In vitro activity of ferroquine (SSR 97193) against Plasmodium falciparum isolates from the Thai-Burmese border
© Barends et al; licensee BioMed Central Ltd. 2007
Received: 01 March 2007
Accepted: 27 June 2007
Published: 27 June 2007
On the borders of Thailand, Plasmodium falciparum has become resistant to nearly all available drugs, and there is an urgent need to find new antimalarial drugs or drug combinations. Ferroquine (SSR97193) is a new 4-aminoquinoline antimalarial active against chloroquine resistant and sensitive P. falciparum strains in vivo and in vitro. This antimalarial organic iron complex (a ferrocenyl group has been associated with chloroquine) is meant to use the affinity of Plasmodium for iron to increase the probability for encountering the anti-malarial molecule.
The aim of the present study was to investigate the activity of ferroquine against P. falciparum isolates from an area with a known high multi-drug resistance rate.
Parasite isolates were obtained from patients with acute falciparum malaria attending the clinics of SMRU. In vitro cultures of these isolates were set-up in the SMRU-laboratory on pre-dosed drug plates, and grown in culture for 42 hours. Parasite growth was assessed by the double-site enzyme-linked pLDH immunodetection (DELI) assay.
Sixty-five P. falciparum isolates were successfully grown in culture. The ferroquine mean IC50 (95% CI) was 9.3 nM (95% C.I.: 8.7 – 10.0). The mean IC50 value for the principal metabolite of ferroquin, SR97213A, was 37.0 nM (95% C.I.: 34.3 – 39.9), which is four times less active than ferroquine. The isolates in this study were highly multi-drug resistant but ferroquine was more active than chloroquine, quinine, mefloquine and piperaquine. Only artesunate was more active than ferroquine. Weak but significant correlations were found between ferroquine and its principal metabolite (r2 = 0.4288), chloroquine (r2 = 0.1107) and lumefantrine (r2 = 0.2364).
The results presented in this study demonstrate that the new ferroquine compound SSR97193 has high anti-malarial activity in vitro against multi-drug resistant P. falciparum.
The emergence of drug resistant malaria is a serious threat for malarial control [1, 2]. Currently, chloroquine-resistant Plasmodium falciparum parasites are prevalent in most of the tropics, and in many areas resistance is high grade (i.e. potentially dangerous, with early treatment failures occurring) [1, 2]. The rapid spread of chloroquine resistance has forced clinicians in many regions of the world to abandon classical therapy with chloroquine, in favour of other drugs that are less well tolerated and, importantly, more expensive. The situation in South-east Asia is of particular concern with increasingly frequent cases of 'multi-drug' resistant malaria . On the borders of Thailand, P. falciparum has become resistant to nearly all available drugs [4, 5]. These observations highlight the urgent need to find new antimalarial drugs or drug combinations and to develop optimal treatment protocols.
Publications on the activity of ferroquine against P. falciparum isolates
Drug IC50 (Geometric mean)
No. of isolates
Pradine et al. 
Atteke et al. 
Pradine et al. 
Chim et al. 
The aim of the present study was to confirm the efficiency of ferroquine against P. falciparum isolates from patients treated for malaria in one of the SMRU clinics along the Thai-Burmese border. This area has a known high multi-drug-resistance rate, with very high resistance against chloroquine .
Isolates of Plasmodium falciparum
Between November 2004 and May 2005 parasite isolates were obtained from patients with acute falciparum malaria attending the clinics of the Shoklo Malaria Research Unit (SMRU). The clinics are open for both migrants and refugees and are situated in an area of forested hills on the northwestern border of Thailand. Isolates were collected if the parasitaemia was at least 0.1%, by taking 5 cc whole blood by venopunture into sterile Vacutainer® tubes containing 0.05 ml K3 EDTA. The blood samples were transported within 4–6 hours at room temperature and then set up in continuous culture upon their arrival in the SMRU laboratory in Mae Sot, not more than one-hour drive from the study sites. Samples were only provided by patients who gave written, informed consent following written and oral explanations given in their own language. This study was part of a series of treatment trials approved by the Ethical Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand.
