INTRODUCTION
Malaria is the most important parasitic disease and kills ~1% of children from sub-Saharan Africa each year.1 Despite major efforts to develop alternative control strategies, chemotherapy remains the only successful intervention in areas of high endemicity. Thus far, the most widely used drug is chloroquine, introduced for chemotherapy of malaria in 1943. It remained the most effective and safe therapeutic option for almost 15 years but, unfortunately, chloroquine-resistant parasites emerged in the 1960s and are common in most endemic regions today.2,3 Several approaches to “rescue” the therapeutic effect of chloroquine have been undertaken, because it proved to be one of the best anti-malarials, when effective. The partial success of structural relatives of chloroquine, such as amodiaquine, motivated the development of structural modifications of the original drug to circumvent resistance mechanisms of the parasite. One such modification is the addition of a covalently bound transition-metal molecule to a candidate molecule.4 Ferroquine is a chloroquine derivative that contains a ferrocene molecule at the lateral carbon chain. Ferrocene alone neither shows anti-malarial activity, nor does it affect chloroquine resistance when co-administered as a separate molecule,5 but one possible derivative of covalently linked chloroquine-ferrocene [7-chloro-4-2-(N′,N′-dimethylaminomethyl)-N-ferrocenylmethylamino-]quinoline; ferroquine] has high anti-malarial activity against chloroquine-resistant Plasmodium falciparum strains in vitro and against P. berghei and chloroquine-resistant P. yoelii in vivo.6 The mechanism of resistance reversion is not known but, most probably, involves enhanced retention of the drug in the food vacuole, the compartment where chloroquine analogs exert their action. One major drawback when using derivatives of a drug against which resistance has developed is the possibility of enhanced selection for resistance against the new drug in a parasite population that has acquired resistance against the original drug. Unfortunately, all studies on field isolates support this idea because they show a marked correlation between chloroquine and ferroquine sensitivities.7–9
This study assessed the susceptibility of P. falciparum to ferroquine, chloroquine, and artesunate in an extensively studied area in Central Africa, where all isolates are chloroquine resistant. In addition, we tested ferroquine and chloroquine activity in well-characterized laboratory isolates with different chloroquine sensitivities to study the effect of strain and initial parasitemia on ferroquine sensitivity.
MATERIALS AND METHODS
Plasmodium falciparum field isolates.
Eighty-one blood samples were collected from patients with uncomplicated malaria at the Medical Research Unit of the Albert Schweitzer Hospital, Lambaréné, Gabon, between August and November 2004. Inclusion criteria were P. falciparum monoinfection proved by Giemsa thick blood smear, parasitemia between 1,000 and 120,000/μL of blood, no history of anti-malarial medication during the preceding month, and age between 1 and 15 years. Venous blood (0.5 mL), taken aseptically in heparinized tubes, was processed immediately. Informed consent and assent were obtained from the legal representative and the participating child, respectively. The studies were approved by the ethics committee of the International Foundation for the Albert Schweitzer Hospital in Lambaréné.
Drugs.
Ferroquine (MW: 435; Sanofi-Synthelabo, Paris, France) was dissolved in dimethyl sulfoxid, chloroquine (MW: 320; Sigma-Aldrich, Seelze, Germany) was resuspended in double distilled water, and artesunate (MW: 384) was prepared in 70% ethanol. The maximum amount of either solvent per well did not exceed 0.0007%. Preliminary experiments showed no effect of the highest solvent concentrations on parasite growth (data not shown). Ninety-six-well flat-bottomed plates (Becton Dickinson, Heidelberg, Germany) were pre-dosed with ferroquine, chloroquine, and artesunate in seven 3-fold serial dilutions and kept at −20°C until use. All dilutions were done in parasite culture medium [RPMI 1640, 25 mmol/L 4-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid), 2 mmol/L l-glutamine, 50 μg/mL gentamycin, and 0.5% wt/vol albumax]. Final drug concentrations ranged from 0.1 to 70 nmol/L (43.5 pg to 30.5 ng) for ferroquine, from 1.4 to 1,000.4 nmol/L (0.4–320 ng) for chloroquine and from 0.1 to 52 nmol/L (38.4 pg to 20.0 ng) for artesunate. All drugs were pre-tested in a chloroquine-sensitive laboratory strain (D10) and in field isolates to determine the appropriate concentration range for each study drug.
