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Antimicrobial Agents and Chemotherapy, February 1999, p. 418-420, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
In Vitro Activities of Benflumetol against 158 Senegalese Isolates of Plasmodium falciparum in Comparison
with Those of Standard Antimalarial Drugs
Bruno
Pradines,1,*
Adama
Tall,2
Thierry
Fusai,1
Andre
Spiegel,2
Remi
Hienne,1
Christophe
Rogier,1
Jean Francois
Trape,3
Jacques
Le
Bras,4 and
Daniel
Parzy1
Unité de Parasitologie, Institut de
Médecine Tropicale du Service de Santé des Armées, Le
Pharo, Marseille,1 and
Centre
National de Référence de la Chimiosensibilité du
Paludisme, Hôpital Bichat, Paris,4 France,
and
Service d'Epidémiologie, Institut
Pasteur,2 and
Laboratoire de
Paludologie, ORSTOM,3 Dakar, Sénégal
Received 26 August 1998/Returned for modification 20 October
1998/Accepted 13 November 1998
 |
ABSTRACT |
The 50% inhibitory concentration (IC50s) of
benflumetol (range, 12.5 to 240 nM; mean, 55.1 nM) for 158 Senegalese
isolates were evaluated. Ten isolates (6%) showed decreased
susceptibility to benflumetol. Benflumetol was slightly more potent
against chloroquine-resistant isolates (P < 0.025).
No correlation or weak correlations in the responses to benflumetol and
pyrimethamine, chloroquine, amodiaquine, artemether, quinine, and
pyronaridine were observed, and these correlations are insufficient to
suggest cross-resistance. Benflumetol may be an important alternative
drug for the treatment of chloroquine-resistant malaria.
| |
TEXT |
Despite considerable efforts to
eradicate or control malaria, the disease continues to be a major cause
of human morbidity and mortality in the tropics. Malaria is a major
cause of death in children (20), usually within the first
24 h of treatment in a hospital (6). Although quinine
is effective in most patients in Africa, its onset of action is slow
and it has been postulated that a faster-acting drug would be more
effective for the sickest children. In addition, clinical failures with
quinine have been observed in Africa (5, 7). This has led to
a search for an effective alternative antimalarial drug with minimal
side effects.
Benflumetol is a racemic fluorene derivative with the chemical name
2-dibutylamino-1-[2,7-dichloro-9-(4-chlorobenzylydene)-9H-fluoren-4-yl]-ethanol. It conforms structurally and in mode of action to the structure and
mode of action of the aryl amino alcohol group of antimalarial drugs,
including quinine, mefloquine, and halofantrine. Investigations confirm
the accepted view that benflumetol exerts its antimalarial effect as a
consequence of its interaction with heme, a degradation product of
hemoglobin metabolism (17). A recent approach has been to
combine an artemisinin-derived antimalarial agent with proven efficacy,
artemether, with benflumetol. This approach is being jointly developed
by the Chinese Academy of Military Medical Sciences and Novartis
(Basel, Switzerland).
This combination offered a rapid and highly effective treatment for
acute uncomplicated falciparum malaria in China (21), Thailand (14), and Gambia (4, 16). No evidence of
substantial differences in activity between enantiomers of benflumetol
and racemic benflumetol were observed in vitro (18).
Benflumetol was also shown to be highly active against 61 Cameroonian
isolates (1).
The aim of this study was to assess the in vitro activity of
benflumetol against 158 Senegalese Plasmodium falciparum
field isolates and to compare its activity with those of chloroquine, quinine, amodiaquine, pyronaridine, artemether, and pyrimethamine.
Isolates of P. falciparum.
Between October and December
1996, 158 fresh P. falciparum isolates were obtained in
Dielmo and Ndiop (280 km southeast of Dakar), which are in the Fatick
Region of Senegal, and in Pikine, Senegal. Patients were treated with
chloroquine (the first-line drug in that area) and
sulfadoxine-pyrimethamine (the second-line drug). Venous blood was
collected before treatment and placed in Vacutainer ACD tubes (Becton
Dickinson, Rutherford, N.J.), and within 84 h the tubes were
transported at 4°C to our laboratory in Marseille, France. Thin blood
smears were stained by using an RAL kit (Réactifs RAL, Paris,
France) and were examined to determine the P. falciparum
density. Samples with parasitemia ranging from 0.01 to 9.4% were used
to test drug sensitivity.
Drugs.
