Amodiaquine Metabolism is Impaired by
Common Polymorphisms in CYP2C8:
Implications for Malaria Treatment in Africa
S Parikh1, J-B Ouedraogo2, JA Goldstein3, PJ Rosenthal1and DL Kroetz4
Metabolism of the antimalarial drug amodiaquine (AQ) into its primary metabolite, N-desethylamodiaquine, is mediated
by CYP2C8. We studied the frequency of CYP2C8 variants in 275 malaria-infected patients in Burkina Faso, the
metabolism of AQ by CYP2C8 variants, and the impact of other drugs on AQ metabolism. The allele frequencies of
CYP2C8*2 and CYP2C8*3 were 0.155 and 0.003, respectively. No evidence was seen for influence of CYP2C8 genotype on
AQ efficacy or toxicity, but sample size limited these assessments. The variant most common in Africans, CYP2C8*2,
showed defective metabolism of AQ (threefold higher Kmand sixfold lower intrinsic clearance), and CYP2C8*3 had
markedly decreased activity. Considering drugs likely to be coadministered with AQ, the antiretroviral drugs efavirenz,
saquinavir, lopinavir, and tipranavir were potent CYP2C8 inhibitors at clinically relevant concentrations. Variable CYP2C8
activity owing to genetic variation and drug interactions may have important clinical implications for the efficacy and
toxicity of AQ.
Malaria, in particular that caused by Plasmodium falciparum,
remains among the leading causes of morbidity and mortality
in the developing world.1Recent estimates suggest that more
than 500 million episodes of P. falciparum malaria occurred
in 2002, leading to one to three million deaths.2,3The burden
of malaria is heaviest in sub-Saharan Africa, where resistance
to the most commonly employed antimalarials, in particular
chloroquine and sulfadoxine–pyrimethamine (PYR), is wide-
spread. In addition, given the level of transmission in many
areas, individuals may receive several short courses of
antimalarial therapy every year.2To combat the emergence
and spread of resistance, the World Health Organization
(WHO) has recommended the use of combination anti-
malarial therapy for P. falciparum malaria. Amodiaquine
(AQ), a 4-aminoquinoline similar in structure to chloro-
quine, is included in two of five recommended regimens,
AQþartesunate and AQþsulfadoxine–PYR.4In addition,
AQ monotherapy is still widely used to treat malaria in
AQ was introduced as an antimalarial in the 1940s. In the
mid-1980s, AQ administration increased largely owing to
increased prophylactic use in Western travelers. However,
several reports soon emerged suggesting an unacceptable level
of toxicity of AQ, in particular agranulocytosis (estimated
1:2,100 users with a fatality rate of 1:31,000)5,6
hepatotoxicity (1:15,600 with numerous fatalities).5,7Re-
dropped, and the WHO removed AQ from its Essential
Drugs List in 1990.8However, subsequent review has
suggested that AQ toxicity was primarily seen in non-
Africans receiving long-term chemoprophylaxis and the drug
was reinstated in 1996 by the WHO as an option for treating
malaria, with millions of dosages subsequently given each
The metabolism of AQ has been characterized in studies
using human liver microsomes and recombinant enzymes.
AQ is metabolized to its primary metabolite, N-desethyla-
modiaquine (DEAQ), by the cytochrome P450 (CYP) 2C8
enzyme,11,12which accounts for 7% of the total microsomal
CYP content of the liver13and is estimated to carry out
oxidative metabolism of at least 5% of drugs.14Three
relatively common sequence-altering variants are denoted as
nature publishing group
Received 10 November 2006; accepted 20 December 2006; published online 14 March 2007. doi:10.1038/sj.clpt.6100122
1Department of Medicine, San Francisco General Hospital, University of California, San Francisco, USA;2Institut de Recherche en Science de la Sante, Bobo-
Dioulasso, Burkina Faso;3Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research
Triangle Park, North Carolina, USA;4Department of Biopharmaceutical Sciences, School of Pharmacy, University of California, San Francisco, USA. Correspondence:
S Parikh (firstname.lastname@example.org)
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 82 NUMBER 2 | AUGUST 2007197
CYP2C8*2, CYP2C8*3, and CYP2C8*4, with the wild-type
denoted as CYP2C8*1 (Table 1, www.imm.ki.se/CYPal-
leles).15CYP2C8*2 is most prevalent in those of African
descent, whereas CYP2C8*3 is more prevalent among
Caucasians.15Several other single nucleotide polymorphisms
have been described, including coding polymorphisms and
two translational stop codons in Japanese, but at much lower
Both CYP2C8*2 and CYP2C8*3 are
defective in metabolism of the anticancer agent paclitaxel,
and CYP2C8*3 has reduced activity towards the endogenous
substrate arachidonic acid.15The aim of this study is to assess
the impact of CYP2C8 polymorphisms on AQ response and
toxicity in a cohort of malaria infected patients in Burkina
Faso and to examine the in vitro metabolism of AQ by
CYP2C8 variants. Additionally, we investigate the potential
for significant CYP2C8 inhibition by other drugs likely to be
coadministered with AQ in sub-Saharan Africa.
