Comparison of the physicochemical, antifungal, and toxic properties of two liposomal amphotericin B products

Abstract

Small unilamellar amphotericin B liposomes can reduce the toxicity of amphotericin B. In this study, we compared the physical, antifungal, pharmocokinetic, and toxic properties of two liposomal amphotericin B products, AmBisome and Anfogen, that have the same chemical composition but are manufactured differently. In vitro tests included determinations of the MICs and the concentrations causing the release of 50% of the intracellular potassium from red blood cells (K50 values) to assess toxicity. The 50% lethal dose (LD50) was evaluated by using uninfected C57BL/6 mice and single intravenous (i.v.) doses of 1 to 100 mg/kg of body weight. Multiple i.v. dosing over 18 days was performed with 0.5, 1.0, or 5.0 mg of Anfogen/kg or 1.0, 5.0, or 25 mg of AmBisome/kg to evaluate chronic toxicity. DBA/2 mice were infected intranasally with 2.5 x 10(6) Aspergillus fumigatus conidia, treated for 3 or 4 days with 3.0, 5.0, or 7.5 mg of Anfogen/kg or 3, 5, 7.5, or 15 mg of AmBisome/kg, and evaluated to assess the toxicity of the drugs to the kidneys (by measurement of blood urea nitrogen and creatinine levels and histopathology) and the drug efficacy. The median particle size was 77.8 nm for AmBisome and 111.5 nm for Anfogen. In vitro K(50) values were significantly lower for Anfogen (0.9 mug/ml) than for AmBisome (20 microg/ml), and the LD50 of AmBisome was >100 mg/kg, versus 10 mg of Anfogen/kg. There was significant renal tubular necrosis in uninfected and infected mice given Anfogen but no tubular necrosis in AmBisome-treated mice. AmBisome at 7.5 or 15 mg/kg was also more efficacious than 7.5 mg of Anfogen/kg for the treatment of pulmonary aspergillosis, based on survival and weight loss data and numbers of CFU per gram of lung. In conclusion, the efficacy and toxicity of these two liposomal amphotericin B products were significantly different, and thus, the products were not comparable.

FIG. 1.

Potassium release from rat RBCs measured after 4 h of incubation at 37°C with serial dilutions of AmBi, Anfo, or D-AMB. K₅₀ values were determined for AmBi (K₅₀ = 20 μg/ml), Anfo (K₅₀ = 0.9 μg/ml), and D-AMB (K₅₀ = 0.4 μg/ml).

FIG. 2.

Single-dose toxicity of Anfo and AmBi for uninfected female C57BL/6 mice. (A) Survival of C57BL/6 female mice (n, 5 per group) given a single i.v. injection of D5W; AmBi at 5.0, 10.0, 50.0, or 100 mg/kg; or Anfo at 1.0, 5.0, 10.0, or 20.0 mg/kg (P = 0.134 for D5W versus Anfo at 10 mg/kg; P < 0.003 for D5W versus Anfo at 20 mg/kg). (B) Mean percent weight changes in groups receiving a single i.v. injection of D5W or AmBi at 5.0, 10.0, 50.0, or 100 mg/kg. (C) Mean percent weight changes in groups receiving a single i.v. injection of D5W or Anfo at 1.0, 5.0, 10.0, or 20.0 mg/kg.

FIG. 3.

Kidney histopathology of Anfo- and AmBi-treated mice. (A) Appearance of normal renal tubules in a kidney from a representative uninfected female C57BL/6 mouse treated i.v. with 18 daily doses of D5W (control; H&E-stained sample shown at a magnification of ×200). Bar, 30 μm. (B) H&E-stained kidney from a representative uninfected female C57BL/6 mouse treated i.v. with 18 daily doses of 5 mg of AmBi/kg. Rare tubules lined by regenerative epithelium with intraluminal protein casts (asterisk) are visible. Bar, 30 μm. (C) H&E-stained kidney from a representative uninfected female C57BL/6 mouse treated i.v. with 18 daily doses of 5 mg of Anfo/kg. Shown are clusters of dilated renal tubules lined by attenuated epithelium, often filled with cellular debris or granular casts (asterisks) and surrounded by small numbers of inflammatory cells. Bar, 30 μm.

