Potent Plasmodicidal Activity of a Heat-Induced Reformulation of Deoxycholate-Amphotericin B (Fungizone) against Plasmodium falciparum

Abstract

The emergence and spread of drug-resistant Plasmodium falciparum continue to pose problems in malaria chemotherapy. Therefore, it is necessary to identify new antimalarial drugs and therapeutic strategies. In the present study, the activity of a heat-treated form of amphotericin B (HT-AMB) against P. falciparum was evaluated. The efficacy and toxicity of HT-AMB were also compared with those of the standard formulation (AMB). HT-AMB showed significant activity against a chloroquine-resistant strain (strain K-1) and a chloroquine-susceptible strain (strain FCR-3) in vitro. The 50% inhibitory concentrations of HT-AMB were 0.32 ± 0.03 μg/ml for strain K-1 and 0.33 ± 0.03 μg/ml for strain FCR-3. In the presence of 1.0 μg of HT-AMB per ml, only pyknotic parasites were observed after 24 h of incubation of early trophozoites (ring forms). However, when late trophozoites and schizonts were cultured with 1.0 μg of HT-AMB per ml, those forms multiplied to ring forms but the number of infected erythrocytes did not increase. These results indicate that HT-AMB possesses potent antiplasmodial activity and that the drug is more effective against the ring-form stage than against the late trophozoite and schizont stages. HT-AMB was observed to have little cytotoxic effect against a human liver cell line (Chang liver cells). In conclusion, the results suggest that HT-AMB has promising properties and merits further in vivo investigations as a treatment for falciparum malaria.

Malaria infection due to Plasmodium falciparum is a major public health problem in many tropical and subtropical areas. Sporozoites, the infective form of the parasite, are transferred to the human host during a bite by female Anopheles mosquitoes, invade hepatocytes, and develop into liver schizonts, which contain large numbers of merozoites. The asexual blood-stage cycle of P. falciparum commences when the merozoites released from hepatocytes enter the blood circulation and invade red blood cells (RBCs). In this phase of the cycle merozoites initially develop within RBCs as ring forms and then progress to trophozoites and eventually to schizonts, which rupture and release a new wave of merozoites that invade a new batch of RBCs. Chloroquine (CQ), a blood-stage schizonticidal drug, has been the drug of choice for the treatment of falciparum malaria for several decades, but its clinical utility has been greatly reduced in most areas where CQ-resistant malaria is endemic (1, 22). However, CQ remains the most widely used first-line antimalarial drug because it is well tolerated, safe for pregnant women and young children, efficacious against susceptible strains of P. falciparum and the other three human malaria species, and inexpensive. At present, the development of new antimalarial drugs and the use of preexisting drugs in combination are the most important approaches to overcoming the problem of drug resistance.

Amphotericin B (AMB) is a heptaene macrolide antibiotic that is active against fungi and yeasts. Fungizone, the commercially available deoxycholate salt form of AMB, is the drug that is the most widely used for the treatment of deep-seated mycotic infections. This drug is also the recommended second-line treatment for visceral leishmaniasis when conventional tetravalent antimony therapy is inappropriate or ineffective (11, 15). Unfortunately, intravenously (i.v.) administered AMB causes acute side effects, which limit its more extensive clinical use.

One approach to decreasing the toxicity of AMB has been to develop new derivatives or formulations with greater aggregation. Some investigators have reported that heat treatment of Fungizone leads to an increase in the size of aggregated AMB. Heating of AMB at 70°C for 20 min (heat-treated AMB [HT-AMB]) induces a superaggregated form that leads to a new equilibrium. This novel formulation has been associated with reduced toxicity in mammalian cells, while its antifungal activity is retained in vitro and in vivo (4, 10). This formulation is inexpensive and can be used to improve the therapeutic index of AMB against candidiasis and cryptococcosis and to encourage the more widespread use of AMB (13).

The aim of this study was to evaluate the antimalarial activity of HT-AMB against blood-stage parasites of P. falciparum in vitro. The efficacy and toxicity of HT-AMB were compared with those of the standard AMB formulation.

MATERIALS AND METHODS

Cultivation of P. falciparum.