A total of 280 subjects in the AQ monotherapy arm
completed the clinical trial and had efficacy outcomes; 275
of these subjects had DNA available and were genotyped for
three nonsynonymous CYP2C8 variants (Table 2). All
genotypes were in Hardy–Weinberg equilibrium. The allelic
frequency for CYP2C8*2 (805A4T) was 0.115 and 25% of
the population were heterozygotes. Only five (2%) indivi-
duals were homozygotes for the CYP2C8*2 variant. The
CYP2C8*3 allele (416G4A and 1196A4G) was much less
common in this African population (allele frequency 0.004).
The CYP2C8*4 (792C4G) allele was not detected.
Association with treatment outcome and adverse events
Overall, 82.2% of participants in the AQ treatment arm of the
study responded successfully to treatment, with no evidence
of clinical malaria or parasitemia over 28 days of follow-up.25
Efficacy outcomes did not vary between CYP2C8*1 homo-
zygotes and CYP2C8*2 heterozygotes (Table 3). In addition,
time to therapeutic failure did not vary between these two
groups (data not shown). All five CYP2C8*2 homozygotes
responded to therapy.
Adverse events were uncommon in the AQ monotherapy
arm.25There was an increase in the self-reported rate of
abdominal pain in both heterozygotes and homozygotes for
the variant CYP2C8*2 genotype compared with wild-type
genotype (52 vs 30%, Po0.01). No other associations were
seen between CYP2C8*2 genotype and specific adverse
events, including nausea, vomiting, fatigue, and jaundice.
AQ metabolism by recombinant CYP2C8 proteins
High-performance liquid chromatography (HPLC)/UV ana-
lysis for the metabolites of AQ after incubation with
recombinant CYP2C8 revealed a single metabolite. The
metabolite was identified as DEAQ by comigration with a
reference standard. Retention times for DEAQ, primaquine
diphosphate, and AQ were 14.8, 16.9, and 18.1min,
respectively (Figure 1). Formation of DEAQ in CYP2C8
incubations was linear with time and protein concentration.
Under the assay conditions used in this study, metabolites
such as N-bisDEAQ, 2-hydroxyDEAQ, or the M2 metabo-
lite, were not seen. This is consistent with the available li-
terature suggesting that metabolites such as M2 are formed
extrahepatically and the lack of evidence that CYP2C8
can catalyze the formation of metabolites other than
Formation of DEAQ by CYP2C8*1 exhibited typical
Michaelis–Menten kinetics (Figure 2). For the wild-type
allele, CYP2C8*1, the apparent Vmaxwas 0.2370.09mmol/
min/mmol P450, with a Km of 0.8170.23mM. The corre-
sponding intrinsic clearance (defined as Vmax/Km) was 0.30l/
min/mmol P450. The CYP2C8*2 allele had a significantly
lower Vmaxof 0.1670.06mmol/min/mmol P450 (P¼0.04)
and a threefold higher Km, 2.5571.06mM (P¼0.05). The
intrinsic clearance of AQ for CYP2C8*2 was sixfold lower
than that for CYP2C8*1 (0.05 vs 0.30l/min/mmol P450,
Po0.01). Metabolic activity was not sufficient to estimate
Table 1 Major CYP2C8 alleles in Caucasians and Africans
CYP2C8*3Exon 3, Exon 8416G4A, 1196A4GR139K, K399R
CYP2C8*4 Exon 5792C4GI264M
NA, not available.
