INTRODUCTION

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The drugs most commonly used to treat visceral leishmaniasis (VL) are the pentavalent antimonials sodium stibogluconate (SSG; Pentostam) and meglumine antimoniate (Glucantime). Second-line drugs, used in instances of antimonial-treatment failure, include amphotericin B (AMB), paromomycin (aminosidine), and pentamidine (reviewed by Berman [3]). However, all of these drugs require a multiple-dose regimen, with the attendant problems of cost and patient compliance, the latter exacerbated by political upheaval in regions where VL is endemic (11). Although early reports of the use of the recently introduced, novel, lipid-based formulations of AMB, AmBisome, Abelcet, and Amphocil in VL treatment indicate that they are effective after fewer doses and have toxicities (3) lower than that of the conventional AMB formulation, they are more expensive. There is a pressing need for an inexpensive alternative to the pentavalent antimonials which is effective, ideally, after a single dose.

A nonionic surfactant vesicle formulation of SSG (SSG-NIV), as a single dose, is effective against Leishmania donovani infection in BALB/c mice (2). Treated mice express the immunological responses associated with cure and show no sign of relapse, at least up to day 42 posttreatment. This type of formulation might be used to improve and extend the clinical utility of the pentavalent antimonials. However, although the NIV formulation may be less expensive to produce than the new AMB formulations, it is important to demonstrate that, as a VL treatment, SSG-NIV is at least as effective. In this study the antileishmanial activity of SSG-NIV was compared with those of AMB vesicular or vesicle-like formulations. SSG and AMB formulations were compared in acute- and chronic-infection models, since previous studies have shown that treatment with free SSG is less effective against chronic infections (1). Prophylatic treatments with SSG and AMB formulations were used to compare their abilities to deliver drug to infection sites and to determine the postdosing persistence of the drugs at these sites.

	MATERIALS AND METHODS

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Materials. SSG was provided by Glaxo Wellcome Ltd. Proprietary AMB formulations (AmBisome, Abelcet, and Amphocil) were purchased directly from wholesalers. The nonionic surfactant tetraethylene glycol mono-n-hexadecylether was purchased from Chesham Chemicals Ltd. Dicetyl phosphate and ash-free cholesterol were obtained from Sigma, and all other reagents were of analytical grade.

Animals and parasites. Age-matched 8- to 10-week-old BALB/c mice (in-house inbred males or females) were used in this study. In-house bred or commercially obtained (Harlan Olac) Golden Syrian hamsters (Mesocricetus auratus) were used for maintenance of L. donovani (strain MHOM/ET/67:LV82). Mice were infected (day 0 of the experiment) by intravenous injection (tail vein, no anaesthetic) with 1 × 10⁷ to 2 × 10⁷ L. donovani amastigotes (5).

Drug formulations. Vesicle constituents (150 µmol) consisting of a 3:3:1 molar ratio of mono-n-hexadecylether tetraethylene glycol to cholesterol to dicetyl phosphate were melted by heating them at 130°C for 5 min. The molten mixture was cooled to 70°C, hydrated with 5 ml of preheated (70°C) 100-mg/ml SSG solution, and homogenized at 8,000 ± 100 rpm at 70°C for 15 min with a mixer (model L4R SU; Silverson Machines) fitted with a five-eighth-in tubular work head. Vesicle suspensions were stored at room temperature.

Acute-infection model. Groups of L. donovani-infected mice (n = 5) were treated intravenously, via the tail vein, on day 7 with either a single dose of phosphate-buffered saline (PBS) (controls) or a single dose of one of the following: SSG-NIV (31 to 300 mg of Sb^V/kg of body weight), SSG solution (266 to 300 mg of Sb^V/kg), AmBisome (1 to 8 mg of AMB/kg), or (on days 7 and 8) SSG solution (88 to 1,332 mg of Sb^V/kg). In multiple-dosing experiments, mice were treated on days 7 to 11 with PBS (controls) or one of the AMB formulations (2.5 mg of AMB, AmBisome, Abelcet, or Amphocil per kg per dose) and were sacrificed on day 14 or 18.

Pretreatment. The effect of pretreatment with SSG-NIV or AmBisome on the parasite burdens of mice which were then subsequently infected with L. donovani was also determined. Male BALB/c mice were given a single intravenous dose of PBS (controls), SSG-NIV (300 mg of Sb^V/kg), or AmBisome (8 mg of AMB/kg) and 18 days later infected with 10⁷ L. donovani amastigotes. Animals were sacrificed on day 27 postinfection.

Chronic-infection model. Mice were treated on day 35 or 38 with a single dose of PBS (controls), SSG solution (300 mg of Sb^V/kg), SSG-NIV (300 mg of Sb^V/kg), or AmBisome (AMB concentration, 8 mg/kg). Animals were sacrificed 7 days after the last drug treatment (i.e., day 42 or 45).

