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0066-4804/10/$12.00 doi:10.1128/AAC.01526-09
Copyright 2010, American Society for Microbiology. All Rights Reserved.
* Corresponding author. Mailing address for A. Nzila: Kenya Medical Research Institute/Wellcome Trust Collaborative Research Program, P.O. Box 230, 80108 Kilifi, Kenya. Phone: (254) 417-522-535.
Fax: (254) 417-522-290. E-mail: anzila@kilifi.kemri-wellcome.org.
Mailing address for T. T. Diagana: Novartis Institute for Tropical
Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670.
Phone: 65 67222988. Fax: 65 63047152. E-mail: thierry.diagana
@novartis.com.
Supplemental material for this article may be found at http://aac
.asm.org/.
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NZILA ET AL.
2605
TABLE 1. Results of competitive binding kinetics experiments on DHFR enzymes of P. falciparum WT (WT-PfDHFR) and quadruple
mutant (QM-PfDHFR S108N, N51I, C59R, and I164L) and human (hDHFR) against QN254, WR99210, PM, and cycloguanila
Mean Ki (nM) SD
Agent
CLG
PM
WR99210
QN254
a
Ki ratio
WTPfDHFR
QMPfDHFR
hDHFR
hDHFR/WTpfDHFR
QM-PfDHFR/
WT-PfDHFR
1.51 0.1
0.59 0.05
0.5 0.1
0.39 0.05
454 38
385 163
1.9 0.8
0.58 0.06
55.6 7.8
30.8 1.4
7.7 0.4
10.2 0.6
37
52
15
26
300
652
1.8
1.5
200 for oral dosing and model 201 for i.v. dosing. The oral bioavailability (F) was
calculated as the ratio between the area under the curve (AUC) after p.o.
administration and the AUC after i.v. administration corrected for dose (F
AUC p.o. dose i.v./AUC i.v. dose p.o.).
In vivo antimalarial efficacy studies. All in vivo efficacy studies were carried
out at the Swiss Tropical Institute, adhering to the regulations of the veterinary
authorities of Basel-Stadt. The murine P. berghei ANKA was used as previously
described (37). Experimental and control groups of five female NMRI mice (20
to 22 g) were infected i.v. with 108 parasitized erythrocytes/ml, in a volume of 0.2
ml (from a fresh mouse donor). In untreated control mice, parasitemia rises
regularly to 30% by day 3 postinfection and causes death of the animals
between day 5 and day 7 postinfection.
Experimental compounds were prepared in 0.5% carboxymethyl cellulose and
administered orally. Comparators (artesunate, chloroquine, and mefloquine)
were prepared in ETPGS vehicle (10% ethanol, 30% PEG 400, and 60% of a
10% vitamin ETPGS solution) and administered orally. Parasitemia was determined microscopically. The activity was calculated as the difference between
parasitemia for the control and treated groups expressed as a percentage relative
to the control group. The survival time was also recorded up to 30 days after
infection. A compound was considered curative if the animal survived to day 30
after infection with no detectable parasites. The 50% effective dose (ED50) and
ED90 values were assessed at day 3 by plotting the dose as a function of parasitemia, using nonlinear fitting with the Microcal Origin Statistical Program
(OriginLab, Northampton, MA). All data reported are based on 10 mice,
except for the three-times treatment with QN254 which, because of the observed
toxicity, were performed only once with a cohort of five mice.
Rat toxicological study. QN254 was suspended in 10% of a 1% (vol/vol)
aqueous Solutol HS15 solution and 90% of a 0.5% aqueous methylcellulose
M0555 solution and administered to groups of five male rats at daily oral (by
gavage) doses of 50, 150, or 500 mg/kg/day. Animals (Wistar rats; Harlan Laboratories, Ltd., Fu
llinsdorf, Switzerland) were approximately 10 weeks of age
(254 to 292 g) at the start of dosing. Clinical observations, body weight and food
consumption determinations, clinical pathology (hematology and clinical chemistry) evaluations, and gross pathology examinations without organ weight determinations (due to the premature sacrifice of all treated groups) were performed on all groups. Microscopic examinations were conducted on all gross
lesions and on a limited standard list of organs and tissues from animals assigned
to the control and low-dose groups. At day 1 and day 8 blood samples were
collected for toxicokinetic analyses.
RESULTS
Inhibitory activity (Ki): QN254 is a potent inhibitor of both
the WT and quadruple mutant plasmodium DHFR enzymes.
QN254 was compared to the antimalarials PM and cycloguanil
(inactive on QM PfDHFR), as well as the triazine WR99210, a
potent inhibitor of all mutant PfDHFRs, including QM
PfDHFR (20). The results of these experiments are summarized in Table 1. Consistent with previously published results
showing that QN254 is active against the highly PM-resistant
strain V1S, our results show that QN254 displays binding affinities comparable to WR99210 against both WT PfDHFR
(Ki 0.39 0.05 nM) and QM PfDHFR (Ki 0.58 0.06
nM). It is also worth noting that QN254 appears to show a
slightly better selectivity than WR99210, as shown by the
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NZILA ET AL.
DHFR genotypeb
Double mutant*
Triple mutant*
Quadruple mutant
a
PM
QN254
371 (8)
778.28 (24)
3,690.79 (1)
4.48 (6)
11.66 (20)
7.61 (1)
2607
Parasitemia
reduction (%)
Avg mouse
survival (days)
Cure
rate
(%)
Toxicity
rate
(%)c
QN254
1 10
1 15
1 30
1 60
1 100
3 30
3 60
3 100
59
73
99.99
99.99
99.99
99.99
99.99
99.99
6.0
6.0
8.1
12.0
23.4
28.4
NAd
NA
0
0
0
0
50
80
60
20
0
0
0
0
0
0
40*
80*
Artesunate
1 100
3 30
98
98.7
7.0
12.2
0
0
0
0
Chloroquine
1 100
3 30
99.6
99.8
12.5
14.3
0
0
0
0
Mefloquine
1 100
3 30
89
98.8
25.5
22.1
0
0
0
0
a
Results represent the percent inhibition of parasitemia compared to untreated controls. QN254 was prepared in 0.5% carboxymethyl cellulose. The
mean survival of control animals was 5.6 to 6.2 days.
b
The solution formulation for artesunate, chloroquine, and mefloquine consisted of 10% ethanol, 30% PEG 400 (polyethylene glycol 400), and 60% of a
10% vitamin-ETPGS solution.
c
*, Mice died around day 10 and were parasite-free. There were 10 mice per
test group except for the 3 treatment with QN254 because of ethical reasons
due to the observed toxicity.
d
NA, not available.
2608
NZILA ET AL.
17.
ACKNOWLEDGMENTS
We thank the director of the Kenya Medical Research Institute for
permission to publish these data.
S.K. is an international research scholar of Howard Hughes Medical Institute. The Kenya Medical Research Institute received support from the EU Commission under Framework 6 as part of the
AntiMal Integrated Project 018834, the European and Developing
Countries Clinical Trials Partnership, and the Wellcome Trust
WT077092. BIOTEC was supported by grants from Thailand-TDR
(T-2) and the Medicines for Malaria Venture. Novartis Institute for
Tropical Diseases and the Swiss Tropical Institute receive funding
from a joint grant of the Medicines for Malaria Venture and the
Wellcome Trust (WT078285).
18.
19.
20.
21.
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