Table 2 CYP2C8 allele frequencies in this study and other
Burkina Faso275 0.1150.004Not foundThis study
Northern Ghana2000.168Not foundNot found
African Americans820.1830.018Not found
British Caucasians116 0.004 0.1500.075
Sweden 1468Not reported 0.095Not reported
Malaysian Indians 1230.0080.12Not found
Japanese360Not foundNot foundNot found
Table 3 Association of CYP2C8*2 with treatment outcome and
CYP2C8*2 genotype, n (%)
ACPR164 (82) 57 (85) 5 (100)
Recrudescence20 (10)6 (9)0
New infection15 (7.5)4 (6)0
ACPR, adequate clinical and parasitological response.aParasite genotyping results
were not obtained for four specimens.
198VOLUME 82 NUMBER 2 | AUGUST 2007 | www.nature.com/cpt
adequately the kinetic parameters for CYP2C8*3, as no
metabolism was detectable until AQ concentrations of
15–25mM were tested and solubility prevented measurements
within the saturable range for this enzyme.
Interactions between recombinant CYP2C8 and other drugs
We studied six drugs for their impact on the AQ
N-desethylase activity of recombinant CYP2C8 proteins
(Table 4). Trimethoprim (TMP), a widely used dihydrofolate
12.7mM). These results correlated with previous reports.32,34
PYR, another dihydrofolate reductase inhibitor, also inhibited
CYP2C8 at similar concentrations (IC50¼45.1712.2mM).
Efavirenz, an antiretroviral non-nucleoside reverse transcrip-
tase inhibitor that is widely used to treat human immuno
deficiency virus (HIV) infection, was a potent inhibitor of
CYP2C8 at clinically relevant concentrations (IC50¼4.07
2.5mM). HIV-1 protease inhibitors were also potent inhibitors
of CYP2C8-mediated AQ desethylase activity: saquinavir,
lopinavir, and tipranavir all inhibited the metabolism of AQ
at clinically relevant concentrations.
Few studies are available on the impact of genetic variation
on the metabolism of commonly used antimalarial drugs. We
describe the influence of CYP2C8 polymorphisms on the
metabolism of AQ. In our study population in Burkina Faso,
the variant allele CYP2C8*2 was common, with a prevalence
of 11.5% and the variant most prevalent in Caucasians,
CYP2C8*3, was rare.20,21These data correlate well with other
published allelic frequencies from West Africa (Table 2).
Compared with the wild-type enzyme, CYP2C8*2 showed a
threefold higher Kmand sixfold lower intrinsic clearance for
AQ. These results are consistent with but of greater
magnitude than the twofold higher Km of recombinant
CYP2C8*2 for paclitaxel and the twofold increase in intrinsic
clearance for that substrate.15The decreased AQ desethylase
activity of the CYP2C8*3 variant was more profound than
that of the CYP2C8*2 variant, suggesting that effects of
reduced metabolism of AQ will be most pronounced in
CYP2C8*3 carriers. Indeed, no appreciable metabolism by
CYP2C8*3 was detectable until AQ substrate concentrations
nearly 15–20-fold higher than the Km of the wild-type
enzyme were tested. CYP2C8*3 also had extremely low
turnover for paclitaxel (6% of that of wild-type CYP2C8*1)
and a threefold lower turnover number for arachidonic acid.
In vivo studies have shown a 4.5-fold increase in the half-life
of (R)-ibuprofen in subjects homozygous for the CYP2C8*3
Absorbance (UV 340 nm)
Retention time (min)
Figure 1 Chromatographic separation of (1) DEAQ, (2) primaquine internal
standard, and (3) AQ. This chromatogram shows the product formation from
an incubation of AQ with recombinant CYP2C8*1 protein.
Figure 2 Plot of velocity vs AQ concentration for the formation of
desethylamodiaquine by recombinant CYP2C8*1 and CYP2C8*2 proteins.
