Anti-Acanthamoeba Potential of Miltefosine: Evaluation of its In vitro and Ex vivo Efficacy and Safety in Human Cornea

Abstract

Purpose : Miltefosine was recently approved by the US Food and Drug Administration (FDA) for the treatment of systemic Acanthamoeba infections. This study aimed to assess the in vitro and ex vivo efficacy of miltefosine against the clinical isolates of Acanthamoeba keratitis and its safety profile to rationalize its localized ocular application.

Methods : Clinical isolates of Acanthamoeba spp. keratitis (n=17) were made axenic, sequenced for genotype identification and tested for the determination of minimal cysticidal and trophozoicidal concentrations (MCC and MTC) of miltefosine. Cytotoxicity of miltefosine was assessed on human corneal epithelium (HCE) at four incubation time points in different concentrations. A.castellanii (T4) trophozoites and cysts were challenged on confluent monolayers of HCE and ex vivo human corneal models in the presence and absence of miltefosine for 24 hrs. Cytopathic effects and changes in the corneal morphology were evaluated using microscopy and histopathology analysis.

Results : Majority of Acanthamoeba isolates tested were T4 genotype (82.3%). MTC₉₀ and MCC₉₀ values of miltefosine were 125 μg/mL and 4 mg/mL respectively. Miltefosine was found to be safe at 62.5 μg/mL and 125 μg/mL on HCE for 4 hrs and 15 min, respectively. Without miltefosine, A. castellanii trophozoites and cysts destroyed the cellular structures and corneal architecture within 24 hrs while miltefosine pre-treatment completely prevented the infection at both the tested concentrations (62.5 μg/mL and 125 μg/mL).

Conclusions : Miltefosine was effective against Acanthamoeba cysts and trophozoites in vitro and ex vivo, however, cidal concentration was higher by more than 30-fold for cysts compared to trophozoites. At the effective concentration the drug was safe for corneal epithelial cells and exvivo corneal tissue.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.

 

Axenic Acanthamoeba trophozoites and cysts in Peptone-yeast-glucose (PYG) media at 7th and 14th day

 

Microscopy images of monolayer of human corneal epithelial (HCE) cell lines infected with infected with A. castellanii trophozoites (1X 10⁴/ mL) were recorded at baseline (a), 1 hrs (b), 2 hrs (c), 4 hrs (d), 8 hrs and 24 hrs (f). Effect of miltefosine (125 μg/mL) alone on HCE cells at baseline (g), 8 hrs (h) and 24 hrs (i). HCE cells infected with A. castellanii trophozoites (1X10⁴/ mL) preincubated with miltefsoine (125μg/mL) at baseline (j), 8 hrs (k) and 24 hrs (l).