Drug sensitivity assay.
The drug sensitivity assay was done according to published standard procedures,10 with minor modifications. Briefly, blood samples were washed once with RPMI 1640 (Sigma-Aldrich), and parasitemia was adjusted to 0.05% with non-infected 0+-erythrocytes of one healthy blood donor. Erythrocytes were resuspended in parasite culture medium to a hematocrit of 1.5%. Two hundred microliters of blood-medium-mixture was added to each duplicate well of the pre-dosed 96-well test plates. The plates were incubated for 72 hours at 37°C in a candle jar. To assess successful in vitro parasite growth, a thick blood smear of one control well was done after 26 hours. Parasite culture was judged successful when at least 20% parasites matured to schizonts. After 72 hours, plates were freeze-thawed twice. Parasite growth, calculated from histidine rich protein 2 (HRP2) levels, was measured with a commercially available enzyme linked immuno-sorbent assay (MalariaAg CELISA, Cellabs, Sydney, Australia), according to the manufacturer’s specifications. Each drug was tested in duplicate. The dependency of inhibitory concentrations on initial parasitemia was tested in two laboratory strains, 1) D10 (sensitive to chloroquine) and 2) Dd2 (multi-resistant), using exactly the same methods as for the field isolates, except that increasing parasitemias were seeded (initial parasitemia: 0.05%, 0.25%, 0.5%, and 1.25%). Both strains were freshly obtained from the malaria research and reference reagent resource center (MR4) and tested for their phenotype and clonality.
Statistical analysis.
Inhibitory concentrations were determined by non-linear regression analysis of log-dose-response curves using TableCurve 2D v4 (SPSS, Chicago, IL). Median and geometric mean 50%, 90%, and 99% inhibition concentrations (IC50, IC90, and IC99) and 95% confidence intervals (CIs) were calculated with JMP v5.0.1.2 (SAS Institute, Cary, NC). In accordance to the protocols distributed by the World Health Organization, chloroquine resistance was calculated from the theoretical blood volume inoculated, because the drug accumulate in erythrocytes (threshold level for chloroquine: IC99 of > 1 μmol/L blood).11 In our low-hematocrit culture conditions, this corresponds to an IC99 resistance threshold of > 30 nmol/L. The in vitro and in vivo resistance against chloroquine is well documented for Lambaréné.12,13 Ferroquine has not yet been tested clinically. Therefore, no threshold level of clinically important resistance can be defined. Inhibitory concentrations were log-transformed for all parametric analyses because of a better approximation to the normal distribution. Correlations between ferroquine, chloroquine, and artesunate were assessed by Pearson correlation coefficient (r). To assess the effect of the initial parasitemia on drug action, several analyses of covariance were done with the logarithm of IC50 as dependent and strain, log initial parasitemia, log chloroquine IC50, and drug as explanatory variables in a standard least squares model provided with JMP. F tests of the difference of sums of squares with and without the respective effect were used to test the contribution of the independent variables on the model. Level of significance was set at two-sided P = 0.05 for all tests.
RESULTS
In vitro drug sensitivity in field isolates.