Determination of parasite numbers. Parasite burdens in the livers, spleens, and bone marrows of control and drug-treated mice were determined (5). Leishman-Donovan units (LDU) were calculated per organ for the liver and spleen by using the formula (4) LDU = amastigote number per 1,000 host cell nuclei × organ weight (in grams).

Presentation and statistical analysis of data. Parasite suppression (mean percentage ± standard error of the mean [SEM] percentage) was determined for a particular site by comparing each experimental parasite burden with the relevant mean control value. For each experiment, the mean control parasite burden (LDU/organ for spleen and liver and number of parasites/1,000 host cell nuclei for bone marrow) is shown. Parasite burdens were analyzed by Student's unpaired t test on the log₁₀-unit-transformed parasite burden data.

	RESULTS

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Acute-infection model. Mean parasite burdens ± SEM on day 14 in PBS-treated, control mice lay in the ranges 35 to 191 LDU in spleen, 2,017 to 3,610 LDU in liver, and 111 to 580 parasites/1,000 host cell nuclei in bone marrow. Treatment of acute L. donovani infections with multiple doses of the AMB formulations or a single dose of SSG-NIV significantly suppressed liver parasite burdens (Table 1), and except for Abelcet, which was significantly less suppressive, these various formulations were equally active. Similar results were obtained against spleen parasites. Against bone marrow parasites, although Abelcet did not give significant suppression, the other three formulations were active, and of these, SSG-NIV was the most active (P < 0.0005). On the basis of their antiparasitic activities at the three infection sites, the drugs could be ranked as follows: SSG-NIV > AmBisome = Amphocil > Abelcet. Comparison of the antiparasitic effects of AmBisome treatment, given either as a single high dose (Table 2) or as five small doses (Table 1), showed that the two regimens were equipotent against the parasites at the three infection sites examined. SSG-NIV, SSG solution, and AmBisome treatments were equally active against liver parasites (Table 2). Against spleen or bone marrow parasites, treatment with SSG-NIV was more effective than treatment with AmBisome and free SSG was inactive. The ranking efficacy against murine VL on a milligram-per-kilogram basis was AmBisome > SSG-NIV > free SSG (Fig. 1). All three preparations had good activity against liver parasite burdens, but only SSG-NIV cleared spleen and bone marrow of parasites. Treatment with a high dose of AmBisome (>8 mg/kg) or free SSG (>1,332 mg of Sb^V/kg) was toxic.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Dose-response curves for SSG solution (FD), SSG-NIV, and AmBisome. Groups of L. donovani-infected BALB/c mice were treated on day 7 with a single dose of PBS (controls), SSG-NIV (31 to 300 mg of SbV/kg), or AmBisome (1 to 8 mg of AMB/kg) or on days 7 and 8 with PBS (controls) or free-SSG solution (88 to 1,332 mg of SbV/kg). On day 14, parasite burdens in the liver, spleen, and bone marrow of control and drug-treated mice were determined. For each tissue site, by comparison with values for the relevant control, the treatment-induced suppression of parasites was calculated. Results for each formulation come from separate experiments, and control parasite burdens for these were, with AmBisome, 102 ± 16 LDU in spleen, 3,550 ± 302 LDU in liver, and 457 ± 126 parasites/1,000 host cell nuclei in bone marrow; with SSG-NIV, 191 ± 67 LDU in spleen, 3,103 ± 525 LDU in liver, and 580 ± 85 parasites/1,000 host cell nuclei in bone marrow; with FD, 35 ± 6 LDU in spleen, 2,017 ± 237 LDU in liver, and 111 ± 10 parasites/1,000 host cell nuclei in bone marrow. Asterisks indicate the highest dose level used due to toxicity.

Chronic-infection model. In the chronic-infection model, typical parasite burdens on day 45 in PBS-treated, control mice were 695 ± 186 LDU in spleen, 2,797 ± 630 LDU in liver, and 269 ± 76 parasites/1,000 host cell nuclei in bone marrow. In chronic infection, mice spleen and bone marrow burdens tended to be higher, and liver burdens tended to be lower, than in the acute-infection model and the most obvious difference was the greater hepatosplenomegaly typical of VL. In general, all treatments were less effective against chronic infection than against acute infection. Therapeutic outcomes of the three treatments could be ranked on the basis of parasite suppression at all three sites as follows: SSG-NIV > AmBisome > SSG solution (Table 2). Free SSG was inactive against spleen and bone marrow parasites.

Pretreatment. Pretreatment with either SSG-NIV or AmBisome conferred a measure of protection against an infectious inoculum given 18 days later. The extent of the protection was site dependent (Fig. 2) and was greatest in the liver.

