In Vitro and In Vivo Synergy of Fosmidomycin, a Novel Antimalarial Drug, with Clindamycin

Materials.Blood components were provided by the local Institute of Clinical Immunology and Transfusion Medicine. Chloroquine, quinine, artemisinin, doxycycline, ciprofloxacin, rifampin, and lincomycin were purchased from Sigma. Mefloquine and halofantrine were gifts from Reto Brun (Basel, Switzerland). Atovaquone was a gift from Peter Kremsner (Tübingen, Germany). Proguanil was a gift from Wallace Peters and Brian Robinson (Harrow, Middlesex, United Kingdom). Lumefantrine was provided by Welding GmbH & Co. (Hamburg, Germany). Azithromycin was extracted from Zithromax tablets (Pfizer). Clindamycin was purchased from Sigma, ICN, and Welding GmbH & Co. The P. falciparum laboratory strains used were 3D7 (The Netherlands), HB3 (Honduras), Dd2 (Indochina), and A2 (Gambia). The Plasmodium vinckei strain was provided by Henri Vial (Montpellier, France).

In vitro antimalarial activity. P. falciparum was cultivated in RPMI 1640 medium (Life Technologies) supplemented with 10% human type O⁺ serum and 25 mM HEPES. Human type O⁺ erythrocytes served as host cells (24). Cultures were kept at 37°C under an atmosphere of 5% O₂, 3% CO₂, and 92% N₂. In vitro drug sensitivity assays were carried out on 96-well microtitration plates (1, 4). Fosmidomycin was dissolved in complete culture medium and sterilized by filtration. The other drugs were dissolved in dimethyl sulfoxide and prediluted with complete culture medium. Infected erythrocytes (0.15 ml per well with 2% hematocrit and 0.4% parasitemia) were incubated in duplicate with a twofold serial dilution of each drug for 48 h. After addition of 0.8 μCi of [³H]hypoxanthine (Amersham Pharmacia) in 50 μl of medium per well, the plates were incubated for another 24 h. Parasites were collected on glass fiber filters with a cell harvester (Micromate 196; Packard), and incorporated radioactivity was measured using a β-counter (Matrix 9600; Packard). Growth inhibition was expressed as percent ³H incorporation compared with untreated controls. Values were plotted on semilogarithmic paper for extrapolation of 50% inhibitory concentrations (IC₅₀s).

In vitro drug interaction.Drug interaction studies were performed as previously described (3). Initially, the IC₅₀s of the test drugs alone were determined. Subsequently, drug solutions were diluted with culture medium to initial concentrations of 80 times the predetermined IC₅₀s. These solutions were combined in ratios of 1:5, 1:2, 2:1, and 5:1. Single and combination drug solutions were then introduced into 96-well plates to give duplicate rows of fosmidomycin alone, the test drug, and the four combinations. Finally, the IC₅₀s of the two test drugs alone and in combination were determined. For data interpretation, the IC₅₀s of the drugs in combination were expressed as fractions of the IC₅₀s of the drugs alone normalized to 1. Isobolograms were constructed by plotting the IC₅₀ of one drug against the IC₅₀ of the other for each of the four drug ratios, with a concave curve indicating synergy, a straight line indicating addition, and a convex curve indicating antagonism. To obtain numeric values for the kind of interaction, results were expressed as the sum of the fractional inhibitory concentrations (sum FIC), calculated as (IC₅₀ of drug A in mixture/IC₅₀ of drug A alone) + (IC₅₀ of drug B in mixture/IC₅₀ of drug B alone). Sum FIC values indicate the kinds of interactions as follows: <0.5, synergy; 0.5 to 1, addition; 1 to 2, indifferent interaction; >2, antagonism. Sum FIC values were calculated for the drug ratio resulting in the point closest to the middle of the isobologram.

For determination of growth inhibition by fosmidomycin in the presence of constant clindamycin concentrations, 20-ml aliquots of the suspension of infected erythrocytes in culture medium were adjusted to the desired clindamycin concentration from a 2 mM stock solution in dimethyl sulfoxide before being loaded in triplicated rows onto the 96-well plate. Then a dilution series of fosmidomycin was prepared on the plate.

