Pre-incubation with efungumab increases the sensitivity of Cryptococcus neoformans to amphotericin B and 5-flucytosine combination therapy in vitro

Abstract number: P1365

Al-Akeel R., Eyes T., Nooney L., Matthews R., Burnie J.

Objectives: Cryptococcal meningitis is associated with poor outcome: mortality ranges from 88% to 100% in African countries. Conventional treatment is with amphotericin B (AMB) + 5-flucytosine (5-FC). 5-FC has intrinsic toxicity and the emergence of resistance is common during treatment. Efungumab (EFU; Mycograb^®) is a recombinant antibody derived against the LKVIRKNIV epitope of Candida heat shock protein 90 (Hsp90). This study devised an assay to evaluate sensitivity of EFU pre-treated Cryptococcus neoformans to AMB and 5-FC in vitro.

Methods: Six isolates of C. neoformans were pre-incubated for 2 hours with EFU 4, 8, 16 or 32 ug/mL. Cells were washed and used to inoculate single (AMB or 5-FC) or combination (AMB+5-FC) antifungals. The MIC was determined by microtitre assay at MIC-0 and MIC-2 endpoints and Fractional Inhibitory Concentration Index (FICI) calculated. Biacore analysis was performed on a sensor chip surface covalently coated with 2 ug/ml EFU to assess binding of EFU to cryptococcal Hsp90.

Results: Before EFU pre-treatment, AMB MIC-0 ranged from 0.25 to 1 ug/mL and 5-FC MIC-2 ranged from 0.25 to 4 ug/mL. Pre-treatment with 4 ug/mL EFU reduced AMB MIC-0 range to 0.25–0.5 ug/mL, but the range of 5-FC MIC-2 was unchanged. At 8 ug/mL EFU pre-treatment, AMB MIC-0 was 0.125 ug/mL for 5 out of 6 isolates, and 5-FC MIC-2- ranged from 0.125–2 ug/mL for 4 out of 6 isolates. At 16 or 32 ug/mL EFU pre-treatment, MICs were reduced by 2–6 dilutions for each antifungal (0.0625–0.25 ug/mL for AMB (MIC-0) and 0.125–1 ug/mL for 5-FC (MIC-2)). Combination of AMB+5-FC gave lower FICIs when cells were pre-treated with EFU vs not pre-treated. FICI values decreased as EFU concentration increased (Table). Biacore analysis demonstrated the high affinity of EFU for C. neoformans Hsp90: K_a=1.38×10⁵ M^-1/S^-1, K_d=5.53×10^-4 S^-1 and K_D=4.19×10^-9 M.

Conclusion: The tight-binding affinity of EFU to cryptococcal Hsp90 enabled EFU+AMB+5-FC triple therapy to be assayed in C. neoformans. EFU pre-treatment increased the sensitivity of C. neoformans to AMB, 5-FC and AMB+5-FC in vitro, suggesting potential synergy of these combinations.