiogram (ECG) with QTc <450 msec. 
6. Willingness and ability to comply with the study protocol for the duration of the 
trial. 
5.3. Exclusion Criteria 
Subjects will not be enrolled into the study if they meet any of the following exclusion 
criteria: 
1.  Females who are pregnant, trying to get pregnant, or are lactating.  
2. The subject has evidence of active substance abuse that may compromise safety, 
pharmacokinetics, or ability to adhere with protocol instructions.  
3.  A positive pre-study hepatitis B surface antigen, positive hepatitis C antibody, or 
positive human immunodeficiency virus-1 (HIV-1) antibody result at screening.  
4.  Subjects with a personal history of cardiac disease, symptomatic or asymptomatic 
arrhythmias, syncopal episodes, or additional risk factors for torsades de pointes 
(heart failure, hypokalemia) or with a family history of long QT syndrome, Brugada 
syndrome, or sudden cardiac death. 
5.  Abnormal serum creatinine (Scr) and estimated glomerular filtration rate (eGFR) 
<70 mL/min as determined by CKD-EPI equation  
6.   History of alcohol or substance abuse or dependence within 6 months of the study.   
7.  Use of prescription or non-prescription drugs except paracetamol at doses of up to 
2 grams/day, including vitamins, herbal and dietary supplements (including St. 
John’s Wort) within 7 days (or 14 days if the drug is a potential enzyme inducer) or 
5 times the drug half-life (whichever is longer) prior to the first dose of study 
medication until the completion of the follow-up procedure, unless in the opinion of 
investigator, the medication will not interfere with the study procedures or 
compromise subject safety; the investigator will take advice from the manufacturer 
representative as necessary. 
8.  The subject has participated in a clinical trial and has received a drug or a new 
chemical entity within 30 days or 5 x half-life, or twice the duration of the biological 
effect of any drug (whichever is longer) prior to the first dose of study medication. 
9. The subject is unwilling to abstain from ingesting alcohol within 48 hours prior to the 
first dose of study medication until collection of the final pharmacokinetic sample 
during each regimen.  
10. Subjects who have donated blood to the extent that participation in the study would 
result in more than 300 mL blood donated within a 30-day period. Note: This does 
not include plasma donation. 
11. Subjects who have a history of allergy to the study drug or drugs of this class, or a 
history of drug or other allergy that, in the opinion of the investigator, contraindicates 
participation in the trial. In addition, if heparin is used during pharmacokinetic 
sampling, subjects with a history of sensitivity to heparin or heparin-induced 
thrombocytopenia should not be enrolled. 
12. Lack of suitability for participation in this study, including but not limited to, 
unstable medical conditions, systemic disease manifested by tendency to 
granulocytopenia e.g. rheumatoid arthritis and lupus erythematosus that in the 
opinion of the investigator would compromise their participation in the trial.  

Protocol version 6.0 dated 22 June 2022                                                                             13 of 43  
13. AST or ALT >1.5 times the upper limit of normal (ULN) 
 14. Subjects with history of renal disease, hepatic disease, and/or cholecystectomy 
15. Abnormal methemoglobin level (more than 3 mg/dL). 
16. History of antimalarial drugs use including but not limited to mefloquine, 
chloroquine, primaquine, artesunate, piperaquine and pyronaridine treatment 
within 6 months. 
17. Subject who have received quinacrine in last 30 days. 
18. Subject has been included in other clinical trials in last 6 months. 
 
6. STUDY ASSESSMENTS AND PROCEDURES 
Each subject will have adequate time to provide informed consent of his or her own free will 
and prior to conducting any study specific procedures. Each subject will be provided a copy 
of the signed informed consent prior to the initiation of any study procedures.  
After a subject has provided written informed consent, the investigator or other study 
personnel will determine if the subject is eligible for enrolment in the study. This will be 
done by reviewing the inclusion and exclusion criteria and completing all of the screening 
assessments.  
The pharmacokinetic blood samples for a subject should be obtained at the scheduled times 
relative to when the subject was dosed. 
 
6.1 Study Procedures (Appendix 2) 
6.1.1 Visit 1 (Screening visit)  
Subjects will be screened to assess eligibility.  Full consent will be obtained before any 
screening procedures are conducted.   
• 
Basic demographics including age, date of birth, sex, height, and body weight 
• 
Physical Examination and symptoms questionnaire assessment 
• 
Vital signs (blood pressure (BP), respiratory rate (RR), body temperature (BT) and 
heart rate (HR)) and medical history  
 
ECG 
• 
Clinical laboratory assessment: 
o chemistry: Albumin, ALP, ALT, AST, total and direct bilirubin, BUN, 
creatinine, creatine kinase, potassium, sodium, magnesium and calcium  
o hematology: hemoglobin, hematocrit, white blood cell count with differential 
and platelet count   
o fasting blood sugar (FBS) (at screening and during admission) 
o urinalysis: white blood cell, red blood cell and protein 
o *TSH (at screening visit only) 
o HIV antibody, hepatitis B surface antigens, and hepatitis C antibody tests. 
(Pre and Post-test counseling will be performed for HIV test) (These tests 

Protocol version 6.0 dated 22 June 2022                                                                             14 of 43  
will be done at screening visit only) 
Note: The remaining serum will be frozen and stored for use in an exploratory 
analysis evaluating independent renal markers against standard blood urea nitrogen/ 
creatinine levels as markers of renal toxicity. 
All laboratory assessments (chemistry, hematology, FBS and urinalysis) must be 
drawn in the fasting state (8 hours fast). Blood collection for clinical laboratory 
assessment is approximately 10 mL.  
 
Serum pregnancy testing (if appropriate)  
 
6.1.2 Visit 2-4  
16 healthy subjects who fulfill the eligibility criteria will be recruited and randomized to the 
study. 
Procedures on each admission (Appendix 2): 
 
Visit 2  
Day -1 (admission) 
 Physical Examination and symptoms questionnaire assessment 
 Chemistry, Hematology, FBS and urinalysis assessment  
 24-hour urine for creatinine clearance test 
 Genetic sample collection (Genetic sample can be collected at any of the 
subsequent days or visits, if it is missed today) 
 Vital signs will be checked every 4 hours. 
 ECG monitoring according to table 2 
 
Holter monitoring (~30 mins baseline recording) 
 Urine pregnancy test (if applicable) 
Day 0-3 (admission) 
 
Daily physical examination and symptoms questionnaire assessment 
 
Vital signs will be checked before and every 4 hours after first dose. 
 
ECG monitoring according to table 2 
 
Continuous Holter monitoring during admission. 
 
Pharmacokinetic samples will be collected according to table 2 
 
Study drug administration (Artemether-lumefantrine OR Amodiaquine) 
will be given according to table 1. 
 
24-hour urine for creatinine clearance test (Day 2) 
 
Chemistry, Hematology, FBS and urinalysis assessment before discharge 
Day 4 (+1) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 

Protocol version 6.0 dated 22 June 2022                                                                             15 of 43  
 
Pharmacokinetic sample collection 
 
ECG 
Day 7 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
Pharmacokinetic sample collection 
 
ECG 
 
Holter monitoring (~ 30 mins) 
Day 14 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 
 
ECG 
Day 21 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 
Chemistry, Hematology and urinalysis assessment 
 
ECG 
Day 28 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 
 
ECG  
 
Holter monitoring (~ 30 mins) 
Day 35 (+/-2d) (follow up) (for Amodiaquine only) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
ECG 
Day 42 (+/-2d) (follow up) (for Amodiaquine only) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 

Protocol version 6.0 dated 22 June 2022                                                                             16 of 43  
 
ECG 
Visit 3  
Day -1 (admission) 
 
 
 
 
 Physical Examination and symptoms questionnaire assessment 
 Chemistry, Hematology, FBS and urinalysis assessment  
 24-hour urine for creatinine clearance test 
 Vital signs will be checked every 4 hours. 
 ECG monitoring according to table 2 
 
Holter monitoring (~30 mins baseline recording) 
 Urine pregnancy test (if applicable) 
Day 0-3 (admission) 
 
Daily physical examination and symptoms questionnaire assessment 
 
Vital signs will be checked before and every 4 hours after first dose. 
 