Eighty-one P. falciparum isolates were tested for their in vitro susceptibility to ferroquine, chloroquine, and artesunate. Resistance against chloroquine is highly prevalent in the study area, whereas no artesunate resistance is reported. After 26 hours, 50 (62%) of the parasite cultures fulfilled the criteria for successful culture, and drug sensitivity was tested in 40 (ferroquine) and 43 (chloroquine and artesunate) samples, respectively. Chloroquine resistance was present in all samples analyzed, whereas artesunate was highly active (Table 1). Ferroquine had a considerably higher activity per molecule compared with chloroquine (Table 1). Cross-resistance was measured by pairwise correlation of log-transformed IC50 values of all 40 isolates that were tested for ferroquine, chloroquine, and artesunate. Neither ferroquine versus chloroquine (r = 0.31, P = 0.06) nor ferroquine and artesunate (r = 0.22, P = 0.17) nor chloroquine and artesunate (r = 0.28; P = 0.08) were significantly correlated. On average, chloroquine sensitivity was 18-fold beyond the resistance threshold indicative for treatment failure in vivo, which has been determined for the study area.12
Covariance analysis of parasite genotype, parasitemia, and drug activity.
Chloroquine accumulates within blood cells and reaches peak concentrations in infected red blood cells.14 Consequently, increased hematocrit or parasitemia should lead to lower effective (intraparasitic) drug activities. Most in vitro studies use a constant hematocrit, but parasitemia is generally not adjusted. We analyzed if initial parasitemia has an effect on chloroquine and ferroquine activities. As expected, we observed a profound effect of initial parasitemia on chloroquine and ferroquine activity in well-characterized chloroquine-sensitive (D10) and multi-resistant (Dd2) parasite strains (Figure 1). Despite different sensitivities toward chloroquine, both strains had similar ferroquine IC50s (Figure 1). Analysis of covariance to predict ferroquine IC50 (the dependent variable) with chloroquine IC50, strain, and initial parasitemia (the independent predictors) revealed that only initial parasitemia was a significant (P = 0.0013) predictor, whereas neither chloroquine (P = 0.30) nor strain (P = 0.62) were significant predictors. Consequently, simple correlation of chloroquine and ferroquine activities does not reflect cross-resistance, if parasitemia is not adjusted.
If initial parasitemia of the assay was not adjusted it should be possible to correct statistically for effect of different starting parasitemia. Therefore, we made a covariance analysis (Figure 2), which revealed that the four variables, drug, strain, interaction of drug and strain, and initial parasitemia, were all significant (P < 0.0001) predictors for IC50. In our model, the slope of IC50s is 2.5-fold per 10-fold increase in parasitemia (95% CI, 1.9–3.2) in both strains and for both drugs (Figure 2). The slope represents the effect of initial parasitemia on drug activity, irrespective of strain and drug and can be used to calculate a parasitemia-independent drug effect. Our results prove how important adjusted parasitemias are when drugs partition within the parasite and show a way to reanalyze data from experiments with unadjusted parasitemias.
DISCUSSION
Resistance against an increasing number of anti-malarials is present in the global population of P. falciparum. A major focus of resistance development is Southeast Asia, but in Africa, resistance against all common anti-malarials spreads with a frightening pace, also.15 Thus, resistance of P. falciparum is reported for chloroquine, halofantrine, mefloquine, and sulfadoxine-pyrimethamine in different African countries.16 We found that all isolates from Lambaréné were resistant to chloroquine, although selection of isolates was possibly biased because only isolates from children with no reported drug intake were included in the study. Most importantly, our findings are in accordance with previously published in vitro and in particular in vivo drug sensitivity trials from Lambaréné.12,13 The high degree of chloroquine resistance suggests continuous drug pressure of chloroquine in that area. Ferroquine, a chloroquine derivative, is highly active