Evaluation of in vitro Activity, Cytotoxicity, and in vivo Efficacy of Novel Liposomal Formulations of Amphotericin B in Comparison to Natamycin and Liposomal Amphotericin B (AmBisome) in a Murine Model of Experimental Mycotic Keratitis caused by Aspergillus fumigatus

Author:

Ruta Petraitiene (US)

Abstract:

Background:  Mycotic keratitis (MK) is an important global cause of blindness and ocular loss, with an estimated incidence exceeding 1,000,000 cases/year. There is a critical need for new therapeutics for this sight-threatening disease. Currently, natamycin (NAT), which is a standard of care for MK, often causes pain, conjunctivitis, and corneal injury. Our current clinical experience using AmBisome (AmBi) for the treatment of MK demonstrated that it was comparable in efficacy to NAT but markedly better tolerated. Therefore, we investigated the antifungal activity and cytotoxicity of new broad-spectrum liposomal formulations of amphotericin B (LFAMBs) in in vitro studies and in an immunosuppressed murine model of experimental MK caused by Aspergillus fumigatus.

Methods:  Initially the in vitro antifungal activity of 35 LFAMBs was studied and compared with NAT, deoxycholate amphotericin B (DAMB), and AmBi against A. fumigatus using CLSI reference methods. Based upon the analysis of MIC data obtained for each formulation against the tested isolates, 20 LFAMBs were selected for in vitro cytotoxicity studies using human corneal epithelial cells (HCEpC) by LDH release assay and MTT assay. Three formulations (PCDODAB, PCDOTMA, and PCDODMA), which demonstrated lower toxicity in HCEpC throughout the range of tested concentrations, were examined for in vivo efficacy against mycotic keratitis in immunosuppressed female BALB/c mice after inoculation of the right cornea with A. fumigatus (1×109 to 1×1010 CFU/mL) under general anesthesia.

Treatment groups consisted of LFAMB-1, LFAMB-2, and LFAMB-3 at 1.25, 2.50, and 4mg/mL, AmBi at 4mg/mL, NAT at 50mg/mL, and untreated control (UC) mice. Antifungal therapy started 24 hours after inoculation and continued for 3 days or 7 days. The antifungal agents were administered as topical ophthalmic drops 6×/day. Antifungal in vivo efficacy was evaluated by postmortem examination of the eyes, quantitative fungal cultures, and histological analysis.

Results:  MICs against A. fumigatus were 0.5-1 of LFAMB-1, 0.25 of LFAMB-2, and 0.25-1 µg/mL of LFAMB-3. Formulations LFAMB-1, LFAMB-2, and LFAMB-3 demonstrated lowest cytotoxicity measured. These three formulations showed lower cytotoxicity to HCEpC than NAT and AMBi. The in vivo efficacy was time-dependent being more active after 7 days of treatment. External ocular examination demonstrated improvement after treatment with the three LFAMBs. Each of the three LFAMBs demonstrated significant decreases in CFU/g of A. fumigatus after 7 days of treatment in comparison to UC (P≤0.05) with LFAMB1 being the most active. By external examination and quantitative cultures, all three LFAMBs were comparable in activity to the treatment controls of AmBi and NAT. Histopathological examinations demonstrated no evidence of hyphal elements and no morphological evidence of LFAMB-induced cytotoxicity in LFAMB treatment of Aspergillus keratitis.

Conclusions:  These new LFAMBs demonstrated in vitro activity and in vivo efficacy, and safety profiles that provide a scientific foundation for clinical trials in the treatment of MK in patients afflicted with this terrible corneal disease.

Abstract Number: 90

Conference Year: 2024

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