ECG monitoring according to table 2 
 
Continuous Holter monitoring during admission. 
 
Pharmacokinetic samples will be collected according to table 2 
 
Study drug administration (Amodiaquine OR Artemether-lumefantrine) 
will be given according to table 1.  
 
24-hour urine for creatinine clearance test (Day 2) 
 
Chemistry, Hematology, FBS and urinalysis assessment before discharge 
Day 4 (+1) (follow up) 
 Physical Examination, vital sign and symptoms questionnaire assessment 
 
Pharmacokinetic sample collection 
 
ECG 
Day 7 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
Pharmacokinetic sample collection 
 
Holter monitoring (~ 30 mins) 
 
ECG 
Day 14 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 

Protocol version 6.0 dated 22 June 2022                                                                             17 of 43  
 
Pharmacokinetic sample collection 
 
ECG 
Day 21 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 
Chemistry, Hematology and urinalysis assessment 
 
ECG 
Day 28 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 
 
Holter monitoring (~ 30 mins) 
 
ECG 
Day 35 (+/-2d) (follow up) (for Amodiaquine only) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
ECG 
Day 42 (+/-2d) (follow up) (for Amodiaquine only) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 
 
ECG 
 
Visit 4  
Day -1 (admission) 
 
 
 
 
 Physical Examination and symptoms questionnaire assessment 
 Chemistry, Hematology, FBS and urinalysis assessment 
 24-hour urine for creatinine clearance test  
 Vital signs will be checked every 4 hours. 
 ECG monitoring according to table 2 
 
Holter monitoring (~30 mins baseline recording) 
 Urine pregnancy test (if applicable) 
Day 0-3 (admission) 

Protocol version 6.0 dated 22 June 2022                                                                             18 of 43  
 
Daily physical examination and symptoms questionnaire assessment 
 
Vital signs will be checked before and every 4 hours after first dose. 
 
ECG monitoring according to table 2 
 
Continuous Holter monitoring during admission. 
 
Pharmacokinetic samples will be collected according to table 2 
 
Study drug administration (Artemether-lumefantrine AND Amodiaquine) 
will be given according to table 1. 
 
24-hour urine for creatinine clearance test (Day 2) 
 
Chemistry, Hematology, FBS and urinalysis assessment before discharge 
Day 4 (+1) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 
Pharmacokinetic sample collection 
 
ECG 
Day 7 (+/-2d) (follow up) 
 Physical Examination, vital sign and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
Pharmacokinetic sample collection 
 
ECG 
 
Holter monitoring (~ 30 mins) 
Day 14 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 
 
ECG 
Day 21 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
ECG 
Day 28 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 

Protocol version 6.0 dated 22 June 2022                                                                             19 of 43  
 
ECG 
 
Holter monitoring (~ 30 mins) 
Day 35 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 
ECG 
Day 42 (+/-2d) (follow up) 
 Physical Examination, vital signs and symptoms questionnaire assessment 
 Chemistry, Hematology and urinalysis assessment 
 Urine pregnancy test (if applicable) 
 
Pharmacokinetic sample collection 
 
ECG 
All laboratory assessments (chemistry, hematology, FBS and urinalysis) must be drawn in the 
fasting state (8 hours fast).  Results of these tests are to be available and reviewed prior to each 
subject receiving the study drug on day 0. 
Note: At all visits the remaining blood serum will be frozen and stored for use in an 
exploratory analysis evaluating independent renal markers against standard blood urea 
nitrogen/ creatinine levels as markers of renal toxicity. 
 
6.3 Safety Assessments 
The physician or qualified clinical study staff will review the data at intervals throughout each 
study period.  
6.3.1. Adverse Events 
Adverse events (AEs) and serious adverse events (SAEs) will be monitored. Refer to Section 
on AEs and SAEs.   
6.3.2. Vital Signs (BP, RR, BT and HR) 
Subjects must have been resting quietly for at least 10 minutes prior to taking measurements. 
Blood pressure (BP) and heart rate (HR) for visit 2-4 will be measured in both supine standing 
position in each time-points. 
6.3.3. ECGs  
Subjects must have been resting quietly for at least 20 minutes prior to taking measurements. 
Each ECG measurement will be done in triplicate at each time point to avoid outlier values due 
to random variation.  
6.3.4 Holter monitor  
During each admission, subjects will undergo continuous Holter monitoring to record changes 
in heart rhythm and cardiac conduction times. Simple non-invasive autonomic function tests 
i.e. heart-rate response to postural change (further detailed in an SOP) will be undertaken 
during the time that the Holter monitor is fitted (including during follow-up if a Holter monitor 
is available). 

Protocol version 6.0 dated 22 June 2022                                                                             20 of 43  
6.3.5. Clinical Laboratory Assessments  
The following hematology, clinical chemistry, FBS, and urinalysis tests will be performed at 
each admission.  
• 
Hematology: hemoglobin, hematocrit, white blood cell (WBC) count with 
differential, and platelet count 
• 
Chemistry: Albumin, alkaline phosphatase (ALP), alanine aminotransferase 
(ALT), aspartate aminotransferase (AST), total and direct bilirubin, blood urea 
nitrogen (BUN), creatinine, creatine kinase (CK), potassium, sodium, magnesium 
and calcium 
• 
FBS: Fasting blood sugar (at screening and during admission) 
• 
Urinalysis: white blood cell (WBC), red blood cell count (RBC), and protein. 
• 
*TSH (for screening visit only) 
Note: The remaining blood serum will be frozen and stored for use in an exploratory 
analysis evaluating independent renal markers against standard blood urea 
nitrogen/ creatinine levels as markers of renal toxicity.   
Any results falling outside of the reference range will be repeated at the discretion of the 
investigator. If there is more than one laboratory result for the same analyte, the last value will 
be recorded in the CRF.  
6.3.6. Pregnancy 
For female subjects, serum pregnancy test will be performed at visit 1, and urine pregnancy 
test will be performed before the first dose of each regimen and biweekly during follow up 
visits.  
Action to Be Taken if Pregnancy Occurs in a Female 
If any female subject or the female partner of a male subject who becomes pregnant while 
participating in this study that is after enrolment until the last follow up visit, the following 
procedures will be followed: 
• 
The subject must be immediately discontinued from study drug. 
• 
The subject must be referred to antenatal clinic for follow up. 
• 
The pregnancy will be monitored to determine the outcome of the pregnancy. Any 
premature termination of the pregnancy will be reported.  
While pregnancy itself is not considered to be an AE or SAE, any pregnancy complication or 
elective termination of a pregnancy for medical reasons will be recorded as an AE or SAE (see 
AE/SAE section of the protocol for definitions and a description of monitoring).  
A spontaneous abortion is always considered to be an SAE and will be reported as such. 
Furthermore, any SAE occurring as a result of a post-study pregnancy and is considered 
reasonably related to the investigational product by the investigator, will be reported. All 
pregnancies occurring during the study period and including follow up period should be 
reported to PI as soon as possible.  
  
6.4. Pharmacokinetics 
Blood for pharmacokinetic assessments may be obtained by individual needle stick or IV 

Protocol version 6.0 dated 22 June 2022                                                                             21 of 43  
cannula. If a cannula is used for collection of the whole blood samples, the cannula will be 
inserted into an arm vein within sufficient time before dosing and may be kept patent using a 
dilute heparin solution as a flush. An initial volume of approximately 1.0 mL of blood will be 
collected and discarded prior to collection of each whole blood sample to ensure that the saline 
or heparin solution does not artificially dilute the samples. If PK samples are collected via 
individual needle sticks rather than through a cannula, then no extra blood needs to be collected 
and discarded prior to the collection of the sample.  
Each blood sample will be labeled with subject’s number, schedule date and time, collection 
date and time. The actual time of each blood sample will be recorded on the case report form 
(CRF).  
 
