Iron chelators as antimalarial agents: in vitro activity of dicatecholate against Plasmodium falciparum

B. Pradines1,2,*, J. M. Rolain2,3, F. Ramiandrasoa4, T. Fusai1,2, J. Mosnier1,2, C. Rogier1,2, W. Daries1,2, E. Baret1,2, G. Kunesch4, J. Le Bras5 and D. Parzy1,2

1 Unité de Parasitologie, Institut de Médecine Tropicale du Service de Santé des Armées, Le Pharo, 13007 Marseille; 2 Institut Fédératif de la Recherche 48, 13000 Marseille; 3 Unité des Rickettsies, CNRS-UPRES A 6020, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille; 4 Laboratoire de Chimie Bioorganique et Bioinorganique, CNRS URA 1384, Institut de Chimie Moléculaire d’Orsay, 91405 Orsay; 5 Centre National de Chimiosensibilité du Paludisme, Laboratoire de Parasitologie, Hôpital Bichat-Claude Bernard, 75018 Paris, France

Received 8 October 2001; returned 2 March 2002; revised 16 April 2002; accepted 29 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study was undertaken to explore the antimalarial effect of a series of dicatecholate iron chelators. They may be made more or less lipophilic by increasing or reducing the length of the R substituent on the nitrogen. In vitro activity against the W2 and 3D7 clones of Plasmodium falciparum, toxicity on Vero cells and toxicity on uninfected erythrocytes by measure of the released haemoglobin were assessed for each compound. These findings were compared with the ability of iron(III), iron(II) and ferritin to reverse the inhibitory effect of catecholates. This study shows that increased lipid solubility of catecholate iron chelators does not lead to improved antimalarial activity. However, their activity is well correlated with their interaction with iron and with their toxicity against Vero cells. This study demonstrates a potent antimalarial effect of FR160 (R = C9H19) on five different strains of P. falciparum in vitro. FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 µM. The effects of FR160 on mammalian cells were minimal compared with those obtained with malaria parasites. FR160 acted on parasites at considerably higher rates than desferrioxamine, and at all stages of parasite growth. The drug was more effective at the late trophozoite and young schizont stages, although FR160 affected rings and schizonts as well. Ascorbic acid, a free radical scavenger, reduced the activities of FR160 and artesunate. FR160 might induce formation of free radicals, which could explain why FR160 antagonized the effects of artesunate and dihydroartemisinin.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Two of the current options for reducing the morbidity and mortality related to malaria are chemoprophylaxis and chemotherapy. During the past 20 years in Africa, there has been an emergence of strains of Plasmodium falciparum resistant to chloroquine and other antimalarial drugs.1,2 Failures of antimalarial prophylaxis with chloroquine, with a combination of chloroquine and proguanil,3 and with mefloquine,4 and clinical failures with halofantrine5 and quinine,6 have been observed in Africa. This has led to a search for an effective alternative antimalarial drug with minimal side effects. This emergence and spread of parasite resistance to currently used antimalarial drugs indicates that novel compounds need to be discovered and developed by identification of novel chemotherapeutic targets.7

Iron chelation therapy has been considered as a possible treatment for various infectious diseases, including malaria.8,9 Iron is an essential element for the growth of all living organisms.10 This metal is used in catalysis of DNA synthesis and for a variety of enzymes concerned in electron transport and energy metabolism. The antimalarial action of iron chelators is dictated by three factors:11 iron(III)-binding capacity, chelator ingress into parasitized erythrocytes and chelator egress from parasites after treatment. Various iron chelators were assessed to improve drug lipophilicity leading to increased access of drug to intracellular parasites and to faster speed of action.12,13 Desferrioxamine does indeed penetrate the infected red cell, and its antimalarial activity is dependent on this.14 One would predict that an effective antimalarial iron chelator would have the ability to cross lipid membranes well, have a high affinity for iron, selectively bind iron as compared with other trace metals and selectively bind iron(III) rather than iron(II).15 The hydrophilic/hydrophobic balance or relative lipophilicity of a compound is an important factor in the movement of an agent across a lipid-containing membrane to enter a cell, and in determining its usefulness.16