In vivo drug interaction.For in vivo drug testing, mice were inoculated by intraperitoneal injection with approximately 5 × 10⁷ infected erythrocytes from a donor mouse. Fosmidomycin was dissolved in phosphate-buffered saline and administered orally (75 mg/kg of body weight). Clindamycin hydrochloride was dissolved in distilled water and administered by intraperitoneal injection (5 mg/kg). Four mice were used for each treatment group, and three mice were used for the control group. Parasitemia was monitored by Giemsa staining of blood smears. Mice were sacrificed when parasitemia exceeded 40%. The animal experiments complied with all relevant federal guidelines and institutional policies.

In vitro drug interaction.Before we embarked on the identification of a therapeutic partner for fosmidomycin, a control experiment was performed to validate the methodology through assessment of the interaction between atovaquone and proguanil, a proven synergistic drug combination. As expected, the IC₅₀s of the individual drugs in the four different mixtures were significantly lower than the IC₅₀s of the drugs alone, resulting in a concave curve in the isobologram (Fig. 1). Subsequently, fosmidomycin was tested in combination with most of the currently used antimalarial drugs (Fig. 1). The antifolate drugs, pyrimethamine and cycloguanil, were excluded from the study because of the existence of highly resistant P. falciparum strains in the field (2). The study was performed with four different strains of P. falciparum including the multidrug-resistant strain Dd2 (resistant to chloroquine, pyrimethamine, and cycloguanil). The absolute IC₅₀s of the drugs used in the study for the different strains are listed in Table 1. In the interaction experiments, there was no apparent specific tendency for any strain. Typical isobolograms are shown in Fig. 1, and the sum FIC values for all drug combinations tested are summarized in Table 2. The interaction of fosmidomycin with all quinoline and aryl-amino-alcohol antimalarial drugss was indifferent, with the exception of quinine, for which the interaction was additive. Also, artemisinin, atovaquone, and proguanil had indifferent effects. In addition, a triple combination of fosmidomycin, proguanil, and atovaquone was tested but also resulted in an indifferent effect (data not shown). Among the antibiotics with known antimalarial activity, doxycycline and azithromycin were additive, and ciprofloxacin and rifampin were indifferent. Synergy was observed only with clindamycin and its natural precursor, lincomycin. Remarkably, the shapes of the corresponding isobolograms were asymmetric, in contrast to the curve obtained in the control experiment with atovaquone and proguanil.

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FIG. 1.

Representative isobolograms of the interaction of fosmidomycin with quinine, artemisinin, proguanil, clindamycin, or lincomycin. The interaction of proguanil with atovaquone was assessed as a control experiment (upper left panel). The P. falciparum strain used for each experiment is indicated.

To assess whether the synergy of fosmidomycin and clindamycin is of clinical relevance, it should be noted that clindamycin is an effective but very slow acting antimalarial drug. Therefore, the absolute IC₅₀s of clindamycin obtained under our assay conditions were comparatively high (Table 1). At lower concentrations of clindamycin, the parasites develop normally in the first cycle after exposure, and reinvasion of new host erythrocytes takes place. Growth inhibition finally occurs at the end of the second cycle (the so-called “delayed kill effect”) (6). Consistently, for patients treated with clindamycin, amelioration of symptoms is observed as late as the fourth day of treatment (10). Fosmidomycin, in contrast, kills the parasites at the end of the first cycle. Consequently, we have investigated whether the synergy of fosmidomycin and clindamycin remains apparent under pharmacologically achievable concentrations of clindamycin. It has been reported that during the course of standard low-dose clindamycin therapy with 5 mg/kg every 8 h, minimal plasma drug levels of 150 to 800 ng/ml are achieved (16, 18). The broad range reported possibly depends on the methods used for drug determination. Therefore, we investigated the sensitivity of P. falciparum to fosmidomycin in the presence of clindamycin concentrations between 42 ng/ml (0.1 μM) and 850 ng/ml (2 μM). Parasite growth was not affected by these concentrations of clindamycin alone within the assay time, but the parasites were killed when the incubation time was extended to 4 days (data not shown). In the presence of an 850-ng/ml concentration of clindamycin, the IC₅₀ of fosmidomycin for P. falciparum strain HB3 changed from 82 to 48 ng/ml (Fig. 2). Remarkably, in the presence of a 42-ng/ml concentration of clindamycin, the IC₅₀ of fosmidomycin was still reduced to 55 ng/ml. In an independent experiment using P. falciparum strain A2, a similar shift to lower IC₅₀s was observed in the presence of clindamycin (Fig. 2). These data clearly demonstrate that an increased therapeutic response can be expected from a combination of fosmidomycin and clindamycin.

