and extracted as described above, with the exception that 1 mM
quinine was used as an internal standard. Typically, IC
determinations were performed in triplicate at seven inhibitor
concentrations, ranging from 0 to 100 m
M. Inhibitor concentrations
were adjusted as needed to adequately span the IC
Analysis of AQ and metabolites. AQ and its major metabolites were
detected and quantiﬁed using HPLC with UV detection. The HPLC
system consisted of an Agilent 1100 Series System with an HP
G1311A quaternary pump, an HP G1322A vacuum degasser, an HP
G1314A UV/Vis detector, and an HP G1313A automated liquid
sampler. A reverse-phase Vydac C
column (4.6 250 mm, 10 mM
particle size) was used for analyte separation. The mobile phase
consisted of water with 0.1% triﬂuoroacetic acid (A) and 95%
acetonitrile with 0.08% triﬂuoroacetic acid (B). The gradient was
initiated and maintained at 15% B for 5 min, followed by a linear
gradient to 19% B over 20 min. Chromatography was carried out at
a ﬂow rate of 1.0 ml/min and efﬂuent was monitored at 340 nm.
Statistical analysis. All data points represent the means of triplicate
, and IC
data were determined by
nonlinear regression analysis using Prism 4.0 (GraphPad software).
Kinetic data were analyzed using a paired t-test with two-tailed
signiﬁcance value. Statistical associations between alleles and
treatment outcome or adverse events were assessed by w
We thank the clinical study teams and technicians in the dispensaries of
Colsama, Sarlafao, and Ouezzin-Ville and the study participants and their
parents/guardians. We also thank members of the Kroetz lab for their
assistance. Financial support for this work was provided by the National
Institutes of Health (NIH) (5K23AI060681 and GM61390) and in part by
the Intramural Research Program of the NIH, National Institute of
Environmental Health Sciences.
CONFLICT OF INTEREST
The authors declared no conflict of interest.
& 2007 American Society for Clinical Pharmacology and Therapeutics
1. Lopez, A.D., Mathers, C.D., Ezzati, M., Jamison, D.T. & Murray, C.J.
Global and regional burden of disease and risk factors, 2001:
systematic analysis of population health data. Lancet 367, 1747–1757
2. Breman, J.G., Alilio, M.S. & Mills, A. Conquering the intolerable burden
of malaria: what’s new, what’s needed: a summary. Am. J. Trop. Med.
Hyg. 71, 1–15 (2004).
3. Snow, R.W., Guerra, C.A., Noor, A.M., Myint, H.Y. & Hay, S.I. The global
distribution of clinical episodes of Plasmodium falciparum malaria.
Nature 434, 214–217 (2005).
4. World Health Organization. Guidelines for the Treatment of Malaria
(WHO Press, Geneva, Switzerland, 2006).
5. Phillips-Howard, P.A. & West, L.J. Serious adverse drug reactions to
pyrimethamine-sulphadoxine, pyrimethamine-dapsone and to
amodiaquine in Britain. J. R. Soc. Med. 83, 82–85 (1990).
6. Hatton, C.S. et al. Frequency of severe neutropenia associated with
amodiaquine prophylaxis against malaria. Lancet 1, 411–414 (1986).
7. Raymond, J.M., Dumas, F., Baldit, C., Couzigou, P., Beraud, C. &
Amouretti, M. Fatal acute hepatitis due to amodiaquine. J. Clin.
Gastroenterol. 11, 602–603 (1989).
8. Centers for Disease Control. Agranulocytosis associated with the use
of amodiaquine for malaria prophylaxis. MMWR Morb. Mortal. Wkly.
Rep. 35, 165–166 (1986).
9. Olliaro, P. et al. Systematic review of amodiaquine treatment in
uncomplicated malaria. Lancet 348, 1196–1201 (1996).
10. Olliaro, P. & Mussano, P. Amodiaquine for treating malaria. Cochrane
Database Syst. Rev. 2, CD000016 (2003).
11. Li, X.Q., Bjorkman, A., Andersson, T.B., Ridderstrom, M. &
Masimirembwa, C.M. 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. 300, 399–407 (2002).
12. Li, X.Q., Bjorkman, A., Andersson, T.B., Gustafsson, L.L. &
Masimirembwa, C.M. Identification of human cytochrome P(450)s that
metabolise anti-parasitic drugs and predictions of in vivo drug hepatic
clearance from in vitro data. Eur. J. Clin. Pharmacol. 59, 429–442 (2003).
13. Shimada, T., Yamazaki, H., Mimura, M., Inui, Y. & Guengerich, F.P.
Interindividual variations in human liver cytochrome P-450 enzymes
involved in the oxidation of drugs, carcinogens and toxic chemicals:
studies with liver microsomes of 30 Japanese and 30 Caucasians.
