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ABSTRACT |
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Resistance of Plasmodium falciparum to antimalarials makes chemotherapeutic choices increasingly difficult.1 Combinations with artemisinin derivatives are becoming progressively more used in countries in and outside Africa because these compounds produce a very rapid therapeutic response (reduction of the parasite biomass and resolution of symptoms), are active against multidrug-resistant P. falciparum, are well tolerated by the patients, and reduce gametocyte carriage; thus, they have the potential to reduce transmission of malaria.2 However, it is difficult to comply rapidly with the worldwide demand for artemisinins because the parent compound is extracted from plants (Artemisia annua), which require time to cultivate. One way to overcome this problem would be to produce synthetic peroxide antimalarials for which OZ277 is the lead candidate.3 OZ277 has shown in vitro activity against laboratory-adapted isolates of P. falciparum [IC50 values for the chloroquine-sensitive NF54: 0.91 ± 0.12 ng/mL for OZ277, compared with 1.6 ± 0.1 ng/mL for artesunate (AS), 5.1 ± 0.8 ng/mL for chloroquine (CQ), and 5.8 ± 0.2 ng/mL for mefloquine (MQ)] and rodent malaria parasites in vivo.3 To assess the distribution of inhibitory concentrations in the heterogeneous, natural parasite populations, we compared the in vitro activity of OZ277 with that of chloroquine, mefloquine, artesunate, and methylene blue (a known highly potent antimalarial) in fresh P. falciparum isolates from Colombia and Tanzania.
Clinical isolates of P. falciparum were collected with 
5000/µL parasitemias (~120 asexual parasites per 200 leukocytes) from patients visiting malaria clinics in the municipalities of Buenaventura (B), Cali (C), Tumaco (TC), and Quibdo (QU) in Colombia and 2000/µL (50 parasites per 200 leukocytes) in Ifakara in Tanzania. Patients older than 7 years (in Colombia) or from 0.5 to 5 years of age (in Tanzania) who had a negative urine Dill-Glazco test for CQ4 (in Colombia) and denied the use of any other antimalarial drug were included in the study. Signed written consent was obtained from all patients and parents/guardians in the case of children. The study was approved by the Institutional Review Board of CIDEIM and the Ethics Committee of the Ifakara Health Research & Development Center. Samples in Tanzania were collected from February to June 2004, and those in Colombia were collected from June 2004 to April 2005. Giemsa-stained thick and thin blood smears were examined to determine parasite densities and to confirm P. falciparum monoinfection.
Venous blood (5 mL) was collected into EDTA Vacutainers (Becton Dickinson, Cockeysville, MD) before patient treatment and processed either within 24 h of collection at CIDEIM or immediately at the Ifakara Health Research & Development Center. In vitro susceptibility to OZ277 tosylate (MW: 565), AS (MW: 384), MQ hydrochloride (MW: 415), CQ diphosphate (MW: 515), and methylene blue (MB) (MW: 374) was measured using a classic 48-h [3H]hypoxanthine incorporation assay.5 All compounds were provided by the Swiss Tropical Institute (except for MB, which was kindly provided by J. Vennerstrom, University of Nebraska), dissolved by sonication in DMSO (10 mg/mL), and kept at 4°C until use. The highest final drug concentrations in the plate were 10 ng/mL for OZ277, AS, MQ, and MB and 100 ng/mL for CQ. Because some samples had low susceptibility, drug concentrations were increased during the study for OZ277 to 20 ng/mL (Tanzania), MQ to 100 ng/mL (both sites), and CQ to 200 ng/mL (both sites). Compounds were prediluted in screening medium (RPMI 1640 supplemented with 0.5% ALBUMAX I in Colombia and ALBUMAX II in Tanzania, 25 mM HEPES, 25 mM NaHCO3, pH 7.3) and 100 µg/mL gentamicin in Colombia or neomycin in Tanzania. Infected erythrocytes (100 µL per 96-well plate) with 2.5% hematocrit and parasitemia ranging from 0.07% to 1.1% (Colombia) and from 0.04% to 3.3% (Tanzania) were added in duplicate to each drug solution (100 µL, titrated over a 64-fold range). After 24 h of incubation at 37°C in an atmosphere of 5% CO2, 0.5 µCi of [3H]hypoxanthine in 50 µL of screening medium was added; plates were then incubated for an additional 24 h. Parasites were harvested onto glass-fiber filters, and radioactivity was counted using a Micro-Betaplate liquid scintillation counter (from Wallac, Zurich, Switzerland in Tanzania; from Beckman, CA, in Colombia). The results were recorded as counts per minute (cpm) per well at each drug concentration and expressed as a percentage of the untreated controls. Fifty-percent inhibitory concentrations (IC50) were estimated by linear interpolation.6 To ensure data quality, IC50 values with coefficients of variation of > 20% and those in which incorporation in the positive control well (parasites without drug) was < 4x the background (uninfected red blood cells) were not included in the analysis.
The median IC50 values and the corresponding ranges were calculated for each drug separately for Colombian and Tanzanian samples that were divided into two groups according to initial parasitemia (< 0.5% and 0.5%). No IC50 could be determined in some samples (1/38 for OZ277 in Colombia; 1/38 for AS in Colombia, 4/38 and 11/44 for MQ at both sites; and 18/36 and 6/37 for CQ at both sites) when the starting compound concentrations were too low. These samples could not be reprocessed as they had to be tested fresh. Correlations of IC50 values between OZ277 and the other compounds were investigated using Spearman’s correlation coefficient. A P value < 5% was considered statistically significant.