In vitro drug assay
Ferroquine (FQ) (SSR97193: 7-chloro-4-[(2-N,N-dimethyl-aminomethyl) ferrocenylmethylamino]quinoline), and ferroquine metabolite (FQM) (SR97213A: C22H22ClFeN3) (Figure 1) were obtained from Sanofi-Synthelabo-Recherche, France. Chloroquine diphosphate (CQ), quinine citrate (QN) and doxycyline hydrochloride (DOX) were obtained from Sigma Chemicals, UK. Lumefantrine (LUM) was obtained from Novartis Pharmacia, Basel, Switzerland. Sodium artesunate (AS), piperaquine (PIP) and mefloquine (MFQ) were kindly donated by Dr. Niklas Lindegård, Faculty of Tropical Medicine, Mahidol University, Thailand. FQ and FQM were dissolved in DMSO, QN, MFQ and AS in 70% ethanol, CQ and DOX in deionised water, PIP in 100% methanol and LUM was dissolved in a 1:1:1 (w/v) mixture of ethanol, Triton-X (Sigma), linoleic acid (Sigma). All drugs were dissolved initially at a concentration of 1 mg/ml, and serial dilutions were made in complete RPMI medium. The solvent in the final concentrations had no significant effect on parasite growth when compared to culture media. All concentrations, including drug-free controls, were distributed in duplicate in 96-well tissue culture plates. The drug-plates were made in bulk and stored at -80°C until use (for up to three months).
For each sample, plasma and buffy coat were removed after centrifugation and the red cells washed three times in phosphate buffered saline (PBS). The infected red blood cells were set-up in the pre-dosed drug plates in complete RPMI with 10% heterologous sera, at a parasitaemia of 0.1% parasitized erythrocytes and a haematocrit of 1.5%. The plates were incubated at 37°C in the presence of 5% CO2, 90% N2 and 5% O2 for 42 hours. After culture the plates were frozen down at -20°C.
The chloroquine-resistant P. falciparum laboratory clone K1 was used for quality control of the drug-plates.
The double-site enzyme-linked pLDH immunodetection (DELI) assay was used to assess P. falciparum antimalarial drug susceptibility. The DELI method was performed as described previously [15, 16]. In brief, the culture plates were thawed and frozen three times order to lyse the cells. 100 μl from each well were transferred into 96-well plates (Nunc-Immuno™ plate, maxisorb, Nalgene Nunc International, Denmark) pre-coated with a capture monoclonal antibody 17E4, which specifically recognizes the pLDH, incubated for 1 hour at 37°C. Following washing 3× with PBS/0.5% bovine serum albumin (BSA Fraction V) (Roche Diagnostics, Mannheim, Germany), a second biotinylated anti-pLDH monoclonal antibody 19G7 was added and the plates incubated for 1 hour at 37°C. After removal of unbound antibody by washing 3× with PBS/0.5% BSA, the plates were incubated at room temperature for 30 minutes with a 1:10,000 solution of streptavidin-POD conjugate (Roche Diagnostics). After washing the plates 3× with PBS/0.5% BSA, the plates were incubated for up to 20 minutes at room temperature with a peroxidase substrate solution, 3,3',5,5'-tetramethylbenzidine (KPL, Maryland, USA). The reaction was stopped with 1 M phosphoric acid and colour development was quantified immediately using a spectrophotometer to determine the OD at 450 nm with a reference filter at 690 nm.
Analysis of dose response curves
Dose response curves, IC50 values, and coefficients of variation were calculated by fitting the data to an inhibitory E-max pharmacokinetic model using WINNONLIN Ver 4.1 (Pharsight Corporation). To ensure data quality we rejected all IC50 values with coefficients of variation (Standard Error × 100)/Mean) of estimated IC50 values > 30% and those in which the pLDH production in control wells (parasites, no drug) was < 5 times background (red blood cells only).
Data were analysed using the program SPSS 11.0 for Windows (SPSS Inc., Chicago, Illinois, USA). Prior to analysis, in vitro drug response data were normalized by log transformation. Ferroquine cross-resistance with the other antimalarials (metabolite, chloroquine, artesunate, quinine, mefloquine, lumefantrine, doxycyclin and piperaquine) was estimated by Pearson correlation coefficient (r) and coefficient of determination (r2).
