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. 1998 Apr;42(4):899–902. doi: 10.1128/aac.42.4.899

Comparison of Fungizone, Amphotec, AmBisome, and Abelcet for Treatment of Systemic Murine Cryptococcosis

Karl V Clemons 1,*, David A Stevens 1
PMCID: PMC105563  PMID: 9559804

Abstract

Three lipid-based formulations of amphotericin B have been approved for use in various countries. The aim of this study was to compare Amphotec (ABCD; Sequus), AmBisome (AmBi; Nexstar), Abelcet (ABLC; The Liposome Co.), and conventional deoxycholate amphotericin B (Fungizone; Bristol Meyers Squibb) for the treatment of experimental systemic cryptococcosis. A model was established in 10-week-old female CD-1 mice by intravenous (i.v.) injection of 6.25 × 105 viable Cryptococcus neoformans yeast cells. Therapy began 4 days later, with i.v. administration three times per week for 2 weeks. Mice received either no treatment, 1 mg of Fungizone per kg of body weight, or 1, 5, or 10 mg of ABCD, AmBi, or ABLC per kg. Ninety percent of control mice died between days 15 and 34. All treatment regimens except ABLC at 1 mg/kg prolonged survival compared with no treatment (P < 0.01 to 0.001). All mice receiving 5 or 10 mg of ABCD or AmBi per kg and 90% of mice given 10 mg of ABLC per kg survived, whereas ≤50% of those given other treatment regimens survived. Fungizone was the least effective of the four formulations, with 5 or 10 mg of ABCD, AmBi, or ABLC per kg resulting in a significantly better outcome than Fungizone (P < 0.001). Among the three formulations, ABCD and AmBi were equally effective, both being better than ABLC at equal 5- or 10-mg/kg doses (P < 0.001). Comparison of residual infectious burdens in various organs showed that each drug had some dose-responsive efficacy in three or more organs at escalating doses. In the brain, ABCD or AmBi at 5 or 10 mg/kg or ABLC at 10 mg/kg was more effective than Fungizone at 1 mg/kg or no treatment, while ABCD or AmBi at 1 mg/kg was as effective as ABLC at 10 mg/kg. Similar results were obtained for the kidneys and lungs. In the spleen, ABCD at 10 mg/kg cured all mice of infection and was superior to all other regimens. In the liver, AmBi at 5 mg/kg was superior to an equal dose of ABCD or ABLC. Overall, the efficacies of ABCD and AmBi were equal to that of Fungizone at 1 mg/kg and were about 10-fold better than that of ABLC, particularly in the brain; a comparative rank order of efficacies was ABCD ≅ AmBi > ABLC ≫ Fungizone. This is the first study that compared all four amphotericin B formulations.

The continued increase in the number of serious systemic fungal infections has made the pursuit of safe and effective therapies an area of much activity over the last several years. At the present time, amphotericin B remains the “gold standard” for treatment. However, as has been well documented, amphotericin B therapy is limited by associated toxicities (11). These toxicities can be reduced by the incorporation of amphotericin B into a lipid-based carrier system, which alters the pharmacokinetics and tissue distribution of the drug (13). At the present time, three lipid-based carrier systems for amphotericin B are in development. These are as follows: Amphotec (ABCD; Sequus Pharmaceuticals, Inc.), which is a colloidal dispersion of cholesteryl sulfate and amphotericin B; AmBisome (AmBi; Nexstar Pharmaceuticals, Inc.), which consists of true unilamellar liposomes; and Abelcet (ABLC; The Liposome Co., Inc.), which is a ribbon form of lipid-stabilized amphotericin B aggregates (12, 15, 18). Each of these preparations has been shown to be less toxic than the conventional deoxycholate formulation of amphotericin B and to have efficacy against various fungi (1, 3, 5–9, 14). In addition, each of these formulations has been shown to be useful for the treatment of cryptococcal meningitis in either animal models or clinical studies (1–3, 10, 14–17, 19). However, no study comparing the relative efficacies of the lipid formulations has been done.