against chloroquine-resistant P. falciparum laboratory strains5 and shows a good anti-malarial and toxicity profile in rodent malaria.6 It is therefore an interesting candidate for clinical development. Our aim was to determine the activity of ferroquine against field isolates of P. falciparum, in a region with high-grade chloroquine resistance, where in vivo testing of anti-malarial drugs is routinely done. A major concern for the development of chloroquine derivatives is the possibility of cross-resistance that is present in the local parasite population. Low-level cross-resistance can lead to an increased rate of selection for resistance against the new drug.17 We measured cross-resistance by correlation between chloroquine and ferroquine. Encouragingly, we did not find a significant correlation between both drugs, although a trend toward a positive association was present. This is in contrast to two other Gabonese studies: one from the southeastern part of Gabon7 and the other from the capital, Libreville.8 Both studies showed highly significant correlations between chloroquine and ferroquine activities (r = 0.66 and r = 0.36, respectively). The main differences between the experimental protocols of the studies were the detection of parasite growth and the initial parasitemia used in the assay. The growth detection assays used give highly comparable results10 and therefore are unlikely to lead to the different outcomes. Initial parasitemia is different in all three studies (Pradines and others8 used 0.05% to 0.8% and Atteke and others7 used 0.02% to 1%, whereas we adjusted ours to 0.05% in all samples), and a positive association between range of initial parasitemias and correlation of chloroquine versus ferroquine activities is present. It has been shown previously that chloroquine activity depends on the initial parasitemia, because of preferential uptake in parasitized erythrocytes.14 We reproduced these results, and more importantly, identified initial parasitemia as a potential confounder in cross-resistance measurements that can be accounted for by statistical methods. For that reason, we developed a statistical model that can serve as a tool to estimate the effect of initial parasitemia on the outcome of the assay post hoc. Our findings show that it is essential to standardize and re-assess the methodology for drug sensitivity assays to obtain comparable results between different sites and to deduce in vivo activity from in vitro results more reliably.
In conclusion, ferroquine is a good candidate for clinical testing as an anti-malarial. We do not expect increased development of resistance in chloroquine-resistant strains, and if the toxicity and the pharmacokinetic profiles in humans are as good as for chloroquine, it is a highly promising drug for further clinical development.
Median inhibitory concentrations of study drugs in field isolates from Gabon
| Drug | N | IC50* | IC90* | IC99* |
|---|---|---|---|---|
| * In nmol/L (range). | ||||
| † All isolates are beyond the threshold level of resistance (IC99 > 30 nmol/L). | ||||
| Ferroquine | 40 | 1.94 (0.60–6.73) | 3.61 (0.86–18.7) | 5.75 (1.10–56.9) |
| Chloroquine† | 43 | 113 (12.4–332) | 241 (20.2–737) | 544 (40.2–967) |
| Artesunate | 43 | 0.96 (0.20–5.95) | 2.47 (0.33–29.9) | 5.76 (0.57–49.1) |
Covariance analysis of IC50s, parasite strain, and initial parasitemia. IC50s of ferroquine and chloroquine were measured in a chloroquine-sensitive (D10,○) and a multi-resistant (Dd2, ☆) strain at different starting parasitemias. Covariance analysis of logarithmically transformed values was done for D10 (straight line) and Dd2 (dashed line) to model the interaction of (A) ferroquine IC50 as dependent and chloroquine IC50 and strain as predictor variables (B) chloroquine IC50, or (C) ferroquine IC50 as dependent and initial parasitemia and strain as predictor variables. Note the parallel increase in chloroquine (B) and ferroquine (C) IC50s with increasing parasitemia in both strains. IC50 values are in nmol/L; initial parasitemia is in percent.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 6; 10.4269/ajtmh.2006.75.1178