Each point is the mean velocity from triplicate determinations at a given
concentration and the lines were drawn using the estimated
Table 4 IC50values for inhibitors of recombinant CYP2C8
Systemic concentration (mM)
Drug IC50(mM)7SE Cmax
Saquinavir 1.870.8 5.5e
Lopinavir 4.170.6 15.6f
NA, not available; SE, standard error. IC50 data are means7SD from four
experiments. Serum concentrations are from published information. Cmaxand Cmin
are the mean maximum and minimum serum levels achieved under standard dosing
intervals, respectively.aOn the basis of trimethoprim 160mg q.i.d. dosing in HIV-
infected patients.29 bOn the basis of pyrimethamine 750mg single dose.30 cOn the
basis of efavirenz 600mg q.i.d. in HIV-infected individuals (Brystol–Myers Squibb
prescribing information).dOn the basis of nevirapine 200mg b.i.d. in HIV-1-infected
individuals31and Walsky et al.32 eOn the basis of saquinavir 1200mg t.i.d (as free
base) in HIV-infected individuals, and coadministered saquinavir soft gel capsule.
1,000mg/ritonavir 100mg b.i.d. in HIV-infected individuals (Roche prescribing
information).33 fOn the basis of lopinavir 400mg/ritonavir 100mg b.i.d. in HIV-
infected individuals (Abbott prescribing information).
500mg/ritonavir 200mg b.i.d. in HIV-infected individuals (Boehringer Ingelheim
Pharmaceuticals prescribing information).hOn the basis of ritonavir 600mg b.i.d. in
healthy and HIV-infected individuals (Abbott prescribing information) and Walsky
et al.32 iOn the basis of nelfinavir mesylate 1250mg b.i.d. in HIV-infected individuals;
Cminwas determined before morning dosage (Agouron prescribing information) and
Walsky et al.32
gOn the basis of tipranavir
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 82 NUMBER 2 | AUGUST 2007199
allele compared with the CYP2C9*1 allele.35Our results for
AQ metabolism by CYP2C8 variants and the reported
findings for paclitaxel, arachidonic acid, and ibuprofen
illustrate that the magnitude of the effect of these
polymorphisms depends on the substrate and the relation-
ship of substrate concentration to Km.
AQ is predominantly metabolized into a single major
metabolite, DEAQ.11,12Several other metabolites have been
described, but were not identified in our studies (Figure 3).
Metabolism into DEAQ occurs rapidly, with no AQ
detectable within a few hours of oral intake (terminal half-
life, 5.2h).36,37In contrast, DEAQ is present in the blood for
an extended period after therapy, with a terminal half-life of
9–18 days and wide interindividual variability in plasma
levels.36,38Both AQ and DEAQ have antimalarial activity, but
AQ is up to threefold more potent.39Nonetheless, owing to
its much higher concentrations, DEAQ is considered the
major active component.39Given our findings of impaired
conversion of AQ into DEAQ by the CYP2C8*2 and
CYP2C8*3 variants, it would be predicted that for both
variants AQ and DEAQ, concentration–time profiles would
be significantly altered, possibly contributing to the observed
pharmacokinetic variability.38However, it is not possible that
such alterations in AQ metabolism would affect therapeutic
efficacy, as both the parent drug and metabolite are active.
Indeed, in our study no impact on antimalarial efficacy was
demonstrated for the common CYP2C8*2 variant.
Of possibly greater importance than impacts on ther-
apeutic efficacy are potential effects of CYP2C8 variants on
AQ toxicity. As noted above, long-term usage of AQ for
malaria chemoprophylaxis led to important risks of blood
dyscrasias and hepatic disorders.6,40,41Mechanisms of AQ
toxicity are not known, but both AQ and DEAQ led to
inhibitory effects on bone marrow progenitor cells and
neutrophil function in vitro.42–46Additionally, several lines of
evidence suggest that AQ toxicity is mediated through the
productionof an immunologically
mine,47,48a finding supported by the demonstration of
anti-AQ antibodies in individuals with AQ-associated
toxicity.49–51Importantly, DEAQ is less readily activated to
AQ.21,52,53One would thus expect that individuals with
impaired metabolism of AQ into DEAQ may be at increased
risk of toxicity because of higher levels of AQ. We propose
that the severe toxicity seen with AQ usage as a chemopro-
phylactic may have been influenced by a relatively high
prevalence of the functionally defective CYP2C8*3 variant in
Caucasians (15–20%). Although the enzymatic defect with
the CYP2C8*2 allele prevalent in Africa (15–18% frequency)
is less profound than that for CYP2C8*3, individuals with the
variant gene might experience decreased tolerability and
increased toxicity with AQ. It is reassuring that our study did
not identify common toxicities or diminished tolerability for
AQ in CYP2C8*2 variants. Nonetheless, as AQ is now widely
used, with individuals probably receiving repeated courses of
therapy, careful monitoring of larger samples for adverse
events related to AQ and for associations with the CYP2C8*2
genotype are warranted.