After screening several catecholate iron chelators against clones of P. falciparum, the catechol derivatives 2,3- and 3,4-dihydroxybenzoic acid attached to spermidine and Tris-(dihydroxybenzoyl)triaminoethylaminoethylamine showed the best in vitro activity.17 A catecholate derived from spermidine, the N4-nonyl,N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide, FR160 (R = C9H19), was the most potent against the chloroquine-susceptible clone D6 and chloroquine-resistant clone W2 of P. falciparum. To improve its activity, dicatecholates with different side chains were synthesized. The aim of this study was to compare the in vitro activity of the different compounds against P. falciparum, their toxicity on cells and uninfected erythrocytes, and their capacity to withhold iron(III), iron(II) or ferritin.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Drugs

The catechol derivatives (Figure 1) were synthesized by treatment of diacylated spermidine with 1-bromobutane (C4H9) or 1-bromononane (C9H19) (named FR160), 1-bromododecane (C12H25) or 1-bromooctadecane (C18H37) in dry acetone in the presence of anhydrous potassium carbonate, followed by deprotection of the methoxy groups with boron tribromide. Stock solutions for each catecholate were prepared in methanol, in ethanol and in dimethylsulphoxide (DMSO). Two-fold serial dilutions were prepared in methanol (for stock solutions in methanol), in distilled water and in RPMI (for stock solutions in methanol, ethanol and DMSO).



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Figure 1. Chemical structures of the different N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide derivatives. R = C4H9; R = C9H19 (FR160); R = C12H25; R = C18H37.

 
Desferrioxamine mesylate, chloroquine, doxycycline, primaquine and pyrimethamine were obtained from Sigma Chemical Co. (St Louis, MO, USA), pyronaridine phosphate from the WHO (Geneva, Switzerland), batch 260642, artesunate and dihydroartemisinine were from Rhône Poulenc Rorer (Antony, France) and atovaquone was from the Wellcome Foundation Ltd (Beckenham, UK). Two-fold serial dilutions were prepared in sterile distilled water for all these drugs. Final concentrations were distributed in triplicate into Falcon 96-well flat-bottomed plates (Becton Dickinson, Franklin Lakes, NJ, USA).

Strains of P. falciparum

One chloroquine-susceptible clone 3D7 (Africa) and four chloroquine-resistant clones or strains W2 (Indochina), Palo Alto (Uganda), FCR3 (Gambia) and Bres1 (Brazil) were maintained in culture. When required for drug assays, cultures were synchronized by sorbitol lysis.18 Susceptibilities to the iron chelator and other antimalarial drugs were determined after suspension in RPMI 1640 medium (Life Technologies, Paisley, UK), supplemented with 10% human serum (pooled from different A+ or AB sera from non-malaria-immune donors) and buffered with 25 mM HEPES and 25 mM NaHCO3 (haematocrit of 1.5%, parasitaemia of 0.5%).

In vitro assay

For in vitro isotopic microtests, 200 µL of the suspension of parasitized erythrocytes was distributed in 96-well plates with antimalarial agents. Parasite growth was assessed by adding 1 µCi of [3H]hypoxanthine with a specific activity of 14.1 Ci/mmol (NEN Products, Dreiech, Germany) to each well. Plates were incubated for 42 h at 37°C in an atmosphere of 10% O2, 6% CO2, 84% N2 and 95% humidity. Immediately after incubation the plates were frozen and then thawed to lyse erythrocytes. The contents of each well were collected on standard filter microplates (Unifilter GF/B; Packard Instrument Company, Meriden, CT, USA) and washed using a cell harvester (FilterMate Cell Harvester; Packard). Filter microplates were dried and 25 µL of scintillation cocktail (Microscint O; Packard) was placed in each well. Radioactivity incorporated by the parasites was measured using a scintillation counter (Top Count; Packard).

The IC50, i.e. the drug concentration corresponding to 50% of the uptake of [3H]hypoxanthine by the parasites in drug-free control wells, was determined by non-linear regression analysis of log-dose–response curves. Data were analysed after logarithmic transformation and expressed as the geometric mean IC50 with 95% confidence intervals (CIs).