FIG. 4.

Comparative efficacy of multidose AmBi and Anfo in triamcinolone-immunosuppressed C57BL/6 female mice (n, 7 per group) challenged intranasally with A. fumigatus (2.6 × 10⁶ spores/mouse). (A) Survival of mice given four i.v. doses of D5W; AmBi at 3.0, 5.0, 7.5, or 15 mg/kg; or Anfo at 3.0, 5.0, or 7.5 mg/kg. P, 0.022 for D5W versus 3.0 and 5.0 mg of Anfo/kg and 3.0, 5.0, 7.5, and 15 mg of AmBi/kg; D5W versus 7.5 mg of Anfo/kg, not significant. (B) Percent mean weight changes for mice given four i.v. doses of D5W or AmBi at 3.0, 5.0, 7.5, or 15 mg/kg. (C) Percent mean weight changes for mice given four i.v. doses of D5W or Anfo at 3.0, 5.0, or 7.5 mg/kg. Observed deaths are indicated, and data for mice who died were not included in weight change calculations.

FIG. 5.

Kidney and lung histopathology of Anfo- and AmBi-treated mice. (A) Appearance of normal renal tubules in an H&E-stained kidney from a representative A. fumigatus-infected female DBA/2 mouse treated i.v. with three daily doses of D5W (control). Bar, 30 μm. (B) H&E-stained kidney from a representative A. fumigatus-infected female DBA/2 mouse treated i.v. with three daily doses of 7.5 mg of AmBi/kg. Rare tubules lined by attenuated epithelium with intraluminal protein casts (asterisks) are visible. Bar, 30 μm. (C) H&E-stained kidney from a representative A. fumigatus-infected female DBA/2 mouse treated i.v. with three daily doses of 7.5 mg of Anfo/kg. Shown are clusters of dilated degenerative tubules (D) containing exfoliated necrotic epithelium admixed with necrotic tubules (N) completely devoid of epithelial-lining cells containing intraluminal granular casts surrounded by congested capillaries and interstitial hemorrhage. Bar, 30 μm. (D) H&E-stained lung from a representative A. fumigatus-infected female DBA/2 mouse treated i.v. with three daily doses of D5W (control). The sample shows evidence of pneumonia characterized by histiocytic and neutrophilic inflammation within terminal airways and alveoli. Necrosis is limited, and angioinvasion is absent. Bar, 150 μm. (E) H&E-stained lung from a representative A. fumigatus-infected female DBA/2 mouse treated i.v. with three daily doses of 7.5 mg of AmBi/kg. The sample shows evidence of pneumonia similar in character to that seen in D5W-treated mice but decreased in severity. Bar, 150 μm. (F) H&E-stained lung from a representative A. fumigatus-infected female DBA/2 mouse treated i.v. with three daily doses of 7.5 mg of Anfo/kg. The sample shows evidence of pneumonia similar in character to that seen in D5W-treated mice but decreased in severity. Bar, 150 μm.

FIG. 6.

CFU per gram of lung from triamcinolone-immunosuppressed C57BL/6 female mice (n, 5 per group) infected intranasally with A. fumigatus (2.6 × 10⁶ spores/mouse). Three i.v. treatments with D5W (control); Anfo at 3.0, 5.0, or 7.5 mg/kg; or AmBi at 3.0, 5.0, 7.5, or 15 mg/kg were given, and lungs were collected from mice sacrificed 24 h after the third treatment. Bars indicate median values for each group. (P < 0.01 for D5W versus 3.0 or 5.0 mg of Anfo/kg; P = 0.095 for D5W versus 7.5 mg of Anfo/kg; P < 0.01 for D5W versus all AmBi doses; P = 0.056 for 7.5 mg of Anfo/kg versus 7.5 mg of AmBi/kg; P = 0.016 for 3.0 mg of Anfo/kg versus 3.0 mg of AmBi/kg; P < 0.008 for 15 mg of AmBi/kg versus 3.0, 5.0, and 7.5 mg of AmBi/kg and 3.0, 5.0, and 7.5 mg of Anfo/kg).