A CQ-resistant P. falciparum strain, strain K-1, which was originally from Thailand, and a CQ-susceptible strain, strain FCR-3, which was originally from The Gambia, were grown asynchronously by the modified method of Trager and Jensen (17). We used RPMI 1640 medium with glutamine supplemented with 10% human type O serum, 25 mM HEPES, 25 μg of gentamicin (Sigma-Aldrich, St. Louis, Mo.) per ml, sodium bicarbonate, and human type O RBCs in disposable sterile dishes and a controlled atmosphere of 5% CO₂-5% O₂-90% N₂ at 37°C.

Antifungal agents.

AMB (injectable Fungizone) was purchased from Bristol Pharma Co. (Tokyo, Japan). A stock solution of AMB was reconstituted in sterile water according to the instructions of the manufacturer. HT-AMB was prepared by heating AMB solutions for 20 min in a water bath at 70°C, as described by Petit et al. (13).

Evaluation of in vitro plasmodicidal effect of HT-AMB.

The following procedures were used to evaluate the antimalarial activities of HT-AMB and AMB. Asynchronously cultivated malaria parasites were used. RPMI 1640 medium was supplemented with 0 (control), 0.5, 1.0, 5.0, or 10.0 μg of HT-AMB or AMB per ml. The antifungal drug-supplemented medium was changed every 24 h. Five hundred microliters of a parasitized RBC (pRBC) suspension was placed in each well of a 24-well flat-bottom culture plate (Sumiron; Sumitomo Bakelite Co., Ltd., Tokyo, Japan) with a hematocrit of 5% and an initial parasitemia level of 0.1%. Thin-smear specimens stained with Giemsa solution were made every 24 h, and the level of parasitemia was determined by counting the number of parasites per 10,000 RBCs.

Determination of HT-AMB and AMB IC₅₀s.

In vitro drug susceptibility tests were performed as described previously (16). Briefly, synchronous pRBCs showing a parasitemia level of 1% were placed in 24-well culture plates. Synchronization was achieved by treating the pRBCs with 5% d-sorbitol for 30 min at room temperature. Twenty microliters of drug-supplemented medium was added to each well to give a series of doubling dilutions from 0.10 to 100.00 μg/ml. After 24 h of incubation in an atmosphere of 5% CO₂-5% O₂-90% N₂ at 37°C, the control wells were checked for parasite growth. When the schizonts in the control wells were fully grown, the culture plates were removed from the incubator. Thin-smear specimens were prepared and stained with Giemsa solution. The numbers of RBCs in the control smears were counted under a microscope until 50 schizonts were encountered. The effects of the drugs on parasite growth were evaluated by the observation of decreased numbers of schizonts per equal numbers of RBCs counted previously in the control cultures. The growth inhibition effect (in percent) was calculated as follows: (test well schizont count/control well schizont count) × 100. The 50% inhibitory concentrations (IC₅₀s) of AMB and HT-AMB were calculated by the probit method.

Effect of HT-AMB on a hepatic cell line.

Cells of the Chang human liver cell line were a kind gift from Takeaki Nagamine, Gunma University School of Health Sciences (Gunma, Japan). The cells were grown continuously in complete Dulbecco modified Eagle’s medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum in a 5% CO₂ atmosphere at 37°C. Prior to exposure to the drugs, the cells were seeded at 10⁵ cells/ml in 96-well culture plates and incubated for 72 h in 0.2 ml of Dulbecco modified Eagle medium supplemented with AMB or HT-AMB. Cell death was evaluated by a lactate dehydrogenase release assay (CytoTox 96 assay kit; Promega Corp., Madison, Wis.), according to the protocol recommended by the manufacturer (5). All of the test compounds were assayed at each concentration in triplicate.

Detection of hemolysis caused by treatment with HT-AMB.

The level of hemolysis was determined by measuring the amount of hemoglobin that eluted into the medium by the sodium lauryl sulfate method (hemoglobin B test; Wako Pharmaceuticals, Osaka, Japan) described previously (16). Briefly, after exposure of pRBCs or RBCs to 1.0 to 100.0 μg of HT-AMB per ml, the samples were centrifuged at 1,000 × g for 5 min at 20°C, and the supernatant was collected and analyzed.

Data analysis.

The data are presented as the means ± standard errors of the means from at least three sets of independent experiments. Student's t test was used for statistical analysis. A P value of less than 0.05 was considered statistically significant.