View larger version (28K): [in this window] [in a new window]   FIG. 2.   Effects on liver, spleen, and bone marrow parasite burdens of treatment of mice with SSG-NIV or AmBisome before infection with L. donovani. Groups of male BALB/c mice (n = 5) were given single doses of PBS (controls), SSG-NIV (300 mg of SbV/kg), or AmBisome (8 mg of AMB/kg) and infected 18 days later with 107 L. donovani amastigotes. At 27 days postinfection, parasite burdens in the livers, spleens, and bone marrows (bm) of control and drug-treated mice were determined. For each tissue site, by comparison with values for the relevant control, the treatment-induced suppression of parasites was calculated. Mean control parasite burdens were as follows: 49 ± 24 LDU in spleen, 1,245 ± 186 LDU in liver, and 21 ± 6 parasites/1,000 host cell nuclei in bone marrow.

	DISCUSSION

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The results of this study confirm those of previous studies (5, 6, 12), namely, that NIV delivery can enhance the antileishmanial activity of SSG and that the several novel formulations of AMB also have good activities against experimental VL (10). If the overall ability to suppress burdens of L. donovani parasites in liver, spleen, and bone marrow is taken as the measure of antileishmanial activity, then in the acute-infection model, the SSG-NIV treatment was most effective in this study. It is apparent that on a milligram-per-kilogram basis, there is a discrepancy between the doses of SSG and AMB and it must be emphasised that the intention here was not to compare absolute antileishmanial activities but rather VL treatments employing one drug dose or a few drug doses. The choice of the doses used for the AMB formulations was based on those used by Berman (3) in his clinical studies. There is no doubt that increasing the drug dose for the various treatments used in this study would tend to increase antileishmanial activity, although Gangneux et al. (8) have shown that with meglumine antimoniate, treatment with high doses (up to 2,200 mg of Sb^V/kg) failed to clear Leishmania infantum parasites from the spleen, liver, and lungs. However, the aim of this study was to compare the efficacy of SSG-NIV treatment with the efficacies of AMB treatments at clinically used dose levels.

The highest tolerated single dose of AMB was found to be 8 mg/kg, and with doses above this, the mice showed signs of distress. In this respect, our findings are at variance with those of Gangneux et al. (9), who apparently administered single doses of 50 mg of AMB/kg, up to a total of 300 mg/kg, with no adverse effects. Apart from this observation, the results of this study are, in general, in agreement with those of Gangneux et al. (8, 9). Although treatment with Ambisome was more effective than with Abelcet against spleen, liver, and bone marrow parasites in this study, Gangneux et al. (8) found that treatments with either Ambisome or Abelcet (total AMB dose, 72 mg/kg), if started early (day 7 postinfection), were equally effective and that at this dose, they cleared parasites from liver, spleen, and lungs. We have previously shown (10) that, in the dose range used here, Ambisome is more effective than Abelcet against murine VL. In a systemic murine model of cryptococcosis (7), the same activity ranking found here for the three proprietary lipid formulations of AMB was recently described.

The efficacies of most VL treatments appear to be dependent on the duration of infection. For example, treatment with a total Ambisome dose of 30 mg of AMB/kg started on day 7 postinfection cleared the spleen, liver, and lungs of L. infantum parasites (8) but if treatment was delayed until day 60 postinfection, parasites were still found in the liver and spleen (9). In the present study, the effect of duration of infection on the antileishmanial activity of AmBisome was more apparent in the spleen and bone marrow than in the liver. A similar effect of duration of infection on the antileishmanial effects of SSG have previously been described (1). By using a total Ambisome dose of 300 mg of AMB/kg, Gangneux et al. (9) could clear liver, lungs, and spleen of parasites in their chronic-infection model. This AMB dose suggests that tolerance to AmBisome has been underestimated. In the present study, the greater activity of the single dose of SSG-NIV (compared to that of AmBisome) observed against acute infection was more pronounced in the chronic-infection than in the acute-infection model, which suggests different effects of the duration of infection on the two treatments.

The protection against infection conferred by pretreatment, with SSG-NIV or AmBisome, suggests the persistence of antileishmanial concentrations of residual drug at a site of parasite multiplication, perhaps within tissue macrophages. Such pretreatment protection has been observed before, with niosomal and liposomal SSG (6). In view of the widely disimilar physicochemical and pharmacokinetic properties of SSG and AMB, it is surprising that both were active at 18 days after administration and it appears that vesicular delivery is an important common factor in this protective effect. It is of significance that the pretreatment protective effect has not been observed with free SSG (6). The fact that, for both SSG-NIV and AmBisome, protection was higher in the liver than in the spleen or bone marrow is also in accord with the involvement of vesicles in this prophylactic effect.

In summary, the results of this study show that the SSG-NIV formulation is highly active and that its efficacy compares well with those of other vesicular and vesicle-like formulations of AMB, which also have good antileishmanial activities. Data from the murine VL model suggests that as a clinical treatment, SSG-NIV would require fewer doses (perhaps one to three doses) than free SSG. Toxicology studies and preclinical trials would, however, be required to determine the optimal treatment protocol. Studies are under way to lower the antimony dose without compromising the high efficacy of the SSG-NIV formulation.