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FIG. 2.

Dose response of P. falciparum growth to fosmidomycin in the presence of different constant clindamycin concentrations. P. falciparum-infected erythrocytes were incubated with a serial dilution of fosmidomycin in the absence (filled circles) or in the presence of clindamycin at 43 ng/ml (diamonds), 210 ng/ml (inverted triangles), or 850 ng/ml (triangles). Parasite growth was monitored by radioactive hypoxanthine incorporation. The clindamycin concentrations selected did not have intrinsic antimalarial activity under the assay conditions. Results obtained by independent experiments with P. falciparum strains HB3 (upper panel) and A2 (lower panel) are presented.

In vivo drug interaction.Next, the efficacy of fosmidomycin plus clindamycin was investigated in the P. vinckei mouse model. Mice were treated with each drug, administered in doses that were calculated to result in a partial reduction of parasitemia. In parallel, mice were treated with a combination of these drugs in subtherapeutic doses. A relatively high dose of fosmidomycin was chosen because of the anticipated short half-life in plasma (25). In the first experiment, the mice were infected on day 0 and treated on days 1 and 2. To assess efficacy, parasitemia was monitored on days 3, 4, and 5. On day 3, the parasitemia of mice treated with 75 mg of fosmidomycin/kg or 5 mg of clindamycin/kg was 7.8 or 20%, respectively, in comparison to 42% in untreated control mice (Fig. 3). When the mice were treated with a combination of 75 mg of fosmidomycin/kg and 5.0 mg of clindamycin/kg, the parasitemia was approximately 0.1% on day 3, increasing to 0.2% on day 5. A combination of 75 mg of fosmidomycin/kg and 2.5 mg of clindamycin/kg was equally efficacious (data not shown). However, efficacy decreased significantly when the clindamycin dose was further reduced to 1.3 mg/kg (data not shown).

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FIG. 3.

In vivo efficacy of fosmidomycin plus clindamycin in P. vinckei-infected mice. (A) For suppressive treatment, drugs were administered on days 1 and 2 postinfection and parasitemia was monitored on days 3 to 5. (B) For curative treatment starting with high parasitemia, drugs were administered on days 3 and 4 and parasitemia was monitored from days 3 to 8. Geometric mean values and ranges of observed values are indicated. Fos, fosmidomycin; Cli, clindamycin.

An additional study was designed to investigate whether a combination of fosmidomycin and clindamycin would be effective when treatment is initiated in the presence of high parasitemia. This was of particular interest because clindamycin alone, even at high doses, is not able to rescue mice under such conditions. Therefore, treatment was started on day 3 after infection at a parasitemia of approximately 20%. Following treatment with fosmidomycin alone, parasitemia continued to increase for 24 h but then fell to 3.9% by day 5. By day 6, parasitemia had reached a range between 3.6 and 74%, and all mice, even those with only moderate parasitemia, had symptoms of severe anemia, a condition known as postschizontal anemia. Treatment with clindamycin alone did not stop the rise of parasitemia. When fosmidomycin and clindamycin were administered in combination, there was a constant reduction in the level of parasitemia to 0.5% on day 6. Again, a combination of 75 mg of fosmidomycin/kg plus 2.5 mg of clindamycin/kg was as effective as 75 mg of fosmidomycin/kg plus 5.0 mg of clindamycin/kg (data not shown).