In vitro and in vivo antifungal activity of amphotericin B lipid complex: are phospholipases important?

Abstract

Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids L-alpha-dimyristoylphosphatidylcholine (DMPC) and L-alpha-dimyristoylphosphatidylglycerol. ABLC is less toxic than amphotericin B deoxycholate (AmB-d), while it maintains the antifungal activity of AmB-d. Active amphotericin B can be released from ABLC by exogenously added (snake venom, bacteria, or Candida-derived) phospholipases or by phospholipases derived from activated mammalian vascular tissue (rat arteries). Such extracellular phospholipases are capable of hydrolyzing the major lipid in ABLC. Mutants of C. albicans that were resistant to ABLC but not AmB-d in vitro were deficient in extracellular phospholipase activity, as measured on egg yolk agar or as measured by their ability to hydrolyze DMPC in ABLC. ABLC was nevertheless effective in the treatment of experimental murine infections produced by these mutants. Isolates of Aspergillus species, apparently resistant to ABLC in vitro (but susceptible to AmB-d), were also susceptible to ABLC in vivo. We suggest that routine in vitro susceptibility tests with ABLC itself as the test material may not accurately predict the in vivo activity of ABLC and that the enhanced therapeutic index of ABLC relative to that of AmB-d in vivo may be due, in part, to the selective release of active amphotericin B from the complex at sites of fungal infection through the action of fungal or host cell-derived phospholipases.

FIG. 1

Comparison of ABLC and AmB-d for the treatment of systemic C. albicans infections in immunosuppressed mice. (A) Infection (intravenous) with 2.5 × 10⁴ CFU of a phospholipase-producing strain of C. albicans (strain SC5314) per mouse. The median survival time for saline-treated mice was 11.5 days. (B) Infection (intravenous) with 5 × 10⁵ CFU of a phospholipase-deficient mutant of C. albicans (mutant SC15183) per mouse. The median survival time for saline-treated mice was 12.5 days. (C) Infection (intravenous) with 10⁷ CFU of a phospholipase-deficient mutant of C. albicans (mutant SC15184) per mouse. The median survival time for saline-treated mice was 4.5 days.

FIG. 2

Comparison of ABLC and AmB-d for the treatment of systemic Aspergillus infections in normal mice. (A) Infection (intravenous) with 5 × 10⁶ CFU of A. fumigatus 10-AF per mouse. The median survival time for saline-treated mice was 8.5 days. (B) Infection (intravenous) with 2 × 10⁶ CFU of A. flavus 89-158 per mouse. The median survival time for mice treated with saline was 6.5 days. (C) Infection (intravenous) with 2 × 10⁷ CFU of A. terreus 92-62 per mouse. The median survival time for mice treated with saline was 18 days.

FIG. 3

Percentage of total radioactivity from ¹⁴C-DMPC formulated as ABLC that was recovered in the organic layer as myristic acid or lysophosphatidylcholine (lyso PC) following incubation with supernatants from culture media exposed or not exposed to activated rat artery sections.