(3× more active than
DEAQ, terminal t1/2 5 h)
terminal t1/2 >100 h
Mount DL 1986;
Jewell H, 1995)
(Li Q, 2002)
Mount DL, 1986;
Jewell H, 1995)
More readily formed by amodiaquine
(Harrison 1992; Jewell 1995)
Figure 3 Metabolic pathway for AQ showing the formation of the active DEAQ, other minor metabolites, and the reactive quinoneimine implicated in
immunotoxicity of AQ.
200VOLUME 82 NUMBER 2 | AUGUST 2007 | www.nature.com/cpt
We evaluated the impact of several drugs on CYP2C8-
mediated AQ metabolism. TMP is a component of
TMP–sulfamethoxasole, which is widely used to prevent
secondary infections in HIV-infected individuals and recom-
mended for daily usage in all HIV-1-infected Africans by
some authorities.54The related dihydrofolate reductase
inhibitor, PYR, is coformulated with sulfadoxine and
commonly administered with AQ to treat malaria.55In our
study, both TMP and PYR inhibited CYP2C8, but only at
concentrations not achieved by standard dosing of these
drugs. Of particular interest for interactions with AQ are
antiretroviral drugs that are increasingly available to treat the
more than 25 million HIV-infected individuals in Africa.56
Considering non-nucleoside reverse transcriptase inhibitors,
nevirapine did not appreciably inhibit CYP2C8,28but
efavirenz was a potent inhibitor, with an IC50 below the
mean minimum serum levels achieved under standard dosing
intervals (Cmin) achieved with standard dosing. Considering
antiretroviral protease inhibitors, previous studies showed
that ritonavir (at high doses), but not nelfinavir, were potent
CYP2C8 inhibitors.28In our study, saquinavir, lopinavir, and
tipranavir were potent CYP2C8 inhibitors at clinically
relevant concentrations. In particular, tipranavir had an
IC50 against CYP2C8 approximately 20-fold below the
minimum serum concentration achieved during standard
dosing. Given the tremendous burden of HIV–malaria
coinfection in Africa, such drug interactions are an important
In addition, the inhibition of CYP2C8 by
efavirenz, saquinavir, lopinavir, and tipranavir has implica-
tions not only for AQ administration but also for other
substrates of CYP2C8 such as paclitaxel, thiazolidinediones,
repaglinide, amiodarone, arachidonic acid, loperamide, and
morphine.14,57It will be of interest to examine the potential
impact of antiretroviral–antimalarial interactions in clinical
We have described a more significant impact of a CYP2C8
variant on the metabolism of AQ than previously described
for any drug. These findings may explain, at least in part, AQ-
associated toxicity seen with long-term chemoprophylactic
usage in primarily Caucasian populations and they suggest
that differences in AQ efficacy and toxicity may also be
associated with different CYP2C8 genotypes in Africans.
With the high prevalence of the CYP2C8*2 variant in Africa,
and with increasing use of multiple drugs that may affect
CYP2C8-mediated metabolism, additional study of the
metabolism of AQ and careful monitoring for AQ-associated
toxicity are warranted.