Covariance analysis of IC50s, parasite strain, and initial parasitemia. IC50s of ferroquine and chloroquine were measured in a chloroquine-sensitive (D10,○) and a multi-resistant (Dd2, ☆) strain at different starting parasitemias. Covariance analysis of logarithmically transformed values was done for D10 (straight line) and Dd2 (dashed line) to model the interaction of (A) ferroquine IC50 as dependent and chloroquine IC50 and strain as predictor variables (B) chloroquine IC50, or (C) ferroquine IC50 as dependent and initial parasitemia and strain as predictor variables. Note the parallel increase in chloroquine (B) and ferroquine (C) IC50s with increasing parasitemia in both strains. IC50 values are in nmol/L; initial parasitemia is in percent.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 6; 10.4269/ajtmh.2006.75.1178
Covariance analysis of IC50s, parasite strain, and initial parasitemia. IC50s of ferroquine and chloroquine were measured in a chloroquine-sensitive (D10,○) and a multi-resistant (Dd2, ☆) strain at different starting parasitemias. Covariance analysis of logarithmically transformed values was done for D10 (straight line) and Dd2 (dashed line) to model the interaction of (A) ferroquine IC50 as dependent and chloroquine IC50 and strain as predictor variables (B) chloroquine IC50, or (C) ferroquine IC50 as dependent and initial parasitemia and strain as predictor variables. Note the parallel increase in chloroquine (B) and ferroquine (C) IC50s with increasing parasitemia in both strains. IC50 values are in nmol/L; initial parasitemia is in percent.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 6; 10.4269/ajtmh.2006.75.1178
Analysis of covariance with IC50 as dependent and strain, drug, log initial parasitemia, and interaction between strain and drug as independent variables (r2 = 0.99; P < 0.0001). Depicted are means of measured chloroquine (▪) and ferroquine (□) IC50s and modeled interactions for chloroquine (dashed line) and ferroquine (straight line). Note the similar slope of IC50–parasitemia interactions for all strains and drugs. IC50 in are nmol/L; initial parasitemia is in percent.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 6; 10.4269/ajtmh.2006.75.1178
Analysis of covariance with IC50 as dependent and strain, drug, log initial parasitemia, and interaction between strain and drug as independent variables (r2 = 0.99; P < 0.0001). Depicted are means of measured chloroquine (▪) and ferroquine (□) IC50s and modeled interactions for chloroquine (dashed line) and ferroquine (straight line). Note the similar slope of IC50–parasitemia interactions for all strains and drugs. IC50 in are nmol/L; initial parasitemia is in percent.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 6; 10.4269/ajtmh.2006.75.1178
Analysis of covariance with IC50 as dependent and strain, drug, log initial parasitemia, and interaction between strain and drug as independent variables (r2 = 0.99; P < 0.0001). Depicted are means of measured chloroquine (▪) and ferroquine (□) IC50s and modeled interactions for chloroquine (dashed line) and ferroquine (straight line). Note the similar slope of IC50–parasitemia interactions for all strains and drugs. IC50 in are nmol/L; initial parasitemia is in percent.
Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 75, 6; 10.4269/ajtmh.2006.75.1178
Address correspondence to Benjamin Mordmüller, Medical Research Unit, Hôpital Albert Schweitzer, BP118 Lambaréné, Gabon and University of Tübingen, Department of Parasitology, Wilhelmstr. 27, 72074 Tübingen, Germany. E-mail: benjamin.mordmueller@uni-tuebingen.de
Authors’ addresses: Andrea Kreidenweiss, Peter G. Kremsner, and Benjamin Mordmüller, Medical Reserach Unit, Hôpital Albert Schweitzer, BP118 Lambaréné, Gabon and University of Tübingen, Department of Parasitology, Wilhelmstr. 27, 72074 Tübingen, Germany, Telephone: 49-7071-2980240, Fax: 49-7071-295189, E-mail: benjamin.mordmueller@uni-tuebingen.de. Klaus Dietz, University of Tübingen, Department of Medical Biometry, Westbahnhofstr. 55, 72070 Tübingen, Germany.
Acknowledgments: The authors thank all patients for participation in the study and the staff of the General Hospital, the dispensary of Isaac, and the Albert Schweitzer Hospital for assistance. Francis Babila Ntumngia and Rolf Fendel gave valuable comments during preparation of the manuscript.