In vitro toxicity assay against erythrocytes

Red blood cells (A+) were obtained from the Blood Transfusion Centre (Military Hospital, Toulon, France). Erythrocytes were washed three times in RPMI 1640 medium (Life Technologies). Erythrocytes were resuspended in RPMI 1640 medium supplemented with 10% human serum and buffered with 25 mM HEPES and 25 mM NaHCO3 to a haematocrit of 1.5%. The suspension was distributed as 200 µL/well into Falcon 96-well plates (Becton Dickinson). Final concentrations of antimalarial drugs (25 µL), which ranged from 10 mM to 1 µM, were distributed in triplicate into plates. Water (25 µL) was distributed in triplicate (negative control). In addition, 225 µL of a suspension of erythrocytes in saponine solution 5% at a haematocrit of 1.5% was distributed in triplicate (positive control) and the plate was gently shaken. Plates were then incubated for 42 h at 37°C in an atmosphere of 10% O2, 6% CO2, 84% N2 and 95% humidity (optimum requirements for isotopic, micro-antimalarial drug susceptibility in vitro test). Immediately after incubation, plates were centrifuged at 2200g for 10 min and the supernatant of each well was collected and transferred on to a new plate. Serial four-fold dilutions in glacial acetic acid were carried out. A solution (100 µL) of TMB (0.5 mg/mL glacial acetic acid) was distributed in a new plate, then 5 µL of pure and diluted supernatant and 5 µL of haemoglobin standard (30 mg/dL) were added. Next, 100 µL of hydrogen peroxide solution (0.03%) was added to each well. Exactly 10 min after addition of hydrogen peroxide, the optical density of each well was measured using an automatic plate reader (Optimax; Molecular Devices, Sunnyvale, CA, USA) at 455 nm.

The data are expressed as haemoglobin concentrations. The 50% haemolytic concentration (HC50), i.e. the drug concentration corresponding to 50% lysis of erythrocytes in saponine control wells, was determined by non-linear regression analysis of log-dose–response curves.

Mammalian cell cultures

Mammalian cells [Vero cells (ATCC)], were maintained in Falcon culture flasks (Becton Dickinson) and grown at 37°C in a CO2 incubator (5% CO2), as monolayers in Medium 199 (Sigma) supplemented with 5% fetal calf serum (Sigma), L-glutamine 100 µg/L (Sigma) buffered with NaHCO3 2.2 g/L (Sigma). Cells were inoculated into 96-well flat-bottomed microtitre plates (Becton Dickinson) at a density of 6 x 104 cells in 200 µL culture medium (confluent in 24 h). The cultures were incubated for 24 h. Next, cells in growing phase were exposed for 48 h to final concentrations of antimalarial drugs (25 µL), which ranged from 10 mM to 1 µM, and distributed in triplicate at 37°C and at 5% CO2. Wells without drug served as control (the influence of water or methanol or ethanol vehicle was assessed previously and was considered to be negligible under the assay conditions).

Colorimetric tetrazolium assay

The tetrazolium (MTT) assay was carried out as described by Etievant et al.19 The optical density of each well was measured using an automatic plate reader (Optimax; Molecular Devices) with a 570 nm test wavelength and a 630 nm reference wavelength.

The IC50 was determined by non-linear regression analysis of log-dose–response curves.

Neutral red assay

The cytotoxicity assay was carried out as previously described by Borenfreund & Puerner20 with some modifications. After 48 h of incubation, test agents were removed and cultures washed with PBS at 37°C. Then 200 µL of medium containing 25 µg/mL neutral red (NR) was added to each well and incubation continued for an additional 3 h at 37°C and 5% CO2. At the end of the incubation period, dye medium was removed by suction and each well was washed rapidly with a solution of CaCl2:formaldehyde:water (1:1:98) to remove unincorporated NR and enhance attachment of cells. Each well was then washed rapidly with PBS. A solution of acetic acid:ethanol:water (1:50:49) was added (150 µL). The plate was then vigorously shaken to ensure solubilization of the NR. The optical density of each well was measured using an automatic plate reader (Optimax; Molecular Devices) with a 540 nm test wavelength.

The IC50 was determined by non-linear regression analysis of log-dose–response curves.

Inhibition of the activity by iron salts and ferritin

A range of concentrations of FeCl3, FeSO4 and ferritin (Sigma), prepared in RPMI, were pre-incubated with C4 at 6 µM, C9 (FR160) at 4 µM, C12 at 10 µM and C18 at 10 µM in RPMI (concentrations in final test) for 3 h. For the in vitro microtest, 50 µL of the complex and 150 µL of a suspension of parasitized erythrocytes (parasitaemia of 0.5% and final haematocrit of 1.5%) were distributed in 96-well plates.