The aim of the current study was to compare the efficacies of the three commercially available lipid-based formulations of amphotericin B (ABCD, AmBi, and ABLC) with each other as well as with the conventional deoxycholate formulation, Fungizone, for the treatment of experimental systemic murine cryptococcosis. In this model, the primary target of the infection is the central nervous system, which is exceedingly difficult to treat and for which it is difficult to effect clearance of the fungal burden. Thus, for the first time, the relative efficacies of these amphotericin B preparations were assessed in a single study.

MATERIALS AND METHODS

Infection model.

A murine model of systemic cryptococcosis was established in 10-week-old female CD-1 mice by intravenous injection of 6.25 × 105 viable Cryptococcus neoformans 9759 yeast cells as described previously (4, 14). Therapy was initiated 4 days after infection, with groups of 10 mice each receiving either no treatment, Fungizone at 1 mg/kg; ABCD (Sequus Pharmaceuticals, Inc., Menlo Park, Calif.) at 1, 5, or 10 mg/kg, AmBi (Nexstar Pharmaceuticals, Inc., San Dimas, Calif.) at 1, 5, or 10 mg/kg, or ABLC (The Liposome Co., Inc., Princeton, N.J.) at 1, 5, or 10 mg/kg. Each drug was prepared in accordance with the manufacturer’s instructions and diluted in sterile 5% dextrose water for intravenous administration. ABCD, AmBi, and ABLC were prepared fresh daily. We have demonstrated in prior studies (14) that treatment of mice with the diluent, 5% dextrose water, is equivalent to no treatment and therefore did not include a diluent-treated group in this study. All treatments were based on an equivalent milligrams of amphotericin B per kilogram of body weight. The formulations were administered intravenously in 0.25-ml volumes three times per week for 2 weeks, for a total of six doses.

The number of animals succumbing to infection was tallied through 49 days of infection. There were originally 10 mice in the group receiving 10 mg of ABCD per kg. A single mouse died on day 6 postinfection, 2 days after the first drug dosage, prior to the second dosage, and before any animals showed symptoms of progressive infection. The cause of death was not evident, since neither this animal nor any other receiving the same regimen showed signs of acute toxicity after the first dosage. Because the cause of death could not be determined, although we speculated that it was due to air embolism, this mouse was not included in any statistical analyses. At the end of the 49-day period, all surviving mice were euthanized by CO2-induced asphyxia, and the number of viable CFU of C. neoformans remaining in the brain, spleen, liver, kidneys, and lungs of each animal was determined by quantitative plating of organ homogenates as described previously (4, 14). All CFU were expressed as the log10 number of viable yeast cells per entire organ.

Statistics.

The statistical analyses of survival were done by a life table analysis using a log rank test (SAS version 6.11; SAS Institute, Cary, N.C.), and the analyses of comparative CFU recovered from the organs were done by a nonparametric method using a Mann-Whitney U test (20). To ensure that death was considered a worse outcome than survival with any amount of burden, a log value of 8 was assigned to data points missing due to death from infection (5–9, 14). In previous studies, this value has been determined to be the approximate number of CFU in an organ just prior to death.

RESULTS

Survival comparisons.

The model established proved to be lethal to untreated control mice, with 90% succumbing to infection between days 15 and 34 postinfection (Fig. 1). Deaths also occurred among treated mice, with 50 to 80% of mice treated with Fungizone, ABCD, or AmBi at 1 mg/kg or with ABLC at 1 or 5 mg/kg dying between days 21 and 49 of infection (Fig. 1). All mice that had been given 5 or 10 mg of ABCD or AmBi per kg and 90% of the mice given 10 mg of ABLC per kg survived through the 49-day experiment. By life table analyses, all treatment regimens except ABLC at 1 mg/kg had significant efficacy in the prolongation of survival compared with the controls (P < 0.008 to 0.0001, depending on the comparison). However, the use of a nonparametric Wilcoxon rank sum test for the comparison of survival rates showed that ABLC at 1 mg/kg provided significant protection compared to no treatment (P < 0.05).