RESULTS

HT-AMB inhibits P. falciparum growth in vitro.

To confirm the plasmodicidal activities of AMB and HT-AMB, a CQ-resistant P. falciparum strain (strain K-1) and a CQ-susceptible strain (strain FCR-3) were exposed to medium containing 0.5 to 10.0 μg of AMB or HT-AMB per ml for 72 h, and parasite growth and multiplication were monitored (Fig. 1). AMB at concentrations equal to or greater than 1.0 μg/ml induced marked decreases in the levels of parasitemia. HT-AMB at concentrations equal to or greater than 1.0 μg/ml also induced decreases in the levels of parasitemia, but the decrease was slower than that obtained with AMB. At these concentrations, complete inhibition of parasite multiplication was attained within 72 h of incubation.

FIG. 1.

Effect of HT-AMB on P. falciparum parasitemia in vitro. The time- and concentration-dependent effects of continuous incubation with AMB (closed symbols) or HT-AMB (open symbols) on levels of parasitemia caused by CQ-resistant strain K-1 (A) and CQ-susceptible strain FCR-3 (B) of P. falciparum are shown. All cultures were started with asynchronized parasites. Parasitemia was measured at the beginning of incubation (0 h) and every 24 h thereafter for 72 h. Parasites were incubated in the presence of drug at concentrations of 0 (diamonds), 0.5 (squares), 1.0 (triangles), and 5.0 (circles) μg/ml.

The results of the in vitro drug susceptibility assay were as follows. The IC₅₀s of AMB were 0.95 ± 0.10 μg/ml for K-1 and 0.89 ± 0.26 μg/ml for FCR-3. The IC₅₀s of HT-AMB were 0.32 ± 0.03 μg/ml for K-1 and 0.33 ± 0.03 μg/ml for FCR-3. The IC₅₀s of HT-AMB were threefold lower than those of AMB. There were no significant differences between the IC₅₀s for CQ-resistant strain K-1 and those for CQ-susceptible strain FCR-3 (P > 0.05).

HT-AMB alters P. falciparum morphology and interferes with parasite development.

The effects of HT-AMB on the morphology and development of P. falciparum parasites were evaluated with synchronized cultures of the two strains (CQ-resistant strain K-1 and CQ-susceptible strain FCR-3). The effects against both strains were similar, and the parasites grew to mature stages after 24 h of incubation without HT-AMB (Fig. 2A). When 1.0 μg of HT-AMB per ml was added to synchronized ring-form parasites, pyknotic parasites inside and outside of the RBCs were observed after 24 h of incubation (Fig. 2B). When HT-AMB was added to synchronized late-stage trophozoites and schizonts in culture, parasites that had multiplied but that had altered morphologies at the ring stage were observed after 24 h of incubation (data not shown).

FIG. 2.

Morphology of P. falciparum K-1 after 24 h of incubation with HT-AMB. Parasites were synchronized at the ring stage. The morphologies of cells in Giemsa-stained thin blood smears from drug-free cultures (A) and cultures incubated with 1.0 μg of HT-AMB per ml (B) for 24 h are shown. Note the parasite pyknotic changes and the prevalence of ring forms in the HT-AMB-treated culture (arrows in panel B). Magnifications, ×1,000.

Effect of HT-AMB on Chang liver cells.

As shown in Fig. 3, AMB at 12.5 μg/ml showed slight cytotoxicity for Chang liver cells (P < 0.05), and AMB at concentrations equal to and greater than 25.0 μg/ml showed strong cytotoxicity, whereas HT-AMB at the same concentrations showed no cytotoxicity (P < 0.01).

FIG. 3.

Effect of HT-AMB on Chang liver cells in vitro. Chang liver cells were cultured for 72 h in the presence of HT-AMB. The viability of the cells was determined with a CytoTox 96 assay kit, which quantitatively measures the amount of lactate dehydrogenase released into the culture medium upon cell death. *, P < 0.05; **, P < 0.01.

Hemolysis of pRBCs by HT-AMB.