Financial support: This study was supported by the fortüne program of the University of Tübingen (project 1195-0-0).
REFERENCES
- 1↑
Snow RW, Trape JF, Marsh K, 2001. The past, present and future of childhood malaria mortality in Africa. Trends Parasitol 17 :593–597.
- 2↑
Mordmüller B, Graninger W, Kremsner PG, 1998. Malaria therapy in the era of chloroquine resistence. Wien Klin Wochenschr 110 :321–325.
- 3↑
Payne D, 1987. Spread of chloroquine resistance in Plasmodium falciparum. Parasitol Today 3 :241–246.
- 4↑
Paschke R, Paetz C, Mueller T, Schmoll HJ, Mueller H, Sorkau E, Sinn E, 2003. Biomolecules linked to transition metal complexes—new chances for chemotherapy. Curr Med Chem 10 :2033–2044.
- 5↑
Domarle O, Blampain G, Agnaniet H, Nzadiyabi T, Lebibi J, Brocard J, Maciejewski L, Biot C, Georges AJ, Millet P, 1998. In vitro antimalarial activity of a new organometallic analog, ferrocene-chloroquine. Antimicrob Agents Chemother 42 :540–544.
- 6↑
Biot C, Glorian G, Maciejewski LA, Brocard JS, 1997. Synthesis and antimalarial activity in vitro and in vivo of a new ferrocene-chloroquine analogue. J Med Chem 40 :3715–3718.
- 7↑
Atteke C, Ndong JM, Aubouy A, Maciejewski L, Brocard J, Lebibi J, Deloron P, 2003. In vitro susceptibility to a new antimalarial organometallic analogue, ferroquine, of Plasmodium falciparum isolates from the Haut-Ogooue region of Gabon. J Antimicrob Chemother 51 :1021–1024.
- 8↑
Pradines B, Fusai T, Daries W, Laloge V, Rogier C, Millet P, Panconi E, Kombila M, Parzy D, 2001. Ferrocene-chloroquine analogues as antimalarial agents: in vitro activity of ferrochloroquine against 103 Gabonese isolates of Plasmodium falciparum. J Antimicrob Chemother 48 :179–184.
- 9↑
Pradines B, Tall A, Rogier C, Spiegel A, Mosnier J, Marrama L, Fusai T, Millet P, Panconi E, Trape JF, Parzy D, 2002. In vitro activities of ferrochloroquine against 55 Senegalese isolates of Plasmodium falciparum in comparison with those of standard antimalarial drugs. Trop Med Int Health 7 :265–270.
- 10↑
Noedl H, Wernsdorfer WH, Miller RS, Wongsrichanalai C, 2002. Histidine-rich protein II: A novel approach to malaria drug sensitivity testing. Antimicrob Agents Chemother 46 :1658–1664.
- 11↑
Wernsdorfer WH, Payne D, 1991. The dynamics of drug resistance in Plasmodium falciparum. Pharmacol Ther 50 :95–121.
- 12↑
Borrmann S, Binder RK, Adegnika AA, Missinou MA, Issifou S, Ramharter M, Wernsdorfer WH, Kremsner PG, 2002. Reassessment of the resistance of Plasmodium falciparum to chloroquine in Gabon: Implications for the validity of tests in vitro vs. in vivo. Trans R Soc Trop Med Hyg 96 :660–663.
- 13↑
Ramharter M, Wernsdorfer WH, Kremsner PG, 2004. In vitro activity of quinolines against Plasmodium falciparum in Gabon. Acta Trop 90 :55–60.
- 14↑
Gluzman IY, Schlesinger PH, Krogstad DJ, 1987. Inoculum effect with chloroquine and Plasmodium falciparum. Antimicrob Agents Chemother 31 :32–36.
- 17↑
Mordmüller B, Kremsner PG, 2006. Malarial parasites vs. anti-malarials: Never-ending rumble in the jungle. Curr Mol Med 6 :247–251.