 
 
Table 2 Pharmacokinetic collection schedule for each study regimens 
Regimen 
Blood sampling 
ECG measurements 
Regimen 1: 
Artemether-
lumefantrine 
(AL) 
 
Dosing: 0h, 8h, 
24 h, 36 h, 48 h, 
60 h 
 
Day 0: Hr. 0 (pre-dose), 0.25, 0.5, 1, 2, 
3, 4, 6, 8 (pre-dose), 10, 12, 14 
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 48.25, 48.5, 
49, 50, 51, 52, 54, 56, 60 (pre-dose), 
61, 62, 64, 66 
Day 3: Hr. 72  
Day 4, Day 7, Day 14, Day 28 
Day -1: Hr. 0, 1, 2, 4, 6, 8, 10, 12, 14 
Day 0: Hr. 0 (pre-dose), 1, 2, 4, 6, 8 
(pre-dose), 10, 12, 14  
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 49, 50, 52, 
54, 60 (pre-dose), 61, 62, 64, 66 
Day 3: Hr. 72  
Day 4, Day 7, Day 14, Day 21, Day 28 
Regimen 2: 
Amodiaquine 
(AQ)  
 
Dosing: 0h, 8h, 
24 h, 36 h, 48 h, 
60 h 
 
Day 0: Hr. 0 (pre-dose), 0.25, 0.5, 1, 2, 
3, 4, 6, 8 (pre-dose), 10, 12, 14 
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 48.25, 48.5, 
49, 50, 51, 52, 54, 56, 60 (pre-dose), 
61, 62, 64, 66 
Day 3: Hr. 72  
Day 4, Day 7, Day 14, Day 28, Day 42 
Day -1: Hr. 0, 1, 2, 4, 6, 8, 10, 12, 14 
Day 0: Hr. 0 (pre-dose), 1, 2, 4, 6, 8 
(pre-dose), 10, 12, 14  
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 49, 50, 52, 
54, 60 (pre-dose), 61, 62, 64, 66  
Day 3: Hr. 72  
Day 4, Day 7, Day 14, Day 21, Day 
28, Day 35, Day 42 
Regimen 3: 
Artemether-
lumefantrine + 
amodiaquine 
(AL+AQ) 
Day 0: Hr. 0 (pre-dose), 0.25, 0.5, 1, 2, 
3, 4, 6, 8 (pre-dose), 10, 12, 14 
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 48.25, 48.5, 
Day -1: Hr. 0, 1, 2, 4, 6, 8, 10, 12, 14 
Day 0: Hr. 0 (pre-dose), 1, 2, 4, 6, 8 
(pre-dose), 10, 12, 14  
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 

Protocol version 6.0 dated 22 June 2022                                                                             22 of 43  
 
Dosing: 0h, 8h, 
24 h, 36 h, 48 h, 
60 h 
49, 50, 51, 52, 54, 56, 60 (pre-dose), 
61, 62, 64, 66 
Day 3: Hr. 72  
Day 4, Day 7, Day 14, Day 28, Day 42 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 49, 50, 52, 
54, 60 (pre-dose), 61, 62, 64, 66  
Day 3: Hr. 72  
Day 4, Day 7, Day 14, Day 21, Day 
28, Day 35, Day 42 
The time-window for the visit on Day 4 is + 1 day and for the visits on Days 7 - 42 is + 2 or - 2 days  
Total blood collection for pharmacokinetic analysis will be around 165 mL.  
The total blood collections for both laboratory assessments and pharmacokinetic sampling 
on each visit was summarized in Appendix 4. 
6.4.1. Pharmacokinetic Samples (Plasma Matrix)  
Blood samples (1 mL for regimen 1, 2 and 2 mL for regimen 3) will be collected into properly 
labeled 2 mL sodium heparin blood collection tubes at the study times shown in Table 2. 
Sample Handling 
Upon collection, blood samples will be centrifuged for 7 minutes at 2000g at 4C. After 
centrifugation, the plasma of each sample will be transferred via pipette to a pre-labeled sample 
storage tube and stored upright until analyzed.  
Sample Storage 
Samples will be immediately frozen in the storage tubes in the upright position in a non-self-
defrosting freezer at -70°C or lower.  
6.5. Storage of Genetic Samples 
If the subject consents for genetic sample collection and testing, the genetic samples (10 mL) 
will be collected.  
In case of any abnormal metabolisms in individuals, genotyping for the identification of 
polymorphisms of Cytochrome 450 (e.g. CYP2D6, CYP3A4) and other enzymes related to 
drug metabolism will be performed. DNA will be extracted from blood and PCR will be 
performed. SNP typing will be used to determine the gene mutation by comparing subject 
with normal metabolism activities to subject with abnormal metabolism activities. These tests 
will determine if there is any genetic abnormality. Such data may be useful in predicting 
those who may not obtain the full therapeutic or prophylactic benefit of study drugs.  
Genetic samples will be stored (approximately 10 years) and tested at the Molecular Tropical 
Medicine Laboratory, Faculty of Tropical Medicine, Mahidol University. In case that 
particular genetic test is unable to be performed at Molecular Tropical Medicine Laboratory, 
Faculty of Tropical Medicine, Mahidol University, genetic sample may be transferred to 
another institute with subject consent. 
A MTA (Material Transfer Agreement) will also be in place before any samples are sent to 
another institution for testing. 
Any proposed plans to use this sample other than for the purposes of this study will be 
submitted to the IRB/EC prior to any genetic testing being performed. 

Protocol version 6.0 dated 22 June 2022                                                                             23 of 43  
7. LIFESTYLE AND/OR DIETARY RESTRICTIONS 
• Subjects should refrain from all illicit drugs throughout the study.  
• Subjects should refrain from the ingestion of grapefruit containing products throughout the 
study.  
• Subjects must fast from all food and drink for at least 8 hours prior to clinical laboratory 
assessment. 
• Subject should not consume alcohol within 48 hours prior to study drug administration and 
throughout the study. 
• Subjects will be allowed to eat normally during out-patient/washout time and will receive 
standard meal during inpatient stay. All drugs will be taken with light meal. They will be 
given a similar light standard meal within 30 minutes before each drug dosing (similar 
calories, carbohydrate, protein and fat ratio). Subjects will be allowed to eat 4 hours after 
study drug administration. Water or soft drinks without caffeine may be consumed 2 hours 
post-dose (after autonomic function testing). All subjects will receive the same meal.  
• Total fluid intake may not exceed 3L per day during the dosing period.  
8. PRODUCTS 
• 
Artemether-lumefantrine  
Artemether-lumefantrine is in the form of dispersible or standard tablets of 20 mg of 
artemether and 120 mg of lumefantrine in a fixed-dose combination formulation. The 
weight-based dosing schedule in appendix 3.  
• 
Amodiaquine  
Amodiaquine is in the form of dispersible tablets containing 76.5 mg amodiaquine 
co-blistered with sulfadoxine/pyrimethamine 250 mg/12.5 mg dispersible tablet 
(SPAQ-CO® Disp).  
Amodiaquine dispersible tablets are tablets with yellow core debossed with “AM” on 
one side and a score line on the other side.  
Sulfadoxine/pyrimethamine dispersible tablets are white round tablets, debossed with 
“SP” on one side and a score line on the other side.  
This study will only use the amodiaquine. The weight-based dosing schedule in 
appendix 3.  
All sulfadoxine/pyrimethamine tablets, unused Amodiaquine and unused Artemether-
lumefantrine will be disposed following hospital standard procedures after study 
completion. 
 