Chemicals. Amodiaquine dihydrochloride (AQ), quinine, PYR, and
dilauroylphosphatidylcholine were obtained from Sigma-Aldrich (St
Louis, MO). Primaquine diphosphate and TMP were obtained from
MP Biomedicals (Solon, OH). The antiretrovirals saquinavir (as free
base), lopinavir, tipranavir, and efavirenz were obtained through the
AIDS Reference and Reagent Program, Division of AIDS, NIAID,
NIH. DEAQ, NADPþ, glucose-6-phosphate, and glucose-6-phos-
phate dehydrogenase were obtained from BD Biosciences Discovery
Labware (San Jose, CA). HPLC-grade acetonitrile was purchased
from Fischer Scientific (Hampton, NH). Recombinant rat CYP
reductase was obtained from Fengyun Xu (University of California,
San Francisco, CA).58
Subjects and clinical study. Details of the clinical study have been
published.25Briefly, residents of Bobo-Dioulasso, Burkina Faso
more than 6 months of age with uncomplicated falciparum malaria
were randomized to receive sulfadoxine–PYR, AQ, or AQ plus
sulfadoxine–PYR in 2004. For this substudy, only samples from
patients treated with AQ monotherapy were analyzed. Patients were
followed for 28 days and treatment outcomes were classified
according to WHO guidelines with parasite genotyping performed
to distinguish true failures (recrudescences) from new infections.4At
each follow-up visit study, clinicians assessed patients for adverse
events, defined as any untoward medical occurrence, following
International Conference on Harmonization guidelines.25The study
was approved by the institutional review boards of the University of
California, San Francisco and the Centre Muraz, Bobo-Dioulasso,
Burkina Faso. All research subjects or their parents or guardians
approved the use of clinical specimens for genetic testing.
CYP2C8 genotyping and sequencing. For genetic analysis, DNAwas
extracted from filter paper with chelex. Genotyping for CYP2C8*2,
*3, and *4 variants was performed using predesigned primers and
probes for the TaqMan 5’nuclease allelic discrimination assay on an
ABI 7500 real-time polymerase chain reaction system (Applied
Biosystems, Foster City, CA). Variant alleles are listed in Table 1.
Reactions were carried out with the following protocol: 951C for
10min, then 50 cycles at 921C for 15s and 601C for 90s. For
confirmation of TaqMan results, random samples were amplified
and sequenced. Polymerase chain reaction primers for sequencing
were as described15and their products were purified with ExoSAP-
IT before direct sequencing (GE Healthcare Bio-Sciences Corp.,
Metabolism of AQ by recombinant CYP2C8. Recombinant wild-type
and variant CYP2C8 allelic proteins were expressed in Escherichia
coli and partially purified as described previously in the laboratory
of author JAG (National Institute of Environmental Health Sciences,
NC). To study enzyme activities, recombinant CYP2C8 proteins
(5pmol) and rat NADPH–CYP reductase (4pmol/pmol P450) were
P450) and incubated at room temperature for 3min. The
reconstituted enzymes were then preincubated in 0.1 M KPO4buffer,
pH 7.4, containing AQ substrate for 5min at 371C. Reactions were
initiated by the addition of 1.3mM NADPþ, 3.3mM glucose-6-
phosphate, and 0.4 U/ml glucose-6-phosphate dehydrogenase in a
final volume of 250ml, incubated at 371C for 15min, and terminated
with the addition of 125ml ice-cold acetonitrile and 1mM
14,000g for 10min, and supernatant was analyzed by HPLC. To
determine enzyme kinetics, AQ was studied at eight different
concentrations ranging from 0 to 100mM. All solvent concentrations
were maintained at o0.1%.
Inhibition of AQ metabolism by selected antibiotics and antiretrovir-
als. Supersomes from baculovirus-infected cells expressing human
CYP2C8 were from BD Biosciences. CYP2C8 (2.5pmol) was pre-
incubated with inhibitor (TMP, PYR, saquinavir, lopinavir,
tipranavir, or efavirenz) and buffer (0.1 M KPO4, pH 7.4) at 371C
for 5min, AQ substrate was added (1mM, approximately the enzyme
Km) and incubated for an additional 5min; the reaction was
initiated by the addition of NADPþ, glucose-6-phosphate, and
glucose-6-phosphate dehydrogenase to a final reaction volume of
250ml and incubated at 371C for 15min. Reactions were terminated
CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 82 NUMBER 2 | AUGUST 2007 201
and extracted as described above, with the exception that 1mM
quinine was used as an internal standard. Typically, IC50
determinations were performed in triplicate at seven inhibitor
concentrations, ranging from 0 to 100mM. Inhibitor concentrations
were adjusted as needed to adequately span the IC50.
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 C18column (4.6?250mm, 10mM
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 5min, followed by a linear
gradient to 19% B over 20min. Chromatography was carried out at
a flow rate of 1.0ml/min and effluent was monitored at 340nm.
Statistical analysis. All data points represent the means of triplicate
determinations. Km, Vmax, and IC50 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 w2test.
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.
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