In addition, we assessed the activity of FR160 against trophozoite stages incubated at the same time with FeCl3 for 3 h, FR160 incubated 3 h before FeCl3 and FR160 incubated 3 h after FeCl3.

Stage and time dependence of FR160

After synchronization by sorbitol lysis,18 infected erythrocytes were exposed to FR160 and desferrioxamine at different concentrations for 6 h at 0 (early rings), 6, 12, 18, 24, 30, 36, 42 and 48 h. Parasitized red blood cells were washed three times with drug-free medium. The cell pellet was resuspended in complete drug-free medium. Radiolabelled hypoxanthine was then added, and plates were incubated until the end of the cycle.

Speed of action was determined for the ring and late trophozoite stages. After exposure for different times to FR160 and desferrioxamine, cells were washed three times with drug-free medium, resuspended in complete drug-free medium and then [3H]hypoxanthine was added. Plates were incubated until the end of the cycle.

The activities of FR160 and doxycycline were assessed after exposure for 48, 96 and 144 h. Parasite growth was assessed by adding 1 µCi of [3H]hypoxanthine with a specific activity of 14.1 Ci/mmol (NEN Products) to each well at 0 h for the 48 h exposure test, at 48 h for the 96 h exposure test and at 96 h for the 144 h exposure test. Media were not changed during longer exposures. Plates were incubated for 48, 96 or 144 h at 37°C in an atmosphere of 10% O2, 6% CO2, 84% N2 and 95% humidity.

Effect of oxygen on in vitro potency of FR160

Activities of FR160, chloroquine, artesunate and doxycycline were assessed at different O2 tensions: 1, 10 and 21%.

Effect of ascorbic acid (vitamin C), an antioxidant, on the activity of FR160

Activities of FR160, chloroquine, artesunate and doxycycline were assessed in combination with different doses of ascorbic acid, ranging from 100 to 800 µM.

Inhibition of haem polymerization

A polymer identical to haemozoin, ß-haematin, can be obtained in vitro from haematin at acidic pH. To identify molecules with inhibitory activity on haem polymerization, we used a quantitative in vitro spectrophotometric microassay of haem polymerization.21

One hundred microlitres of a 4 mM solution of haematin, previously dissolved in 0.1 M NaOH, was distributed in 96-well microplates (0.4 µmol/well). Fifty microlitres of different doses of antimalarial drugs at a drug:haem ratio between 0:1 and 10:1 were added to triplicate test wells. Either 50 µL of water or 50 µL of the solvent used to solubilize the drugs was added to control wells. Haematin polymerization was initiated by adding 0.8 mmol of acetic acid (50 µL) at a final pH of 3, and the suspension was incubated at 37°C for 24 h to allow complete polymerization. Plates were then centrifuged at 3300g for 15 min and the soluble fraction of unprecipitated material collected. The remaining pellets were resuspended with 200 µL of DMSO to remove unreacted haematin. Plates were then centrifuged again at 3300g for 15 min. The DMSO-soluble fraction was collected and the pellets, consisting of a pure precipitate of ß-haematin, were dissolved in 0.1 M NaOH for spectroscopic quantification. A 150 µL aliquot of each fraction was transferred onto a new plate and serial four-fold dilutions in 0.1 M NaOH were carried out. The amount of haematin was determined by measuring the absorbance at 405 nm using an automatic plate reader (Optimax; Molecular Devices). The data are expressed as the molar equivalents of test compounds relative to haematin required to inhibit haem polymerization by 50%.

Drug combinations

Combinations of FR160 with atovaquone, artesunate, dihydroartemisinin, primaquine, pyronaridine, pyrimethamine and cycloguanil were tested. To evaluate drug interactions, isobolograms were constructed by plotting a pair of fractional IC50s for each combination of FR160 and the other drugs. The different drugs’ fractional IC50 was calculated by dividing their fixed concentrations by the IC50 of tested drugs alone and plotting on the x-axis. The corresponding FR160 fractional IC50 was calculated by dividing the IC50 of FR160 combined with fixed concentrations of other drugs and plotted on the y-axis. Points lying above the straight diagonal line (corresponding to the points where there is no interaction between the drugs) are antagonistic, points below the straight diagonal line are considered to be synergic.22


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Activity, toxicity and interaction with iron and length of side chain

The HC50, determined by in vitro erythrocyte toxicity assay, the IC50, determined by colorimetric MTT assay, and the IC50 against P. falciparum for the different dicatecholate compounds are summarized in Table 1. The IC50 values for P. falciparum were similar whatever the solvents used in the stock solution and in two-fold serial dilutions (data not shown).