9. CONCOMITANT MEDICATIONS  
The Principal Investigator must be informed as soon as possible about any medications taken 
by the subject from the time of screening until the last follow up visit or withdrawal visit. If a 
subject takes any concomitant medications taken during the study, it will be recorded in the 
CRF. Drug name, dose, frequency, route of administration and the start and end dates of 
administration are to be recorded 

Protocol version 6.0 dated 22 June 2022                                                                             24 of 43  
9.1. Prohibited Concomitant Medications  
Use of prescription drugs including but not limited to drugs with antimalarial activities and 
any drug contraindicated with the investigational drugs e.g. quinacrine, mefloquine or non-
prescription drug, including, vitamins, herbal and dietary supplements (including St. John's 
Wort) within 7 days (or 14 days if the drug is a potential enzyme inducer) or 5 half-lives 
(whichever is longer) prior to the first dose of study medication and for the duration of the trial 
including follow-up will be prohibited, unless in the opinion of the investigator, the medication 
will not interfere with the study procedures or compromise subject safety; the investigator will 
take advice from the manufacturer representative as necessary, 
Subject should discuss with study team member before using over-the-counter medications 
during the study period except as needed to treat an adverse event. 
If a subject takes a prohibited medication during the course of the study, the investigator and 
the statistician will determine if the subject should be withdrawn from the study or from the 
final pharmacokinetic analysis. 
10. SUBJECT COMPLETION AND WITHDRAWAL 
10.1. Subject Completion 
Subjects who complete all the study visits and follow up per protocol, will be considered as 
having completed the study.  
10.2. Subject withdrawn from the study 
This includes the following: 
• 
Subject voluntarily withdrew consent 
• 
The Principal Investigator, sponsor withdrawing the study subject at his/her discretion for 
the benefit of the subject.  
The reasons for withdrawal, or failure to provide a reason, will be documented by the 
investigator in the Completion/Withdrawal section of the CRF.  
If a subject is prematurely discontinued from participation in the study for any reason, the 
investigator must make every effort to perform the follow up visit assessments, unless they 
have withdrawn consent. 
10.3 Subject discontinues the study drugs   
Such subjects will be followed up for safety data collection. Reasons for this scenario may 
include: 
• 
Pregnancy  
• 
Subject requests to discontinue study drugs 
• 
Abnormal renal and hepatic laboratory result including;  
o Any creatinine above 1.5 x ULN OR increase 0.3 mg/dL above baseline 
o Any ALT or AST above 3 x ULN  
o Any elevation in total and direct bilirubin 2x ULN  
o Any AST or ALT above 2xULN and TBL >1.5xULN   
o Any AST or ALT above 2xULN with the appearance of fatigue, nausea, vomiting, right 
upper quadrant pain or tenderness, fever, rash and/or eosinophilia (eosinophil percent or 
count above the ULN)  

Protocol version 6.0 dated 22 June 2022                                                                             25 of 43  
If the abnormalities are potentially caused by one of the study drug the research physician will 
decide whether the other regimen of the study drugs should be continued. All laboratory 
abnormalities will be assessed by a trained physician. In case of abnormalities, a medical 
history to assess exposure to substances (medication, alcohol and others) with known toxicity 
for instance liver or kidney will be performed. In addition, the physician can perform additional 
investigations that are deemed necessary to explain the laboratory abnormalities (such as 
hepatitis serology or urine analysis). The liver function and creatinine abnormalities will be 
monitored until resolution or the stabilization of the condition. In any case all laboratory values 
should have returned to normal (within the normal range in the case of full blood count values 
and at least <1.5 times the ULN in case of the biochemistry values) before the administration 
of the next treatment round. 
 QTc interval abnormalities;  
 QTc or QT interval longer than 500 milliseconds 
A case by case judgement will be made on whether the subjects should be discontinued from 
the study completely. All other drugs known to prolong the QTc interval will be stopped. QTc 
intervals will be monitored continuously until normalisation of the QTc interval to below 450 
milliseconds. Magnesium and potassium levels will be measured and deficiencies will be 
supplemented through intravenous or oral medication. 
Unevaluable subjects may be replaced, according to the discretion of the investigators with 
another subject of the same population, if either sample size or completeness of dataset is 
compromised. All data will be listed and will be valid for clinical safety and tolerability 
evaluation.  
10.4 Study Halting Rules   
When three subjects experience adverse events of grade 3 or above that are “probably related 
or related” to the study drug, administration of study drug(s) will be halted and the investigator 
team will stop recruiting new study subjects promptly. Enrolled subjects who do not experience 
adverse event of grade 3 or above will receive study drug(s) until the study ends. 
11. ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS 
The investigator is responsible for the detection and documentation of events meeting the 
criteria and definition of an adverse event (AE) or serious adverse event (SAE), as provided in 
this protocol.   
All SAEs and AEs will be promptly reported from first dose of study drug administration to 
discontinuation of the subject from study participation. Any events occurring between 
screening and first dose of study drug administration will be considered as baseline, preexisting 
conditions. 
11.1. Definition of an Adverse Event 
An AE is any undesirable event that occurs to a study subject during the course of the study; 
that is, from the time of administration of study drugs until study ends (i.e., until the follow up 
visit) whether or not that event is considered related to the study drugs, or to a concomitant 
drug or procedure. Therefore, an AE can be absent at baseline but newly develops, or was 
present at baseline and worsens and includes:  
 any unfavorable and unintended symptom 
 physical sign 
 abnormal laboratory result 

Protocol version 6.0 dated 22 June 2022                                                                             26 of 43  
 an illness 
Any new clinical sign or clinical deterioration that occurs between signing the consent form 
and the administration of study drugs is not an AE. This information will be recorded in the 
medical records. 
11.2. Definition of a Serious Adverse Event 
A serious adverse event is any untoward medical occurrence that, at any dose:  
a. Results in death 
b. Is life-threatening 
NOTE:  The term 'life-threatening' in the definition of 'serious' refers to an event in which the 
subject was at risk of death at the time of the event. It does not refer to an event, which 
hypothetically might have caused death, if it were more severe.  
c. Requires hospitalization or prolongation of existing hospitalization 
NOTE:  In general, hospitalization signifies that the subject has been detained (usually 
involving at least an overnight stay) at the hospital or emergency ward for observation and/or 
treatment that would not have been appropriate in the physician’s office or out-patient setting. 
Complications that occur during hospitalization are AEs. If a complication prolongs 
hospitalization or fulfills any other serious criteria, the event is serious. When in doubt as to 
whether “hospitalization” occurred or was necessary, the AE should be considered serious.  
Hospitalization for elective treatment of a pre-existing condition that did not worsen from 
baseline is not considered an AE.  
d. Results in disability/incapacity 
NOTE:  The term disability means a substantial disruption of a person’s ability to conduct 
normal life functions. This definition is not intended to include experiences of relatively minor 
medical significance such as uncomplicated headache, nausea, vomiting, diarrhea, influenza, 
and accidental trauma (e.g., sprained ankle) which may interfere or prevent everyday life 
functions but do not constitute a substantial disruption.  
e. Results in a congenital anomaly/birth defect 
Medical or scientific judgment should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not be immediately 
life-threatening or result in death or hospitalization but may jeopardize the subject or may 
require medical or surgical intervention to prevent one of the other outcomes listed in the above 
definition. These should also be considered serious.  
Examples of such events are invasive or malignant cancers, intensive treatment in an 
emergency room or at home for allergic bronchospasm, blood dyscrasias, or convulsions that 
do not result in hospitalization, or development of drug dependency or drug abuse.  
 