To detect hemolysis as an index of cytotoxicity, the concentration of hemoglobin in the pRBC or RBC culture medium was determined. In the presence of 1.0 μg of HT-AMB per ml, the concentration of hemoglobin in the pRBC culture medium was significantly higher (0.949 ± 0.192 g/dl) than that in the RBC culture medium (0.138 ± 0.048 g/dl) (P < 0.05). Similar results were observed in the case of AMB treatment.

DISCUSSION

Identification of the antimalarial effects of drugs that have been used for other purposes is an attractive approach to overcoming the increasing threat of drug-resistant malaria. AMB is one of the antifungal agents that is the most effective and widely used for the treatment of systemic fungal infections commonly found in immunocompromised patients. However, it can show dose-dependent renal toxicity, which is not predictable by monitoring of the serum drug concentration (19, 20). Several lipid AMB formulations have been developed to decrease this toxicity.

As an inexpensive alternative, simple AMB was treated with moderate heat (70°C for 20 min) to produce a new, self-aggregated state. It has been reported that in vitro HT-AMB exhibits significantly lower levels of toxicity for mammalian renal cells and fewer hemolytic effects against RBCs than the standard formulation and that HT-AMB also shows increased toxicity for fungal cells (4, 10, 13). Our study showed that in vitro HT-AMB has a greater plasmodicidal effect than AMB against both CQ-resistant and CQ-susceptible P. falciparum strains. The growth curves of asynchronous parasites showed that AMB has a greater inhibitory effect than HT-AMB, but significant differences were observed only at high concentrations that would not be applicable to treatment for malaria. We confirmed that HT-AMB showed no cytotoxicity for Chang liver cells, as expected, and that HT-AMB showed much less hemolytic activity than AMB. In fact, we conducted in vivo studies of the efficacies of AMB and HT-AMB against malaria parasites using Plasmodium berghei NK65 and 20 female ICR/Jcl mice (age, 6 weeks), which consisted of 5 control mice, 5 mice treated with 0.5 mg/kg of body weight i.v., 5 mice treated with 1.0 mg/kg i.v., and 5 mice treated with 2.0 mg/kg i.v. However, no significant difference in parasite growth or the survival rate of the mice was observed (data not shown). AMB and HT-AMB were also not observed to have hemolytic activity. We learned from these experiments that maintenance of effective drug concentrations in the peripheral blood is very important (10), but we were not able to maintain effective drug concentrations by the administration of a single i.v. dose to mice. Further in vivo experiments are still needed before our findings on the effectiveness of AMB and HT-AMB in in vitro studies can be applied to the treatment of both drug-resistant and -susceptible human P. falciparum malaria.

Most antimalarial drugs, including CQ, have been reported to show schizonticidal activity in blood. One of the schizonticidal mechanisms of CQ is inhibition of heme polymerization in vitro (2). Another antimalarial drug, quinoline, also inhibits heme polymerization (2, 7, 8, 12). In this study, pyknotic parasites were observed inside and outside of RBCs when HT-AMB was added to synchronized ring-form cultures at 1.0 μg/ml for 24 h (Fig. 2B). In contrast, when HT-AMB was added to synchronized mature-stage (late trophozoite and schizont) cultures, ring-form parasites that had multiplied successfully invaded and remained inside the RBCs for 12 to 24 h of incubation, indicating that HT-AMB has a greater hemolytic effect against pRBCs than it does against non-pRBCs.

It is generally assumed that the permeabilizing effects of AMB are related to its ability to form transmembrane channels, whereas the lytic effect is due to the peroxidative action of AMB at the membrane level (3, 14, 18). The oxidation of unsaturated fatty acids leads to a change in the membrane, which becomes more sensitive to the osmotic shock induced by channel formation. Autoxidation of AMB in solution as well as AMB-induced peroxidation of unsaturated fatty acids in the RBC membrane is assumed to be triggered by the reactive oxygen species that may be produced by AMB. It has also been reported that increased amounts of reactive oxygen species are generated during malaria infection, leading to RBC membrane damage (6, 9). This may explain the higher levels of plasmodicidal activity and hemolytic activity of HT-AMB against pRBCs, the greater effect of HT-AMB against ring forms than against late trophozoites and schizonts, and the apparently different antimalarial mechanism of HT-AMB compared with those of quinoline antimalarial drugs.

In conclusion, the results of the present study suggest that HT-AMB has promising properties and merits further in vivo investigations for the treatment of falciparum malaria.