FIG. 1.

FIG. 1

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Cumulative mortality of mice treated with ABCD (A), AmBi (B), or ABLC (C). In each panel, the control and Fungizone data are presented for comparison.

Fungizone was the least-effective treatment with regard to prolonging survival, with 5 or 10 mg of ABCD, AmBi, or ABLC per kg proving to be significantly more effective (P < 0.002 to 0.0001, depending on the comparison). Only higher doses of ABCD or AmBi or ABLC at 10 mg/kg proved more efficacious than 1 mg of ABCD or AmBi per kg (P < 0.05 to 0.01). ABCD and AmBi were superior to ABLC at equivalent 5-mg/kg doses (P < 0.01). However, all three drugs were equally effective at 1- or 10-mg/kg doses (P > 0.05). Thus, ABCD and AmBi showed equivalent efficacies and were more effective than ABLC. No toxicities were observed, either acutely or on gross examination at necropsy, for any treatment regimen.

Clearance of infectious burden.

The second parameter examined for the determination of comparative efficacies was that of infectious burden remaining in the organs of surviving mice 49 days after infection. The mean burdens of C. neoformans recovered from the organs of mice in groups with 50% or more survivors showed that each of the lipid-based formulations exhibited some dose responsiveness in the reduction of the fungal burdens in three or more organs with escalating doses (Table 1). For each formulation, the 10-mg/kg dose caused a greater reduction of the infectious burden in each organ than did the 5-mg/kg dose. This reduction of infection was up to 100-fold in some organs (Table 1).

TABLE 1.

Recovery of C. neoformans from the organs of surviving mice treated with Fungizone, ABCD, AmBi, or ABLC

Formulation and concn (mg/kg) No. of survivors/no. cured Geometric mean log10 CFU/organ (no. free of infection) [95% confidence interval]
Brain Spleen Liver Kidney Lung
None 1/0 1.74 (0) 0 (1) 0.86 (0) 0 (1) 1.03 (0)
Fungizone, 1 1/0 1.52 (0) 0.71 (0) 0 (1) 0 (1) 0 (1)
ABCD
  1 5/0 5.92 (0) [4.0–7.9] 2.60 (0) [0.9–4.3] 3.79 (0) [3.0–4.5] 3.07 (0) [2.3–3.8] 4.03 (0) [3.0–5.0]
  5 10/0 5.55 (0) [4.7–6.4] 1.30 (3) [0.3–2.3] 1.62 (3) [0.6–2.7] 1.86 (3) [0.6–3.1] 2.60 (1) [1.6–3.6]
 10a 9/0 4.05 (0) [2.8–5.3] 0 (9) 0.16 (8) [0–0.5] 0.31 (7) [0–0.9] 0.70 (6) [0–1.5]
AmBi
  1 3/1 3.72 (1) [0–11.7] 1.5 (1) [0–4.8] 1.81 (1) [0–5.7] 1.10 (1) [0–3.7] 1.67 (1) [0–5.3]
  5 10/0 5.52 (0) [4.2–6.8] 1.53 (3) [0.6–2.4] 0.27 (9) [0–0.9] 1.53 (3) [0.6–2.5] 2.37 (2) [1.1–3.7]
 10 10/0 4.12 (0) [3.0–5.2] 0.87 (3) [0.2–1.5] 0 (10) 0.44 (7) [0–1.0] 1.54 (2) [0.7–2.4]
ABLC
  1 2/1 2.45 (1) [0–33] 0 (2) 0 (2) 0 (2) 0 (2)
  5 5/0 6.59 (0) [5.8–7.3] 1.81 (1) [0.04–3.6] 2.13 (0) [1.1–3.1] 1.99 (1) [0.2–3.8] 2.54 (0) [2.2–4.1]
 10 9/0 6.48 (0) [5.8–7.1] 1.10 (4) [0–2.3] 0.51 (7) [0–1.5] 2.68 (0) [1.8–3.6] 3.19 (0) [2.2–4.1]
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a

There were only nine mice in this group. 