Laboratory Abnormalities and Other Abnormal Assessments as Adverse Events or 
Serious Adverse Events 
Abnormal laboratory findings (e.g., biochemistry, hematology, urinalysis) or other abnormal 
assessments (e.g., BP, RR, BT, HR and ECGs) detected during the study or are present at 
baseline and worsen following the start of the study will be reported as AEs or SAEs. 
Subjects with an adverse event will be managed clinically. This may include stopping study 

Protocol version 6.0 dated 22 June 2022                                                                             27 of 43  
drug, further investigations, treatment, referral to a specialist, etc.  
11.3 Prompt Reporting of Serious Adverse Events 
All SAEs should be reported promptly to medical monitor for the study within 24 hours for all 
SAEs to e-mail address: ALAQsafety@tropmedres.ac.  
If all information is not yet available then an initial report must be made within the timeframe. 
Further interim reports may be made if necessary or a final report when all relevant information 
is available. 
Faculty of Tropical Medicine Ethic committee (FTM EC) will be notified within 5 official days 
for all SAEs.  
11.4 Evaluating Adverse Events and Serious Adverse Events 
11.4.1. Assessment of Intensity  
All adverse events will be evaluated by the investigators and graded according to Common 
Terminology Criteria for Adverse Events (CTCAE) version 5.0. For lab results that are not 
available in Common Terminology Criteria for Adverse Events (CTCAE) version 5.0, the 
investigator should make a determination as to whether or not the laboratory abnormality is 
clinically significant. If PI believes lab abnormality is clinically significant, it should be 
reported as an adverse event and a grade should be identified to the best of their ability. 
If an adverse event is not listed in the Common Terminology Criteria for Adverse Events 
(CTCAE) version 5.0, the Investigator will assess the severity using the following guidelines: 
1 = Mild: asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated. 
2 = Moderate: minimal, local or non-invasive intervention indicated; limiting age 
appropriate instrumental ADL* 
3 = Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self-
care ADL** 
4 = Life-Threatening consequences; urgent intervention indicated 
5 = Death related to AE 
Activities of Daily Living (ADL)  
*Age-appropriate instrumental ADL which will be defined in study SOPs.  
**Age-appropriate self-care ADL which will be defined in study SOPs.  
 
Clarification of the difference in meaning between “severe” and “serious” 
The term “severe” is often used to describe the intensity (severity) of a specific event (as in 
mild, moderate, or severe myocardial infarction); the event itself, however, may be of relatively 
minor medical significance (such as severe headache).  This is not the same as “serious”, which 
is based on the outcome or criteria defined under the serious adverse event definition. An event 
can be considered serious without being severe if it conforms to the seriousness criteria, 
similarly severe events that do not conform to the criteria are not necessarily serious.  
Seriousness (not severity) serves as a guide for defining regulatory reporting obligations. 
11.4.2. Assessment of Relatedness  
The investigator is obligated to assess the relationship between study drug and the occurrence 

Protocol version 6.0 dated 22 June 2022                                                                             28 of 43  
of each AE/SAE using the following categories of relatedness: 
 Definite: clear-cut temporal association, with a positive re-challenge test or laboratory 
confirmation. 
• Probable: clear-cut temporal association, with improvement upon drug withdrawal, and 
not reasonably explained by the subject’s known clinical state. 
• Possible: less clear temporal association; other etiologies are possible. Other possible 
etiologies should be recorded on the CRF. 
• None: no temporal association with the study drug; related to other aetiologies such as 
concomitant medications or conditions, or subject’s known clinical state. 
The investigator will provide the assessment of causality as per the instructions for completion 
of the AE/SAE data collection tool.  
11.4.3. Action Taken 
 No action taken 
• Study drug is changed 
• Study drug is discontinued  
• No concomitant medication required 
• Concomitant medication discontinued 
• Concomitant medication is required 
• Hospitalization required or prolonged 
• Other  
11.4.4. Outcome 
The investigator will follow-up the AE until resolution or until no further medically relevant 
information can be expected. AE outcome will be classified as follows: 
 Resolved 
 Resolved with sequelae 
 Continuing  
 Death 
12. DATA ANALYSIS AND STATISTICAL CONSIDERATIONS 
The primary focus of statistical analysis is to verify the pharmacokinetic interactions of 
artemether, lumefantrine and amodiaquine in terms of AUC0-∞, AUC0-LAST and 
CMAX.  Comparisons across groups will be made using an analysis of variance (ANOVA) 
of logarithmically transformed pharmacokinetic parameters (i.e. AUC0-LAST, AUC0-∞, CMAX). 
The criteria for assuming no interaction or equivalence when co-administered is met if 
confidence interval (90% CI) values for geometric mean ratios of AUC0-∞, AUC0-LAST and 
CMAX fall within 80% to 125% for the two groups. The point estimate of the geometric mean 
ratio and the residual variability from the ANOVA will be used to calculate the 90% confidence 
intervals around the mean. 
12.1 
General Considerations 
All data will be summarized descriptively for the appropriate analysis sets defined below. 
12.2 
Analysis Sets  
Safety Set 
All subjects who receive study drug(s) will be included in the safety population. 

Protocol version 6.0 dated 22 June 2022                                                                             29 of 43  
Full Analysis Set  
Subjects who receive the full dose of study drug without vomiting with enough samples for 
characterization of the concentration-time profile will be included in the pharmacokinetic 
analysis.  
Subjects who vomit study drug or who do not have enough time points for characterization of 
their PK profile or with protocol violations will be replaced at the discretion of the study 
investigator so that the study sample size is retained for analysis.   
12.3 
Subject Disposition  
The number of subjects recruited and receiving study drug(s) will be presented by study drug 
groups, as will be the number of subjects included in the various analysis populations.    
Protocol violations will be listed per subject, describing the nature of the violation and 
specifying whether it is a major or a minor violation.   
12.4 
Determination of sample size 
A sample size of at least 12 is recommended sample size for determining bioequivalence test 
(22).Therefore, we will recruit 16 healthy volunteers to ensure that we have higher power of 
the bioequivalence test. 
12.5 
Methods of statistical analysis  
12.5.1 Pharmacokinetic assessments  
Individual concentration-time data will be evaluated using a non-compartmental analysis 
approach as implemented in WinNonlin (Pharsight Corporation, California, USA). Total 
exposure up to the last measured concentration (AUC0-LAST) will be calculated using the linear 
trapezoidal method for ascending concentrations and the logarithmic trapezoidal method for 
descending concentrations. Exposure will be extrapolated from the last observed concentration 
to time infinity by CLAST/λZ for each individual subject to compute total drug exposure (AUC0-
∞). The terminal elimination half-life (t1/2) will be estimated by log-linear regression of the 
terminal phase. Maximum plasma concentration (CMAX) and time to maximum concentration 
(TMAX) will be taken directly from the observed data. Apparent volume of distribution (VD/F) 
and oral clearance (CL/F) will be computed individually according to standard procedures and 
summarized for each treatment arm.  
Pharmacokinetic parameters (i.e. AUC0-LAST, AUC0-∞, CMAX, TMAX, CL/F, VD/F, and t1/2) will 
be described using means (SD or 95% CI) and medians (range) as appropriate.   
12.5.2 Safety Analysis  
Safety analyses will be based on the safety set. Safety and tolerability of artemether, 
lumefantrine and amodiaquine will be assessed by comparing the frequency (%) of adverse 
events and serious adverse events, with particular attention to abdominal pain, appetite 
perturbation and QT interval prolongation, using the Fisher’s exact test.  Safety data will be 
presented in tabular and/or graphical format and summarized descriptively. Any clinically 
relevant abnormalities or values of potential clinically concern will be described. Subjects will 
be analysed as treated. 
An additional exploratory analysis evaluating independent renal markers against standard 
blood urea nitrogen/ creatinine levels will be conducted using appropriate statistical methods.   
Adverse Events 
All adverse event summaries will refer to treatment emergent adverse events, i.e. adverse 

Protocol version 6.0 dated 22 June 2022                                                                             30 of 43  
events that newly started or increased in intensity after the study drug administration. AE 
summaries will be generated for all AEs that occurred after study drug administration, until the 
end of the study. 
12.6 Assessment of the relationship between pharmacokinetic parameters and ECG 
findings.  
In addition to the results of the safety analysis, an exploratory analysis of the ECG data will be 
conducted.  The ECG parameters will be compared with the PK properties of the drugs using 
appropriate statistical methods.  
12.7. Interim analyses  
No formal interim analysis is planned.  
 