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Table 1.  In vitro IC50 of a series of dicatecholate iron chelators against the chloroquine-susceptible clone 3D7 and chloroquine-resistant clone W2, IC50 against Vero cells determined by colorimetric MTT assay and HC50 against uninfected erythrocytes
 
The differences in IC50 between the chloroquine-susceptible 3D7 and chloroquine-resistant W2 strains are not statistically significant for C4H9 (P > 0.25), C12H25 (P > 0.05) and C18H37 (P >0.15). The iron chelator FR160 (C9H19) is more active against the chloroquine-susceptible clone (P < 0.005). Correlations are found between the activities on chloroquine-susceptible 3D7 and chloroquine-resistant W2 clones (r = 0.995), between activity against 3D7 and toxicity on Vero cells (r = 0.826), and between activity against W2 and toxicity on Vero cells (r = 0.843). There is no correlation between activities against 3D7 and W2, and toxicity on red blood cells (r = 0.168 and 0.260). Toxicity on erythrocytes is weakly correlated with cytotoxicity (r = 0.544). Activities against P. falciparum 3D7 and W2 or toxicities on cells and erythrocytes are not correlated to lipophilicity (r = 0.039, 0.111, 0.187 and 0.349, respectively).

The in vitro activity of C12 was not decreased in the presence of iron(III), iron(II) or ferritin (Figures 2 and 3). The other compounds were more susceptible to iron(III) than iron(II) (Figure 2). The drug most sensitive to iron or ferritin was C9 (FR160), followed by C18, C4 and C12. Activities and interaction with iron are correlated.



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Figure 2.  In vitro inhibition of P. falciparum growth (W2) by the different N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide derivatives in the presence of iron(III) (FeCl3) (grey bars) and iron(II) (FeSO4) (black bars).

 


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Figure 3. In vitro inhibition of the P. falciparum growth (W2) by the different N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide derivatives in the presence of ferritin. Hatched bars, C9H19; white bars, C4H9; black bars, C12H25; grey bars, C18H37.

 
In vitro growth inhibition of FR160 and desferrioxamine against P. falciparum and toxicity on Vero cells

The effect of FR160 was tested on five P. falciparum strains: one chloroquine-susceptible (3D7) and four chloroquine-resistant (W2, FCR3, Palo Alto, Bres1). FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 µM (Table 2). The activity of FR160 was similar to that reported previously for a P. falciparum W2 clone.17 IC50s for desferrioxamine were 10- to 20-fold higher. The IC50s of cytotoxicity for FR160 were 200- to 300-fold higher than the values for activity (Table 2).


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Table 2.  In vitro activity of FR160 (R = C9H19) and desferrioxamine against the various P. falciparum clones, and in vitro cytotoxicity of FR160 and desferrioxamine against Vero cells by NR and MTT assays
 
Stage and time dependence

The results presented in Figure 4 show a statistical difference of action between FR160 and desferrioxamine according to the developmental stage of the parasite. The effects of FR160 and desferrioxamine were assessed on synchronized cultures. The sensitivities of different stages of parasite development to the inhibitory action of FR160 and desferrioxamine were examined by adding various drugs to the incubation medium at different times during the parasite cycle (Figure 4). Addition of desferrioxamine (40–100 µM) had no effect on the development of rings to trophozoites and schizonts to rings. Desferrioxamine was potent on the late trophozoite stages and on young schizonts. The trophozoites were identified as the most drug-sensitive stage for FR160. However, rings and schizonts show vulnerability to FR160. At all times in the cycle, exposure to FR160 results in growth inhibition. A growth inhibition superior to 50% was reached after 6 h of exposure to FR160 on rings, whereas desferrioxamine was not active (Figure 5). A growth inhibition superior to 70% was reached after 3 h of exposure to FR160 and after 6 h for desferrioxamine on trophozoites. The antimalarial action of FR160 was rapid. Prolonged exposure to FR160 (96 or 144 h) did not alter IC50s, in contrast to doxycycline (Table 3).