For the brain, comparisons showed that ABCD and AmBi at 5 or 10 mg/kg were efficacious compared to either Fungizone at 1 mg/kg or no treatment (P < 0.01) whereas only the 10-mg/kg dose of ABLC showed efficacy (P < 0.01). Comparisons among the three preparations showed that ABLC was the least efficacious, with 1 mg of ABCD or AmBi per kg being equivalent to 10 mg of ABLC per kg and 5 or 10 mg of ABCD or AmBi per kg being superior to the same dose of ABLC (P < 0.01 to 0.001). Dose escalation effectively increased the efficacy of a formulation, with the 10-mg/kg dose of either ABCD or AmBi causing a significant reduction of the burden when compared with lower doses of the same drug (P < 0.05 to 0.01). However, ABLC at 10 mg/kg was superior to only the 1-mg/kg dose (P < 0.05) and ABLC at 5 mg/kg was as effective as the 10-mg/kg dose. It should be noted that none of the formulations, when given at 5 or 10 mg/kg, cleared any of the surviving mice of cryptococcosis in the brain (Table 1).

The results of the comparisons of mean infectious burdens recovered from the kidneys and lungs were similar to those for the brain. The 5- and 10-mg/kg doses of ABCD and AmBi were superior to 1 mg of Fungizone per kg (P < 0.01 to 0.001). In the kidneys, these two doses were also superior to the same doses of ABLC (P < 0.05 to 0.001). In the lungs, ABCD or AmBi at 10 mg/kg was superior to the same dose of ABLC (P < 0.01 to 0.001) and AmBi at 5 mg/kg was superior to the same dose of ABLC (P < 0.05) (Table 1). In the kidneys, increasing doses of either ABCD or AmBi, but not ABLC, caused a greater number of animals to be free of detectable infection (Table 1). In contrast, only ABCD at 10 mg/kg cured more than 50% of treated mice of lung infection (Table 1).

Clearance of the fungal burden from the liver and spleen showed some differences in comparative efficacies that might be due to differences in pharmacokinetics. For example, ABCD at 10 mg/kg was found to be superior (P < 0.05 to 0.001) to all other dosing regimens. This dose of ABCD cleared all animals of infection in the spleen, whereas 10 mg of AmBi per kg cured only three animals and 10 mg of ABLC per kg cleared four (Table 1). ABLC showed somewhat better efficacy in the clearance of the infectious burden from the liver, with the 10-mg/kg dose being equivalent to 10 mg of either ABCD or AmBi per kg (P > 0.05). However, 5 mg of ABCD or AmBi per kg was superior to 5 mg of ABLC per kg (P < 0.05 to 0.001) in the treatment of disease in the liver, as well as in the spleen (P < 0.05). In addition, AmBi at 5 mg/kg was more efficacious than ABCD at 5 mg/kg (P < 0.05) in the treatment and cure of cryptococcal infection in the liver. At equivalent 10-mg/kg doses, ABCD cleared eight mice of infection in the liver, AmBi cleared 10, and ABLC cleared seven mice of infection.

DISCUSSION

The incorporation of amphotericin B into lipid carrier systems has proven to reduce the toxicities associated with the administration of the conventional deoxycholate formulation of amphotericin B. This reduction in toxicities occurs primarily through avoidance of renal clearance. However, experimental studies have demonstrated that higher dosages of the drug must be administered for it to retain its therapeutic activity against most fungal infections, because the efficacy has been reduced by severalfold (1, 3, 5–9, 14). Previous studies have examined the efficacy of each of the three lipid-carried formulations against C. neoformans but have done so singly (1–3, 14, 17). Although each study reported an efficacy equal to or just less than that of Fungizone, clearance of infection from the brain, which is the key target of cryptococcal infection, was not achieved in most instances (1–3, 14, 17). Given these results, it was of interest to us to do a single study in which the comparative efficacies of these formulations, including Fungizone, were examined.

