Abstract
Purpose: :
Alpha-toxin is a key mediator of corneal damage in S. aureus keratitis. Cyclodextrin complexes containing cholesterol have been shown to significantly reduce alpha-toxin activity in vitro and in a keratitis model. This study seeks a potent inhibitory effect in toxin-mediated corneal pathology by various sterols complexed with methyl- or hydroxypropyl- forms of beta-cyclodextrin.
Methods: :
Eleven sterols were allowed to complex with either cyclodextrin via equilibration in an ethanolic solution followed by lyophilization and reconstitution in sterile phosphate buffered saline. For quantitative determinations of alpha-toxin inhibition in vitro, the hemolytic activity of alpha-toxin was determined in the presence of two-fold dilutions of each complex using rabbit erythrocytes (107/well).The ability of each sterol complex to inhibit alpha-toxin in vivo was tested by incubating the complex (1% w/v) with 12 hemolytic units of alpha-toxin for 1 hr prior to intrastromal injection (10 µL) of rabbit corneas (n ≥ 5). Topical drops of the inhibitors (1%) were applied to rabbit eyes at 60, 45, 30, and 15 minutes before toxin injection. Epithelial erosions were measured (mm2) at 2, 4, 6, and 8 hours after injection. Methyl-cyclodextrin complexed with cholesterol served as a positive control of inhibition for both the in vitro and in vivo assays.
Results: :
Of the 22 complexes tested in vitro, three (methyl-cyclodextrin:lanosterol, hydroxypropyl-cyclodextrin:desmosterol, hydroxypropyl-cyclodextrin:squalene; 0.1% solution) showed greater than 32-fold reduction of toxin activity. Of these three, lanosterol was similar to the methyl-cyclodextrin:cholesterol complex, demonstrating a ≥8000 fold reduction in lytic activity. However, in vivo, hydroxypropyl-cyclodextrin:demosterol (P ≤ 0.004), methyl-cyclodextrin:lanosterol (P ≤ 0.022), hydroxypropyl-cyclodextrin:squalene (P ≤ 0.021) had significant reductions in the size of the toxin-induced epithelial erosion.
Conclusions: :
The methyl-cyclodextrin:cholesterol complex produces the most potent inhibition of S. aureus alpha-toxin in vitro and in vivo. The cyclodextrin complex with lanosterol had potent in vitro inhibitory activity and three sterol complexes tested produced significant in vivo inhibition of alpha-toxin mediated corneal damage.
Keywords: keratitis • Staphylococcus • microbial pathogenesis: experimental studies