Benflumetol was obtained from Novartis; chloroquine
diphosphate, quinine hydrochloride, amodiaquine, and pyrimethamine
dihydrochloride were from Sigma Chemical Co. (St. Louis, Mo.);
artemether was from Rhône Poulenc Rorer (Antony, France); and
pyronaridine phosphate (batch no. 210642) was from the World Health
Organization. Stock solutions were prepared in ethanol for benflumetol,
in sterile distilled water for chloroquine diphosphate, pyronaridine
phosphate, and amodiaquine dihydrochloride, and in methanol for
quinine, artemether, and pyrimethamine (methanol and ethanol had no
cytotoxicity for parasite growth at the methanolic and ethanolic
dilutions used). Twofold serial dilutions were prepared in ethanol for
benflumetol and in sterile distilled water for the other antimalarial
drugs (there was no evidence of precipitation of the antimalarial
agents in aqueous medium). The final concentrations, which ranged from 6.25 to 800 nM for benflumetol, 25 to 3,200 nM for chloroquine, 50 to
3,200 nM for quinine, 3.1 to 400 nM for amodiaquine, 0.8 to 100 nM for
pyronaridine, 0.8 to 100 nM for artemether, and 50 to 40,000 nM for
pyrimethamine, were distributed in triplicate into Falcon 96-well
flat-bottom plates.
In vitro assay.
The isotopic, micro drug susceptibility test
used in this study was described previously (11). The 50%
inhibitory concentration (IC50), i.e., the drug
concentration resulting in 50% of the uptake of
[3H]hypoxanthine by the parasites in drug-free control
wells, was determined by nonlinear regression analysis of log
dose-response curves. Data were expressed as the geometric mean
IC50, and 95% confidence intervals (CIs) were calculated.
The unpaired t test was used to compare the
IC50s for chloroquine-susceptible and chloroquine-resistant
isolates. Assessment of the cross-resistance of benflumetol with the
other antimalarial agents was estimated with the Pearson correlation
coefficient (r) and the coefficient of determination
(r2). A positive correlation in the response to
two drugs may be interpreted as resistance to the first drug that
facilitated resistance to the other drug (19). The cutoff of
reduced susceptibility to benflumetol in vitro has not yet been
determined. Isolates were considered chloroquine-resistant if the
IC50 was greater than 100 nM. Cutoff values for resistance
to quinine, amodiaquine, pyronaridine, artemether, and pyrimethamine
were 500, 80, 15 (11), 10.5 (10), and 2,000 nM,
respectively. The in vitro threshold value for resistance to
antimalarial agents has been defined statistically (>2 standard
deviations above the mean). Only in vitro resistance to chloroquine,
evaluated statistically, has been confirmed by correlation with
therapeutic effectiveness in vivo (13).
Results.
The IC50s of benflumetol for the 158 Senegalese wild isolates ranged from 12.5 to 240 nM (mean
IC50, 55.1 nM), and the 95% CI was 48.1 to 62.1 nM. Ten
isolates (6%) showed decreased susceptibility to benflumetol in vitro
(IC50, >150 nM). This in vitro threshold value for reduced
susceptibility to benflumetol was defined statistically (>2 standard
deviations above the mean). On the basis of our criterion for
resistance (IC50, >100 nM), 78 of 158 fresh isolates of
P. falciparum studied were considered to be chloroquine
resistant (Table 1). Benflumetol was
slightly more potent against chloroquine-resistant isolates than
against chloroquine-susceptible parasites (P < 0.025). There was a significant positive correlation between the responses to
benflumetol and artemether (r = 0.40; P < 0.001),
benflumetol and quinine (r = 0.37; P < 0.001),
and benflumetol and pyronaridine (r = 0.32; P < 0.001) and a nonsignificant positive correlation between the
responses to benflumetol and amodiaquine (r = 0.22; P < 0.05) (Table 2). A negative
nonsignificant correlation in the response to benflumetol and
chloroquine (r = 
0.11; P > 0.1) was found. No
correlation between the responses to benflumetol and pyrimethamine was
observed (r = 0.04; P > 0.1).
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TABLE 1.
In vitro susceptibilities of 158 Senegalese isolates of
P. falciparum to benflumetol, chloroquine, quinine,
amodiaquine, pyronaridine, artemether, and pyrimethamine
|
|
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TABLE 2.
Correlation of in vitro responses of 158 Senegalese
isolates of P. falciparum to benflumetol, artemether,
chloroquine, quinine, amodiaquine, pyronaridine, and pyrimethamine
|
|
Discussion.