13. STUDY CONDUCT CONSIDERATIONS 
13.1. Regulatory and Ethical Considerations, Including the Informed Consent Process 
The study will be conducted in accordance with all applicable regulatory requirements. The 
study will also be conducted in accordance with good clinical practice (GCP), all applicable 
subject privacy requirements, and, the guiding principles of the Declaration of Helsinki.  
13.2. Risks  
Artemether-lumefantrine  
Reported Artemether-lumefantrine side effects have generally been mild. The common side 
effects are headache (56%), anorexia (40%), dizziness (39%), asthenia (38%), GI disorders 
(loss of appetite 40%, nausea 26%, vomiting 17%, abdominal pain 17%), arthralgia (34%), 
myalgia (32%), sleep disorder (22%), palpitations (18%), fatigue (17%). Rare but could be 
serious side effects are hepatomegaly (9%) and anemia (4%).  
 
Amodiaquine  
The main side effects of amodiaquine are nausea, vomiting and fatigue which are mild to 
moderate in nature. Transient sinus bradycardia and QTc prolongation were reported in 
malaria patients treated with amodiaquine (16 and 7 of 20 patients, respectively), but did not 
result in any symptoms (Ngouesse et al., 2001). Transient neurological problems were 
reported in drug overdose. When the drug was used for a long period such as in malaria 
prophylaxis (now not recommended), rare adverse reactions of agranulocytosis (1:2000-
2200), hepatoxicity (1:15600), yellow pigmentation, and ocular changes including 
irreversible retinal abnormalities were observed. 
Risk of Blood Collection 
Sample blood taking may cause some pain, bleeding and bruising. Rarely, a local skin infection 
may develop. Occasionally, a subject may feel lightheaded or faint when blood is drawn. Blood 
for the pharmacokinetic study will be drawn via an indwelling catheter whenever possible to 
minimize the number of venipunctures.  
13.3. Benefit 
There are no anticipated direct benefits to the subjects in this study. Knowledge gained from 
this study is expected to help find the drug dosage for optimal treatment regimen.   

Protocol version 6.0 dated 22 June 2022                                                                             31 of 43  
13.4. Confidentiality 
The information obtained during the conduct of this clinical study is confidential. The results 
of the research study may be published, but subject names or identities will not be revealed. 
Records will remain confidential. To maintain confidentiality, the principal investigators at the 
study site will keep records in locked cabinets and subjects will be only identified by subject 
number and initial to prevent association with the subject’s name.  
The monitor(s), the auditor(s), members of the Ethics Committee, and regulatory authorities 
may be able to access your medical and study records. However, any information is still 
confidential and your name will not be identified in the publications of this research outcome. 
With subject’s consent, subject’s clinical data and results from blood analyses stored in our 
database may be shared with other researchers to use in the future. However, the other 
researchers will not be given any information that could identify the subject. 
13.5. Compensation    
Subjects will receive compensation for traveling costs associated with traveling to the study 
site and loss of work time during their stays in the hospital. The amount of compensation will 
be 500 baht/day for screening visit (visit 1) and each follow up visit after admission visit 2-4 
and 6,000 baht for 4 nights admission (visit 2-4). Total compensation will be around 28,000 
baht for screening, all admission visits and follow up visits.  
If the subject is withdrawn from the study before the study ends, the subject will be 
compensated for the time that the subject is actually enrolled in the study. 
13.6. Data Management  
The data collection tool for this study will be approved case report forms (CRFs). Subject data 
necessary for analysis and reporting will be entered/transmitted into a validated database or 
data system. Clinical data management will be performed in accordance with applicable 
standards and data cleaning procedures.  
Study records will be stored for a period of 10 years after completion of the study, while 
electronic data will be retained indefinitely. 
13.7. Study Monitoring  
The trial will be conducted in compliance with this protocol, International Conference on 
Harmonization (ICH) Guidelines for Good Clinical Practice (GCP) and any applicable 
regulatory requirement(s). Monitors will visit the clinical research sites to monitor all aspects 
of the study in accordance with the appropriate regulations. The objectives of a monitoring visit 
will be: 
1) to verify the prompt reporting of all data points, including reporting SAEs, checking 
availability of signed informed consent 
2) to compare individual subject records and the source documents (supporting data, laboratory 
specimen records and medical records to include physician progress notes, nurses’ notes, 
subjects’ hospital charts) 
3) to ensure protection of study subjects, compliance with the protocol, and accuracy and 
completeness of records 
The monitors will be involved with the site initiation visit, study close out visit and interim 
monitoring visits the number of which will be determined by the quality of the site.  In addition, 
the monitor will inspect the clinical sites regulatory files to ensure that regulatory requirements 
are being followed. During the monitoring visits, the investigator (and/or designee) and other 

Protocol version 6.0 dated 22 June 2022                                                                             32 of 43  
study personnel will be available to discuss the study progress and monitoring visit. 
13.8. Protocol Deviation and Violation 
A protocol deviation is a variance in a research activity between that described in the protocol 
that has been reviewed and approved by the IRB/EC and the actual activities or procedures that 
were performed. A protocol deviation may be classified as minor or significant.  A “significant” 
protocol deviation is also referred to as a protocol violation.   
Only the site Investigator may institute emergency departures from the protocol that eliminate 
an apparent immediate hazard to a subject and that is deemed crucial for the safety and 
wellbeing of that subject.  The site IRB/EC must be made aware of these departures from the 
protocol in accordance with local requirements. 
Protocol Deviation 
A protocol deviation is an activity performed that deviates from the approved protocol but does 
not significantly affect the subject’s safety and/or efficacy assessment of the trials (no 
immediate hazard or data falsification).  These deviations are to be noted on the “Protocol 
Deviation /Violation Form” which will be reviewed by the monitor during monitoring visits.  
The following are examples of protocol deviations: 
• 
Missed tests or study procedures 
• 
Late or missed visit by a subject. 
Protocol Violation 
A protocol violation is an activity that has the potential to or is affecting the safety, rights and 
wellbeing of subjects and/or efficacy assessment of the trial.  If a protocol violation is identified 
by either site staff or the monitor, the site Investigator needs to complete the Protocol 
Deviation/Violation Form.   
The following are some examples of activities that deviate from the protocol that will be 
considered protocol violations that should be reported: 
• 
Omission or inadequate administration of informed consent form 
• 
Error of study drug administration 
• 
Enrolling a subject who does not meet the study criteria for enrollment 
• 
Subject who developed withdrawal criteria during the study but was not withdrawn 
• 
Any activity that has the potential to or is affecting the subject’s rights, safety or welfare 
13.9  Insurance Policy  
The University of Oxford has a specialist insurance policy in place: - Newline Underwriting 
Management Ltd, at Lloyd’s of London – which would operate in the event of any Subject 
suffering harm as a result of their involvement in the research.” 
13.10. Publication Policy  
Any data published in the peer-reviewed medical literature will protect the identity of the 
subjects. This trial will be registered in a web based protocol registration scheme. All those 
who have made a substantial contribution will be co-authors on publications. 
 