J. Pharmacol. Exp. Ther. 270, 414–423 (1994).
14. Totah, R.A. & Rettie, A.E. Cytochrome P450 2C8: substrates, inhibitors,
pharmacogenetics, and clinical relevance. Clin. Pharmacol. Ther. 77,
15. Dai, D. et al. Polymorphisms in human CYP2C8 decrease metabolism
of the anticancer drug paclitaxel and arachidonic acid.
Pharmacogenetics 11, 597–607 (2001).
16. Hichiya, H. et al. Functional characterization of five novel CYP2C8
variants, G171S, R186X, R186G, K247R, and K383N, found in a
Japanese population. Drug. Metab. Dispos. 33, 630–636 (2005).
17. Soyama, A. et al. Five novel single nucleotide polymorphisms in the
CYP2C8 gene, one of which induces a frame-shift. Drug Metab.
Pharmacokinet. 17, 374–377 (2002).
18. Cavaco, I. et al. CYP2C8 polymorphism frequencies among malaria
patients in Zanzibar. Eur. J. Clin. Pharmacol. 61, 15–18 (2005).
19. Rower, S. et al. Short communication: high prevalence of the
cytochrome P450 2C8*2 mutation in Northern Ghana. Trop. Med. Int.
Health 10, 1271–1273 (2005).
20. Cavaco, I., Piedade, R., Gil, J.P. & Ribeiro, V. CYP2C8 polymorphism
among the Portuguese. Clin. Chem. Lab. Med. 44, 168–170 (2006).
21. Bahadur, N. et al. CYP2C8 polymorphisms in Caucasians and their
relationship with paclitaxel 6alpha-hydroxylase activity in human liver
microsomes. Biochem. Pharmacol. 64, 1579–1589 (2002).
22. Yasar, U. et al. Linkage between the CYP2C8 and CYP2C9 genetic
polymorphisms. Biochem. Biophys. Res. Commun. 299, 25–28 (2002).
23. Muthiah, Y.D., Lee, W.L., Teh, L.K., Ong, C.E. & Ismail, R. Genetic
polymorphism of CYP2C8 in three Malaysian ethnics: CYP2C8*2 and
CYP2C8*3 are found in Malaysian Indians. J. Clin. Pharm. Ther. 30,
24. Nakajima, M. et al. Genetic polymorphisms of CYP2C8 in Japanese
population. Drug Metab. Dispos. 31, 687–690 (2003).
25. Zongo, I. et al. Amodiaquine, sulfadoxine-pyrimethamine, and
combination therapy for uncomplicated falciparum malaria: a
randomized controlled trial from Burkina Faso. Am. J. Trop. Med. Hyg.
73, 826–832 (2005).
26. Churchill, F.C., Mount, D.L., Patchen, L.C. & Bjorkman, A. Isolation,
characterization and standardization of a major metabolite of
amodiaquine by chromatographic and spectroscopic methods.
J. Chromatogr. 377, 307–318 (1986).
27. Mount, D.L., Patchen, L.C., Nguyen-Dinh, P., Barber, A.M., Schwartz, I.K.
& Churchill, F.C. Sensitive analysis of blood for amodiaquine and three
metabolites by high-performance liquid chromatography with
electrochemical detection. J. Chromatogr. 383, 375–386 (1986).
28. Jewell, H., Maggs, J.L., Harrison, A.C., O’Neill, P.M., Ruscoe, J.E. & Park,
B.K. Role of hepatic metabolism in the bioactivation and detoxication
of amodiaquine. Xenobiotica 25, 199–217 (1995).
29. Ribera, E. et al. Rifampin reduces concentrations of trimethoprim
and sulfamethoxazole in serum in human immunodeficiency
virus-infected patients. Antimicrob. Agents Chemother. 45,
30. Bustos, D.G. et al. Pharmacokinetics of sequential and simultaneous
treatment with the combination chloroquine and
sulfadoxine-pyrimethamine in acute uncomplicated Plasmodium
falciparum malaria in the Philippines. Trop. Med. Int. Health 7, 584–591
31. van Heeswijk, R.P. et al. The steady-state pharmacokinetics of
nevirapine during once daily and twice daily dosing in HIV-1-infected
individuals. AIDS 14, F77–F82 (2000).
32. Walsky, R.L., Gaman, E.A. & Obach, R.S. Examination of 209 drugs for
inhibition of cytochrome P450 2C8. J. Clin. Pharmacol. 45, 68–78 (2005).
33. Veldkamp, A.I. et al. Steady-state pharmacokinetics of twice-daily
dosing of saquinavir plus ritonavir in HIV-1-infected individuals.
J. Acquir. Immune. Defic. Syndr. 27, 344–349 (2001).
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