The results of our study indicate that some differences in potency and therapeutic efficacy exist among the different formulations of amphotericin B. All three preparations showed efficacy in the prolongation of survival in this model, with the efficacies of ABCD, AmBi, and ABLC being about equal, on a milligram of amphotericin B per kilogram of body weight basis, to that of Fungizone. However, both ABCD and AmBi were superior to ABLC at equivalent doses of 5 mg of amphotericin B per kg. Similarly, all three lipid formulations could be administered at significantly higher dosages of amphotericin B than could Fungizone (1 mg/kg is approaching the lethal toxic dose for Fungizone), which significantly improved survival. Thus, a rank order of efficacy for the prolongation of survival appears to be ABCD = AmBi > ABLC ≫ Fungizone.

In addition to the differences in prolongation of survival, organ-specific differences in efficacy were also noted. Although no preparation administered at the 5- or 10-mg/kg dose cleared any mice of infection in all organs, each showed some dose responsiveness in increased clearance of the burden with escalating doses in three or more organs. In the brain, kidneys, and lungs, ABCD and AmBi showed equivalent efficacies and were superior to ABLC. AmBi showed the greatest activity in the liver, whereas ABCD showed a clear advantage over both AmBi and ABLC in the spleen. Overall, ABCD was better in clearing infection from organs other than the brain.

Clearance of C. neoformans from the brain was similar to that reported in previous studies for each preparation (1–3, 14, 17). None of the preparations cured an infection in the brain at the 10 mg of amphotericin B per kg dose. This may be deemed to be as expected considering the difficulty of central nervous system penetration. However, it should also be noted that each formulation did show efficacy in the reduction of the fungal burden in the brain. This is supported by the fact that the animals die because of parenchymal brain infection. Treatment with ABCD, AmBi, or ABLC significantly prolonged survival, and each of these formulations was more effective than Fungizone. In addition, the mean burdens of C. neoformans recovered from the brains of treated mice were lower with escalating doses. Overall, a rank order of efficacy for the clearance of infection from all organs appears to be ABCD ≥ AmBi > ABLC ≫ Fungizone.

Considering our previous work indicating that 1 mg of Fungizone per kg is approaching the limit of acute lethal toxicity (4–9, 14), ABCD, AmBi, and ABLC were each about 10-fold less toxic than Fungizone, since no acute toxicities were observed nor were gross pathological changes in the organs evident at necropsy. Thus, all three lipid preparations were overall superior to Fungizone because the higher doses permitted produced superior results. Among these formulations, the therapeutic efficacy of ABCD or AmBi proved superior to that of ABLC by up to 10-fold in survival and in clearing infection from the brain. ABCD and AmBi showed therapeutic efficacies equivalent to that of Fungizone on a milligram-per-kilogram basis for the treatment of systemic cryptococcosis, while ABLC proved to be about 10-fold less effective than Fungizone. However, no clear-cut advantage was noted for either ABCD or AmBi when they were compared with each other. These differences in efficacy might change if the number of doses is increased or the dosing schedule is altered. Changes in the duration and schedule of dosing might also improve the efficacy of each formulation. In addition, comparative studies of these formulations in other models of systemic infection would be desirable, since it is possible that changes in the rank order of efficacy could occur or even that no one preparation would prove significantly better than another. Regardless, the results of our present study demonstrate that ABCD and AmBi are about equal in efficacy and more effective than ABLC for the treatment of cryptococcosis, with the efficacies of all three formulations being superior to that of Fungizone for the treatment of systemic and meningeal cryptococcal disease.

ACKNOWLEDGMENT

These studies were funded in part by a grant from Sequus Pharmaceuticals, Inc.

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