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Figure 4. In vitro P. falciparum growth (W2) in the presence of FR160 (5 µM) (grey bars) and desferrioxamine (DFX) (50 µM) (black bars) added to the incubation medium at different times of the parasite cycle.

 


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Figure 5. In vitro speed of action of FR160 (5 µM) (circles) and desferrioxamine (DFX) (50 µM) (diamonds) on rings and trophozoites against P. falciparum W2 clone.

 

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Table 3.  In vitro activities of FR160 and doxycycline against the P. falciparum W2 clone after exposure of 48, 96 and 144 h
 
Effect of oxygen on in vitro potency of FR160

In contrast to chloroquine, artesunate and doxycycline, the activity of FR160 seems not to be altered by O2 tension. The differences of IC50 at 1, 10 and 21% of oxygen for FR160 are not statistically significant (P > 0.05). The differences of the chloroquine IC50 are statistically significant whatever the percentage of oxygen (P < 0.001). For doxycycline, the differences of IC50 are statistically significant between 1% and 10%, and 1% and 21% of oxygen (P < 0.025). When artesunate is used, the differences of IC50 are statistically significant between 1% and 10% (P < 0.001), and 10% and 21% of oxygen (P < 0.005).

Effect of ascorbic acid (vitamin C) on the activity of FR160

As with doxycycline and artesunate, the activity of FR160 was decreased by addition of ascorbic acid. The decrease of the activity of FR160 in the presence of ascorbic acid is statistically significant from 400 µM ascorbic acid (P < 0.025). The activity of artesunate decreases significantly in the presence of 400 µM ascorbic acid (P < 0.005). When doxycycline is used in association with ascorbic acid, doxycycline is significantly less potent from 100 µM ascorbic acid (P < 0.025). Ascorbic acid has no statistically significant effect on the activity of chloroquine (P > 0.20).

Inhibition of the activity by iron salts

No difference has been shown between incubation of FR160 and FeCl3 at the same time, and incubation of FR160 after 3 h of exposure to FeCl3 on trophozoite stages (Figure 6). In contrast, inhibition of the activity of FR160 was less when FeCl3 was added 3 h after exposure to FR160.



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Figure 6. Time-dependent incubation of FR160 (4.8 µM) and iron(III) on trophozoites of P. falciparum W2 clone. Grey bars, FR160 at t = 0 h and iron(III) at t = 0 h; black bars, FR160 at t = 0 h and iron(III) at t = 3 h; white bars, FR160 at t = 3 h and iron(III) at t = 0 h.

 
Inhibition of haem polymerization

No inhibition of haem polymerization was obtained using FR160 and desferrioxamine (the IC50 for haem polymerization being >10 equivalents of FR160 or desferrioxamine, whereas the IC50 for chloroquine was 60 equivalents).

Drug combinations

The combination of atovaquone and FR160 demonstrated a synergic or at least additive antimalarial effect (Figure 7). Endoperoxide drugs like artesunate or dihydroartemisinin antagonized the antimalarial effects of FR160. FR160 antagonized the effects of primaquine and pyronaridine, and cycloguanil and pyrimethamine demonstrated additive effects with FR160 (not shown).



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Figure 7. In vitro combinations of FR160 with atovaquone and artesunate against the P. falciparum W2 clone.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
As the ability of desferrioxamine to penetrate the erythrocyte membrane is limited, it was postulated that other iron chelators with a higher affinity and/or increased lipid solubility would show improved antimalarial activity. The present study was undertaken to explore the antimalarial effects of a series of dicatecholate iron chelators. They may be made more or less lipophilic by increasing or reducing the length of the R substituent on the nitrogen. Increasing lipophilicity is expressed by an increasing value of log P (determined by software Pallas 2.0, CompuDrug Chemistry Ltd, Columbus, OH, USA).