Patients were treated with chloroquine (the
first-line drug in Senegal) and sulfadoxine-pyrimethamine (the
second-line drug). In a study carried out in the Fatick Region of
Senegal in 1995, 29% of isolates exhibited resistance to chloroquine
(9). That study revealed an increase in the prevalence
(49%) of in vitro resistance to chloroquine in 1996. The in vitro
activity of benflumetol was four- to sixfold greater than that of
quinine, and benflumetol was even three times as effective against
chloroquine-resistant isolates. As shown in Table 1, benflumetol was
slightly more potent against chloroquine-resistant isolates than
against chloroquine-susceptible isolates (P < 0.025)
whereas quinine (P < 0.001), pyronaridine (P < 0.002), and amodiaquine (P < 0.025) were less potent against chloroquine-resistant parasites.
Artemether and pyrimethamine were equally effective against
chloroquine-sensitive and chloroquine-resistant isolates. For 10 isolates (6%) benflumetol IC50s were greater than 150 nM,
suggesting reduced susceptibility in vitro. This finding could indicate
that some P. falciparum isolates may be innately less
susceptible to benflumetol. The negative in vitro correlation between
the responses to chloroquine and benflumetol remained below the
threshold of statistical significance (r = 0.11; P > 0.1). These observations led us to believe that benflumetol may be
an important alternative drug for the treatment of
chloroquine-resistant malaria.
A positive correlation between the IC50s of two
antimalarial drugs may suggest in vitro cross-resistance, but the
relationship between in vitro and in vivo resistance depends on the
level of resistance and the coefficients of correlation (r)
and determination (r2). The positive
correlations between the responses to benflumetol and artemether,
benflumetol and quinine, and benflumetol and pyronaridine that we
observed are insufficient to suggest cross-resistance: only 16% of the
variations in responses to benflumetol are explained by variations in
responses to artemether; these proportions are 14% for quinine and
11% for pyronaridine. From these data we can only suggest that common
mechanisms of action may explain the positive correlations between
these drugs. In addition, previous study showed that the susceptibility
of the parasites to artemether was enhanced by the presence of low
levels of benflumetol in the medium (8). This result
suggests that pharmacologically relevant concentrations of benflumetol
do potentiate the antimalarial activity of artemether.
Since the mechanisms underlying the antimalarial actions of
benflumetol, quinine, artemether, pyronaridine, and amodiaquine are
still unclear, one can only speculate about the positive correlations between responses to artemether and benflumetol. Benflumetol seems to
exert its antimalarial effect as a consequence of its interaction with
heme (17). It conforms structurally, physicochemically, and
in mode of action to the aryl amino alcohol group of antimalarial agents, including quinine, mefloquine, and halofantrine. Previous in
vitro studies in Cameroon have demonstrated a high correlation between
benflumetol and mefloquine and between benflumetol and halofantrine
(1). Several findings suggest that heme may be the common
link between quinoline antimalarial agents (2, 3, 12).
Benflumetol may be an important alternative drug for the treatment of
chloroquine-resistant malaria, despite the positive weak correlations
between responses to benflumetol and artemether, quinine, and
pyronaridine for P. falciparum isolates. However, positive
correlations in vitro suggest common features in drug uptake and/or
mode of action or resistance and reinforce the idea that a novel
antimalarial agent should not be used for monotherapy. In addition,
benflumetol and artemether have been reported to act in a synergistic
manner (8). This combination may be an important alternative
drug for the treatment of chloroquine-resistant malaria. Recent studies
have confirmed that the combination of artemether-benflumetol (CGP 56 697; Novartis) is effective in vivo and is very well tolerated in the
treatment of multidrug-resistant falciparum malaria (4,
14-16).
| |
ACKNOWLEDGMENTS |
This work was supported by le Ministère Français de la
Coopération et du Développement, la Direction Centrale du
Service de Santé des Armées, and the Groupement Recherche
en Parasitologie 1077.
We thank F. Ndiaye, L. V. Faye, F. Diene-Sarr, A. B. Ly, A. Badiane, C. Bouganali, J. Faye, M. Gning, E. H. Mbengue, O. Sarr, B. Thiam, and Y. Sonko for technical assistance with and availability for field work; D. Fouard for help in transporting isolates; and the
staff of the Tropical Medicine Institut of the French Army Health
Service (P. Bigot, W. Darries, R. Ges, J. Mosnier, D. Ragot, and Y. Trullemans) for technical support.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: IMTSSA,
Unité de Parasitologie, Blvd. Charles Livon, Parc le Pharo, BP
46, 13998 Marseille Armees, France. Phone: 33 4 91 15 01 50. Fax: 33 4 91 59 44 77. E-mail: imtssa{at}gulliver.fr.
| |
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Antimicrobial Agents and Chemotherapy, February 1999, p. 418-420, Vol. 43, No. 2
0066-4804/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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