The in vitro IC50 responses of the 65 isolates of Plasmodium falciparum to ferroquine, ferroquine metabolite, chloroquine, artesunate and quinine. Ferroquine cross-resistance with the other antimalarials was estimated by Pearson correlation coefficient (r), and coefficient of determination (r2)
Mean inhibitory concentration (nM)
No. of isolates
95% confidence interval
8.69 – 9.96
34.32 – 39.89
304.04 – 381.89
3.06 – 6.28
894.36 – 1154.29
The in vitro IC50 responses of a subset of Plasmodium falciparum isolates (n = 22) to Ferroquine, Ferroquine metabolite, Doxycyxline, Lumefantrine, Mefloquine, and Piperaquine. Ferroquine cross-resistance with the other antimalarials was estimated by Pearson correlation coefficient (r), and coefficient of determination (r2).
Mean inhibitory concentration (nM)
No. of isolates
95% confidence interval
8.17 – 9.72
28.16 – 35.01
2236.38 – 3307.09
10.31 – 13.44
125.82 – 165.33
37.16 – 65.11
This study confirms that ferroquine (SSR97193) is highly active against P. falciparum in vitro. The P. falciparum isolates analysed in this study were highly multi-drug resistant but ferroquine was more active than chloroquine, quinine, mefloquine, piperaquine and doxycycline. Only artesunate was more active than ferroquine. In addition, ferroquine was four times more active in vitro than the tested principal ferroquine metabolite (SR97213A). This underlines the importance to test the biological activity of the plasma and whole blood of treated patients or volunteers, as other metabolites may be active.
Weak but significant correlations between the response to ferroquine and that to chloroquine and lumefantrine were found. The high activity of ferroquine on chloroquine-resistant P. falciparum (the lowest CQ IC50 analysed is 102.4 nM) suggests either that both drugs have different modes of action, or that ferroquine reverses chloroquine resistance . Chloroquine is believed to act by concentrating in the parasite digestive vacuole and preventing the conversion of toxic heme to haemozoin, leading to membrane damage and parasite death [17, 18]. Biot et. al. recently demonstrated that ferroquine, like chloroquine, forms complexes with haematin and is an even stronger inhibitor of β-haematin formation than chloroquine . Chloroquine-resistant parasites expel chloroquine much more rapidly from red blood cells than chloroquine-sensitive parasites. This efflux of chloroquine is catalyzed by a P. falciparum transmembrane protein (Pf CRT) . Ferroquine may block the Pf CRT through its lipophilic properties, acting like a resistance-reversing agent .
So far, no resistance of P. falciparum to ferroquine has been found in vitro either in cultures of patient isolates or in laboratory-adapted strains under drug pressure .
In Table 1, the published studies on the in vitro susceptibility to ferroquine and chloroquine are summarized. The isolates tested in the present study show comparable in vitro sensitivity to ferroquine to that found in Gabon (Libreville)  and Senegal . Isolates from Cambodia have the highest IC50 values for ferroquine. However, caution must be exerted when comparing these results because in the present study a DELI method rather than an isotopic assay is used. Previous reports have demonstrated that DELI does slightly overestimate the IC50 for chloroquine and lumefantrine, and underestimate for artesunate, compared to the isotopic microtest . No data on the direct comparison of DELI with the isotopic microtest for ferroquine levels are available as yet.
The results presented in this study indicate that ferroquine is active in vitro regardless of high grade multi-drug resistance. Still further research is needed to elucidate the mode of action of ferroquine and identify the putative molecular markers of resistance. In addition, since chloroquine resistance is also found in Plasmodium vivax , the activity of ferroquine to P. vivax should be studied.
We would like to thank all the patients and staff of the Shoklo Malaria Research Unit. We are also grateful to Sanofi-Aventis and Dr. Niklas Lindegårdh for providing the drugs used in the in vitro assays. This study received financial support from Sanofi-Aventis. The Shoklo Malaria Research Unit is part of the Wellcome Trust-Mahidol University, Oxford Tropical Medicine Research Programme sponsored by The Wellcome Trust of Great Britain.
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