We show that the antimalarial effect of these iron chelators on P. falciparum (W2 or 3D7) cannot be explained by their lipophilicity. Activity is not directly correlated to ability to penetrate erythrocyte membranes. These observations are in contradiction to those reported by other authors, who have demonstrated that the efficacy of several reversed siderophores or hydroxypyridones was clearly correlated with their lipophilicity.15,23 However, if we analyse, by calculation of the correlation coefficient, the physical properties and the antimalarial activity of iron chelators, of which the inhibitory potency was considered to correlate with the magnitude of their partition coefficient and therefore their lipophilicity,13,16 we note that the coefficients of correlation between partition coefficient and IC50 are weak (r = 0.602 and 0.472). The best correlation between IC50 values and physicochemical properties was obtained when IC50 was plotted against the product of relative iron(III)-binding and partition coefficient.13

We demonstrated that the activity of our dicatecholate iron chelators is clearly correlated with the ability of iron(III), iron(II) and ferritin to reverse their inhibition and therefore their iron withholding. In studies reported by other authors, the compounds used displayed iron(III)-binding affinity of the same order of magnitude as desferrioxamine,13 and this parameter was considered to play a secondary role in determining the difference in their activities.13,16 Ligands that bind iron(III) may have more potential as iron chelators than those that bind iron(II).24,25 In our experiments, iron(III) showed greater reversal of inhibitory activity on P. falciparum than iron(II) and ferritin when combined with any of three dicatecholates. Nevertheless, the reversal of iron chelators’ growth inhibition by iron(III), iron(II) and ferritin are correlated. The inhibition of parasite growth by the C12H25 compound is not modified in the presence of iron(III), iron(II) or ferritin.

This study demonstrates a potent antimalarial effect of FR160 (C9H19) on five different strains of P. falciparum in vitro. FR160 inhibited parasite growth with an IC50 between 0.8 and 1.5 µM. FR160 was more potent against the chloroquine-susceptible 3D7 clone. This observation was similar to that observed with the chloroquine-susceptible D6 clone (0.17 µM).17 Nevertheless, the differential activity of FR160 against the chloroquine-susceptible clones is less than a factor of two and is well below the differential activity of chloroquine, indicating that there is a slight interaction with the resistance process. FR160 is one of the most potent in vitro iron chelator antimalarials compared with: desferrioxamine (3–35 µM),24,26–29 reversed siderophores (0.3–70 µM),13,23,27 deferiprone,15,26 polyanionic amines (5 µM),30 acylhydrazones (18–30 µM),31,32 aminothiols (3–9 µM)33 and dexrazoxane (>200 µM).34 The effects of FR160 on mammalian cells in culture were minimal compared with those obtained with human malaria parasites. Most of the compounds used as antimalarials are potent in a low or middle nanomolar range. The in vitro activity of FR160 is in the low micromolar range. A generally acceptable level of efficacy would be in the low or middle nanomolar range. However, if the mechanisms of action of such a compound are sufficiently new and different from those of the commonly used antimalarial drugs, this compound could warrant further research.

FR160 had time- and stage-dependent effects. The drug was more effective against the late trophozoite and young schizont. However, FR160 affected rings and schizonts as well. Growth inhibition by desferrioxamine occurred only when late trophozoites and early schizonts were exposed. This observation on desferrioxamine is similar to those reported by other authors.35,36 FR160 differs from desferrioxamine in IC50 value and in the profile of antimalarial action. FR160 affected parasites at considerably higher rates and at all stages of parasite growth. So, prolonged exposure to FR160 (96 or 144 h) did not alter IC50 values, in contrast to slow antimalarial drugs such as antibiotics.37 Effects of FR160 were particularly reversible on trophozoites after iron(III) addition, whereas desferrioxamine affected trophozoites in an irreversible manner.12

Ascorbic acid, known for its properties as an antioxidant and free radical scavenger, decreased the activities of FR160 and artesunate. Moreover, FR160 antagonized the effects of artesunate and dihydroartemisinin. Artemisinin and its derivatives are believed to exert their antimalarial effects through the generation of free radicals, because the peroxide functional group is required for activity.38 Iron is required for the antimalarial activity of artemisinin and its derivatives,39 but the specific source of intracellular iron for their activation is still unknown. This is analogous to the Fenton reaction, in which iron catalyses the homolysis of hydrogen peroxide and hydroperoxide to produce free radicals. We showed in this study that ascorbic acid, a free radical scavenger, decreased the activity of artesunate. The reduction of the activity of FR160 in combination with ascorbic acid could be explained by the hypothesis that FR160 might induce formation of free radicals. Certain iron chelators, such as alkylthiocarbamates or lactoferrin, form an extracellular complex with iron, which subsequently enters the parasitized erythrocyte to produce a rapidly lethal free radical-mediated intracellular reaction.40 However, the activity of FR160 was not enhanced by high oxygen tension, as has been reported previously for other free radical-generating compounds.41 However, in our experiment the activity of artesunate was not enhanced by high oxygen tension. The antagonism between FR160 and artesunate or dihydroartemisinin provides a clear indication that FR160 interferes with the mode of action of artemisinin derivatives, possibly by competing for free radical generation.