A total of 97 samples were collected (50 in Colombia and 47 in Tanzania), 83 of which fulfilled criteria for successful culture. In Colombia, 38 of the samples that yielded interpretable results were treated with OZ277, AS, and MQ; 37 with MB; and 36 with CQ. In Tanzania, 45 samples were included in the analysis of OZ277 and AS; 44 samples of MQ; and 40 samples of CQ. The geometric mean parasitemia was 0.2% versus 0.17% in the < 0.5% group and 1.09% versus 0.78% in the 0.5% group in Tanzania and Colombia, respectively.
OZ277 showed IC50 values comparable to those of AS. In our data, within the < 0.5% parasitemia group, the observed OZ277 IC50 values (Table 1
) varied from 0.22 to 10 (45x) in Tanzania and from 0.34 to 8 (23x) in Colombia. This variation is wider than the one (18x) observed in Gabon (0.07 to 1.26 ng/mL), where the HRP2 in vitro test was used and parasitemias of all samples were normalized to 0.05%.7 The effect of the initial parasitemia on the drug susceptibility of P. falciparum in vitro could also be reproduced with laboratory-adapted strain NF54 (data not shown). Adjusting parasitemias to 0.1% to 0.5% for isotopic assays has been recommended.8 Hence, when testing antimalarials in vitro, the use of similar starting parasitemias can minimize experimental variations, especially for drugs that partition within the infected red blood cell like CQ,9 artemisinin,10,11 and OZ277 do.7,12 The samples in the < 0.5% group with an OZ277 IC50 value of 10 ng/mL in Tanzania (N = 1) and > 10 ng/mL in Colombia (N = 1) had a parasitemia of 0.48% and 0.28%, respectively. This may indicate previously existing resistance, although it should be noted that a 10 ng/mL AS IC50 value in Tanzania and > 10 ng/mL in Colombia were observed in different isolates, and no clinical resistance has so far been reported for AS or any other artemisinin derivative.
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, group < 0.5%). MB was recently reconsidered as a useful antimalarial drug and has been explored as a combination agent with CQ and artemisinin derivatives.14 Our data from Colombian isolates indicate that MB might indeed be a promising option for treating drug-resistant malaria. There was a significant positive correlation in the IC50 values of OZ277 with all the other drugs except CQ. The correlation between OZ277 and AS was r = 0.78 (P < 0.001) in Tanzania and r = 0.45 (P = 0.006) in Colombia, and that between OZ277 and MQ was r = 0.48 (P = 0.001) in Tanzania and r = 0.62 (P < 0.001) in Colombia. Correlations between OZ277 and AS are not surprising because both molecules are peroxides, and therefore they might share similar mechanisms of action. Similar correlations have been observed earlier with Gabonese P. falciparum isolates.7 However, the positive correlation between in vitro susceptibility of OZ277 and MQ, which we observed at both sites, was not found in Gabon.7 This observation should be kept in mind and could be potentially of some concern due to the presence of MQ resistance, particularly in southeast Asia. On the other hand, the clinical significance of this correlation could be low because AS and MQ have also shown a positive correlation in vitro but, nevertheless, a high therapeutic efficacy when used in combination.15
We conclude that our in vitro results demonstrate that OZ277 has excellent activity against freshly obtained, CQ-resistant P. falciparum field isolates. This reinforces in vitro data obtained with laboratory isolates and from animal models3 as well as previous work performed with P. falciparum field isolates from another location.7 It is necessary to standardize the initial parasitemias in order to compare the in vitro efficacy of OZ277 in different locations.
Received September 4, 2006. Accepted for publication November 4, 2006.
Acknowledgments: We are grateful to Rubiela Giraldo, Monica Banguero, Claudia Quelal, and Meleny Ramirez, who collected the samples in Colombia, and to Marcel Tanner, Hassan Mshinda, and the diagnostic team from the Ifakara Health Research & Development Centre in Tanzania.
Financial support: This study was supported by the Medicines for Malaria Venture (MMV).
* Address correspondence to Lyda Osorio, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte #3-03, Cali, Colombia. E-mail: lydaosorio{at}cideim.org.co, lyda.osorio{at}lshtm.ac.uk 
Authors’ addresses: Lyda Osorio, Claribel Murillo, and Samanda Aponte, International Center for Medical Research and Training (CIDEIM), Avenida 1 Norte #3-03, Cali, Colombia, Telephone: +57 2 668 2164, Fax: +57 2 667 2989, E-mail: lyda.osorio{at}cideim.org.co, claribel_murillo{at}cideim.org.co, and samanda_aponte{at}cideim.org.co. Valeriana Mayagaya, Ifakara Health Research & Development Centre, P.O. Box 53, Ifakara, Tanzania, Telephone: +255-23-2625164, E-mail: mayagaya6{at}yahoo.com. Christian Scheurer, Reto Brun, and Sergio Wittlin, Swiss Tropical Institute (STI), Socinstrasse 57, CH-4002 Basel, Switzerland, Telephone: +41-61-284-8136, Fax +41-61-284-8101, E-mail: christian.scheurer{at}unibas.ch, reto.brun{at}unibas.ch, and sergio.wittlin{at}unibas.ch. Hugues Matile, F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, CH-4070 Basel, Switzerland, Telephone: +41-61- 688-3191, Fax: +41-61-688-2438, E-mail: hugues.matile{at}roche.com.
Reprint requests: Lyda Osorio, CIDEIM Avenida 1 Norte #3-03 Cali, Colombia.
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