Protocol version 6.0 dated 22 June 2022                                                                             33 of 43  
14. RESEARCH USE OF STORED HUMAN SAMPLES/SPECIMENS 
14.1. Intended Use of the Samples/Specimens 
Samples and data collected under this protocol will be used to study the pharmacokinetic 
parameters of artemether-lumefantrine and amodiaquine. 
14.2. Storage of Samples/Specimens 
Samples will be stored no longer than 10 years using codes assigned by the investigators or their 
designee(s). Access to research samples will be limited using either a locked room or a locked 
freezer.  
In the future, investigators from this protocol and other investigators besides those listed in this 
protocol may wish to study these samples. A proposal of the planned research will be submitted 
to the IRB for their consideration. 
14.3. Storage of Genetic Sample 
Any proposed plans to use this sample other than for the purposes of this study will be 
submitted to the IRB/EC prior to any genetic testing being performed. Genetic samples will 
remain in the country of collection until (if applicable) the IRB/EC approves shipment of 
genetic material for specific testing. A MTA (Material Transfer Agreement) will also be in 
place before any samples are sent to another institution for testing.  
15. REFERENCES 
1. Dondorp, A.M., et al. Artemisinin Resistance in Plasmodium falciparum Malaria. N 
Engl J Med, 2009;361(5): p. 455-67. doi:10.1056/NEJMoa0808859. 
2. World Health Organization (2010). Guideline for the treatment of malaria (second 
edition). 
3. Lubell, Y., et al. Artemisinin resistance – modeling the potential human and economic 
costs. Malar J, 2014, 13:452. 
4. Saunders, D.L., et al., Dihydroartemisinin-piperaquine failure in Cambodia. N Engl J 
Med, 2014. 371(5): p. 484-5. 
5. Na-Bangchang, K., et al. Declining in efficacy of a three-day combination regimen of 
mefloquine-artesunate in a multi-drug resistance area along the Thai-Myanmar 
border. Malar J, 2010, 9:273. 
6. Smithuis, F., et al., Effectiveness of five artemisinin combination regimens with or 
without primaquine in uncomplicated falciparum malaria: an open-label randomised 
trial. Lancet Infect Dis, 2010. 10(10): p. 673-81. 
7. Anvikar, A.R., et al., Artesunate-amodiaquine fixed dose combination for the treatment 
of Plasmodium falciparum malaria in India. Malar J, 2012. 11: p. 97. 
8. Faye, B., et al., Multicentre study evaluating the non-inferiority of the new paediatric 
formulation of artesunate/amodiaquine versus artemether/lumefantrine for the 
management of uncomplicated Plasmodium falciparum malaria in chil-dren in 
Cameroon, Ivory Coast and Senegal. Malar J, 2012. 11: p. 433. 
9. Thanh, N.X., et al., The efficacy and tolerability of artemisinin-piperaquine (Artequick) 
versus artesunate-amodiaquine (Coarsucam) for the treatment of un-complicated 
Plasmodium falciparum malaria in south-central Vietnam. Malar J, 2012. 11: p. 217. 

Protocol version 6.0 dated 22 June 2022                                                                             34 of 43  
10. Sylla, K., et al., Monitoring the efficacy and safety of three artemisinin based-
combinations therapies in Senegal: results from two years surveillance. BMC Infect 
Dis, 2013. 13: p. 598. 
11. Sidhu, A.B., D. Verdier Pinard, and D.A. Fidock, Chloroquine resistance in 
Plasmodium falciparum malaria parasites conferred by Pfcrt mutations. Sci-ence, 2002. 
298(5591): p. 210-3. 
12. Folarin, O.A., et al., In vitro amodiaquine resistance and its association with mutations 
in Pfcrt and Pfmdr1 genes of Plasmodium falciparum isolates from Nigeria. Acta Trop, 
2011. 120(3): p. 224-30. 
13. Sisowath, C., et al., In vivo selection of Plasmodium falciparum pfmdr1 86N coding 
alleles by artemether-lumefantrine (Coartem). J Infect Dis, 2005. 191(6): p. 1014-7. 
14. Venkatesan, M., et al., Polymorphisms in Plasmodium falciparum chloroquine 
resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect 
treatment outcomes for P. falciparum malaria after artemether-lumefantrine and 
artesunate-amodiaquine. Am J Trop Med Hyg, 2014. 91(4): p. 833-43. 
15. Boni, M.F., D.L. Smith, and R. Laxminarayan, Benefits of using multiple first-line 
therapies against malaria. Proc Natl Acad Sci U S A, 2008. 105(37): p. 14216-21. 
16. Takala-Harrison, S., et al., Genetic loci associated with delayed clearance of 
Plasmodium falciparum following artemisinin treatment in Southeast Asia. Proc Natl 
Acad Sci U S A, 2013. 110(1): p. 240-5. 
17. Coartem Prescribing Information, March 2015 
18. Summary of pre-clinical data of Amodiaquine supplied by Guilin Pharma 
19. Lefèvre, G., et al. Pharmacokinetics and electrocardiographic pharmacodynamics of 
artemether-lumefantrine (Riamet) with concomitant administration of ketoconazole in 
healthy subjects. Br J Clin Pharmacol. 2002;54(5):485-92. 
20. Khoo, S., Back, D., and Winstanley, P. The potential for interactions between 
antimalarial and antiretroviral drugs. Aids 2005;19(10): 995-1005. 
21. Li, X.Q., et al. Amodiaquine clearance and its metabolism to N-desethylamodiaquine 
is mediated by CYP2C8: a new high affinity and turnover enzyme-specific probe 
substrate. J Pharmacol Exp Ther. 2002 Feb;300(2):399-407. 
22. Bhupathi C, Vajjha VH. Sample size recommendation for a bioequivalent study. 
Statistica. 2017 Apr 7;77(1):65-71. 
 
 
 
 
 
 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             35 of 43  
Appendix 1 
Protocol Approval and Signature Page 
 
 
I have read and approve protocol titled A Randomized, Open-Label Study to Evaluate 
Potential Pharmacokinetic Interaction of Orally Administered Artemether-lumefantrine 
and Amodiaquine in Healthy Adult Subjects. I confirm that this study will be conducted 
according to all stipulations of the protocol, including all statements regarding confidentiality. 
 
I will conduct this study in accordance with all applicable regulations and Good Clinical 
Practice (GCP). 
 
 
 
_______________________________ 
 
_______________________________ 
(Assistant Professor  Borimas Hanboonkunupakarn)  
 
 
Date 
Principal Investigator   
 
 
 
  
 
  
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             36 of 43  
Appendix 2  
Time and Events Table 
Procedure 
Visit 1 
Within 14 days11 
Visits 2-4 
Follow up visit3,4,5,6,9,10,12 
Screening 
Day -1 
Day 0 
Day 1 
Day 2 
Day 3 
Informed Consent 
× 
 
 
 
 
 
Demographics 
× 
 
 
 
 
 
Medical Hx/ 
Medication Hx 
× 
× 
 
 
 
 
Check in to unit 
 
× 
 
 
 
 
Physical Exam and 
symptoms questionnaire 
× 
× 
× 
× 
× 
× 
Vital signs 
× 
×1 
×1 
×1 
×1 
×1 
Pregnancy test 
×2 
×3 
 
 
 
 
ECG  
× 
×4 
×4 
×4 
×4 
×4 
Holter monitoring  
 
× 
× 
× 
× 
× 
Clinical Lab5 
- Hematology 
- Chemistry 
- FBS 
- Urinalysis 
× 
× 
 
 
 
× 
24-hour urine for 
creatinine clearance test 
 
× 
 
 
× 
 
PK blood collection6 
 
 
× 
× 
× 
× 
Study Drug 
Administration7 
 
 
× 
(×) 
(×) 
 
PGx sample8 
 
(×) 
(×) 
(×) 
(×) 
(×) 
Adverse event9 
 
 
× 
× 
× 
× 
Concomitant 
medication10 
 
 
× 
× 
× 
× 
Discharge from unit 
 
 
 
 
 
× 
1. 
Vital signs will be measured before dosing and every 4 hours after first dose. Blood pressure (BP) 
and heart rate (HR) for visit 2-4 will be measured in both the supine and standing positions in each 
time-points 
2. 
Serum pregnancy test will be done at screening visit  
3. 
Urine pregnancy test will be done at day -1 for each admission visit and biweekly during follow up 
visit 
4. 
ECG (x3) will be performed according to table 2 
5. 
Clinical lab assessment will be performed at screening visit (visit 1), day -1 and day 3 of each 
admission visit (visit 2-4) and weekly during follow up visit (see appendix 4 for the detail) 
6. 
PK sample will be collected according to table 2 and appendix 4 
7. 
Study drug administration will be administered according to table 1 and 2 and appendix 3 
8. 
Genetic sample can be collected at any of the subsequent visit if the blood draw at the visit 2 is missed 
9. 
Adverse events will be monitored until the last follow up visit. 
10. Concomitant medications will be monitored until last follow up visit. 
11. If interval between screening (visit 1) and on day -1 visit 2 is three days or less, the clinical laboratory 
screening test result and serum pregnancy test result can be used for enrolment evaluation on day -1 
visit 2. In such cases, these tests would not need to be repeated at day-1 visit 2. 
12. Holter monitoring will be performed on day -1, admission visit, D7 and D28 of visit 2, visit 3 , and 
visit 4 
 

Protocol version 6.0 dated 22 June 2022                                                                             37 of 43  
Appendix 3 
Drug dosing schedule 
 
Artemether-lumefantrine dosing schedule 
One tablet A/L contains 20 mg artemether and 120 mg lumefantrine. 
 