Free haem may also be responsible for the activation of artemisinin. The interaction of artemisinin and haem has been demonstrated in vitro and in situ.4244 Although most haem is polymerized into haemozoin, some free haem may be transiently present. It was initially believed that this reaction was catalysed by haem polymerase enzyme.45 However, haematin polymerization may be a physical process alone and not necessarily enzyme mediated.46 Moreover, artemisinin has been found to be a potent inhibitor of haem polymerization.47,48 The hypothesis is still debated.49 We report here that FR160, like desferrioxamine, does not lead to inhibition of haem polymerization. This could also provide one possible explanation for the observed antagonism between FR160 and artesunate or dihydroartemisinin. It has been demonstrated that desferrioxamine increased the concentration of soluble forms of haematin, and initiated and enhanced the rate of haematin polymerization.50

The possibility that iron withholding serves as the antimalarial mechanism for FR160 is less probable, but is supported by experiments showing that inhibition of plasmodial growth is completely negated when iron(III) or iron(II) is added to FR160. FR160 could act as an extracellular or intracellular iron scavenger, and cause parasite iron deprivation or act on protein or ligand-bound iron. Moreover, the observation that FR160 was more effective when pigmented trophozoites and early schizonts were exposed is consistent with the idea that a major metabolic pool of iron is presumably mobilized by parasites at the trophozoite–schizont stage.51 The effect of iron withholding has been well documented for desferrioxamine.35,51,52

Many metabolic processes of the erythrocytic malaria parasite are dependent upon iron (iron-dependent enzymes). The withholding of iron from the parasite by iron chelators could conceivably disrupt the metabolism of the parasite. One possible explanation for desferrioxamine action is the inhibition of ribonucleotide reductase (RNR), a key enzyme in de novo pyrimidine synthesis, mainly active in the late trophozoite stage of P. falciparum.53 This hypothesis was indirectly confirmed by an ultrastructural study by Atkinson et al.,36 who showed that desferrioxamine activity leads to nuclear alterations compatible with perinuclear localization of the RNR. FR160 is more effective at the late trophozoite and early schizont stages, like desferrioxamine, although it affects rings and schizonts as well. This might be consistent with the idea that RNR is mainly active at the trophozoite stage. In addition, hydroxyurea and benzohydroxamic acid, which inhibit mammalian RNR with IC50s of 500 and 400 µM, respectively,54 also inhibit P. falciparum parasites with IC50s of 792 and 17 µM, respectively.55 The chemical structure of FR160 has some common structural features with hydroxyurea and benzohydroxamic acid. However, the hypothesis that the RNR of P. falciparum is a target for FR160 seems improbable.

The present study shows that increased lipid solubility of catecholate iron chelators does not lead to improved antimalarial activity, but their activity is well correlated with their interaction with iron and with their toxicity against Vero cells. FR160 holds much promise as an effective antimalarial agent. It seems to act preferentially by formation of free radicals. Efforts are underway to characterize the mechanisms of action of this iron chelator. Resistance to antimalarial drugs reinforces the idea that a novel antimalarial should not be deployed as monotherapy. FR160 and tetracyclines, which are inhibited by iron,56 show additive or synergic activity in vitro against chloroquine-resistant P. falciparum parasites.57 The efficacy of these agents in animals and their pharmacokinetics should be assessed.


    Acknowledgements
 
The authors thank Professor Walliker (Cell Animal and Population Biology, Edinburgh, UK), Professor Milhous (Walter Reed Army Institute of Research, Washington, DC, USA) and Dr Gysin (Laboratoire de Parasitologie Expérimentale, Marseille, France) for clones and strains. This work was supported by la Délégation Générale pour l’Armement (contrat d’objectif no. 9810060), la Direction de la Recherche et de la Technologie/Services Techniques des Recherches et des Développements Technologiques (contract no. 97/2509A), le Groupe de Recherche en Parasitologie 1077 and la Direction Centrale du Service de Santé des Armées.


    Footnotes
 
* Corresponding author. Tel: +33-4-91-15-01-10; Fax: +33-4-91-15-01-64; E-mail: bruno.pradines{at}free.fr Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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