No. of tablets recommended at approximate timing of dosing 
Weight:  
0 h 
8 h 
24 h 
36 h 
48 h 
60 h 
≥ 35 Kg 
4 
4 
4 
4 
4 
4 
 
 
Amodiaquine dispersible tablets dosing schedule 
One tablet contains 76.5 mg Amodiaquine. 
 
No. of tablets recommended at approximate timing of dosing 
Weight:  
0h 
8h 
24h 
36h 
48h 
60h 
35-41 Kg 
2 
2 
2 
2 
2 
2 
42-55 Kg 
3 
3 
3 
3 
3 
3 
≥ 56 Kg 
4 
4 
4 
4 
4 
4 
 
 
 
 
 
 
 
 
 
 
 

Protocol version 6.0 dated 22 June 2022                                                                             38 of 43  
Appendix 4 
Total Blood Collection 
 
visit 
laboratory 
assessments 
PK Sampling 
Blood sampling 
Visit 
1  
Screening visit 
Clinical lab (Chemistry and Serum 
pregnancy test, HIV, Hep B, Hep C 
and TSH 6 mL, Hematology 2 mL, 
FBS 2 mL) (Total 10 mL) 
10 mL 
0 
10 mL 
Visit 
2 
Day-1 (admission) 
Clinical lab (Chemistry 5 mL, 
Hematology 2 mL, and  FBS 2 mL) 
(Total 9 mL) 
9 mL 
0 
85 mL  
(Artemether -
lumefantrine 
only)  
Or 
100 mL (for 
amodiaquine 
only) 
Day 0-3 (admission) 
Clinical lab (Chemistry 5 mL, 
Hematology 2 mL, and FBS 2 mL) 
(Total 9 mL) 
Pharmacokinetic samples at  
Day 0: Hr. 0 (pre-dose), 0.25, 0.5, 
1, 2, 3, 4, 6, 8 (pre-dose), 10, 12, 14 
Day 1: Hr. 24 (pre-dose), 26, 28, 
30, 36 (pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 48.25, 
48.5, 49, 50, 51, 52, 54, 56, 60 (pre-
dose), 61, 62, 64, 66 
Day 3: Hr. 72  
total 35 time-points  (1 mL each 
time- points)  
9 mL 
35 mL 
Day 4 (+1) (follow up) 
Pharmacokinetic samples (1mL) 
0 
1 mL 
Day 7 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1mL) 
7 mL 
1 mL 
Day 14 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
7 mL 
1 mL 

Protocol version 6.0 dated 22 June 2022                                                                             39 of 43  
visit 
laboratory 
assessments 
PK Sampling 
Blood sampling 
(1mL) 
Day 21 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
7 mL 
0 
Day 28 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1 mL) 
7 mL 
1 mL 
Day 35 (+/-2d) (follow up) (for 
Amodiaquine only) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
7 mL 
0 
Day 42 (+/-2d) (follow up) (for 
Amodiaquine only) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1 mL) 
7 mL 
1 mL 
Visit 
3 
Day-1 (admission) 
Clinical lab (Chemistry 5 mL, 
Hematology 2 mL, and FBS 2 mL) 
(Total 9 mL) 
9 mL 
0 
85 mL  
(Artemether -
lumefantrine 
only)  
Or 
100 mL (for 
amodiaquine 
only) 
Day 0-3 (admission) 
Clinical lab (Chemistry 5 mL, 
Hematology 2 mL, and FBS 2mL) ( 
Total 9 mL) 
Pharmacokinetic sample collection 
at  
Day 0: Hr. 0 (pre-dose), 0.25, 0.5, 1, 2, 
3, 4, 6, 8 (pre-dose), 10, 12, 14 
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 48.25, 48.5, 
49, 50, 51, 52, 54, 56, 60 (pre-dose), 61, 
62, 64, 66 
Day 3: Hr. 72  
Total 35 time-points (1 mL each 
time- points) 
9 mL 
35 mL 

Protocol version 6.0 dated 22 June 2022                                                                             40 of 43  
visit 
laboratory 
assessments 
PK Sampling 
Blood sampling 
Day 4 (+1) (follow up) 
Pharmacokinetic sample collection 
(1 mL) 
0 
1 mL 
Day 7 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1 mL) 
7 mL 
1 mL 
Day 14 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1 mL) 
7 mL 
1 mL 
Day 21 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
7 mL 
0 
Day 28 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1mL) 
7 mL 
1 mL 
Day 35 (+/-2d) (follow up) (for 
Amodiaquine only) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
7 mL 
0 
Day 42 (+/-2d) (follow up) (for 
Amodiaquine only) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(1 mL) 
7 mL 
1 mL 
Visit 
4 
Day-1 (admission) 
Clinical lab (Chemistry 5 mL, 
Hematology 2 mL, and FBS 2 mL) 
(Total 9 mL) 
9 mL 
0 mL 
140 mL 
Day 0-3 (admission) 
Clinical lab (Chemistry 5 mL, 
9 mL 
70 mL 

Protocol version 6.0 dated 22 June 2022                                                                             41 of 43  
visit 
laboratory 
assessments 
PK Sampling 
Blood sampling 
Hematology 2mL ,and FBS 2mL) 
(Total 9 mL) 
Pharmacokinetic sample collection 
at  
Day 0: Hr. 0 (pre-dose), 0.25, 0.5, 1, 2, 
3, 4, 6, 8 (pre-dose), 10, 12, 14 
Day 1: Hr. 24 (pre-dose), 26, 28, 30, 36 
(pre-dose), 38, 40, 42 
Day 2: Hr. 48 (pre-dose), 48.25, 48.5, 
49, 50, 51, 52, 54, 56, 60 (pre-dose), 61, 
62, 64, 66 
Day 3: Hr. 72  
Total 35 time-points (2 mL each 
time- points) (1 mL for Artemether 
-lumefantrine and 1 mL for 
Amodiaquine 
Day 4 (+1) (follow up) 
Pharmacokinetic sample collection 
(2 mL) 
0 
2 mL 
Day 7 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and  
Hematology 2mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(2 mL) 
7 mL 
2 mL 
Day 14 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(2 mL) 
7 mL 
2 mL 
Day 21 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2mL) (Total 7 mL) 
7 mL 
0 
Day 28 (+/-2d) (follow up) 
Clinical lab (Chemistry 5 mL and 
Hematology 2mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(2 ml) 
7 mL 
2 mL 
Day 35 (+/-2d) (follow up)  
Clinical lab (Chemistry 5 mL and 
7 mL 
0 

Protocol version 6.0 dated 22 June 2022                                                                             42 of 43  
visit 
laboratory 
assessments 
PK Sampling 
Blood sampling 
Hematology 2mL) (Total 7 mL) 
Day 42 (+/-2d) (follow up)  
Clinical lab (Chemistry 5 mL and 
Hematology 2 mL) (Total 7 mL) 
Pharmacokinetic sample collection 
(2 mL) 
7 mL 
2 mL 
Genetic sampling (10 mL) 
Genetic sample can be collected at any 
schedule visits.  
10 mL 
 
10 mL 
Total 
345  mL