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R. S. Kadam, J. Hejkal, H. F. Edelhauser, U. B. Kompella; An ex vivo Model Using High Throughput Cassette Dosing, LC/MS/MS Analysis, and Microdialysis to Assess Corneal Permeability of Eight Beta-Blockers: Comparison With Isolated Corneal Transport Studies. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3188.
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Beta-blockers and isolated cornea or corneal cell culture models are routinely used to study the influence of lipophilicity on corneal drug permeation. The purpose of this study was to establish an ex vivo bovine eye model employing cassette eye drop dosing, LC/MS/MS analysis, and microdialysis to determine corneal beta-blocker penetration and to compare the results with isolated cornea model.
A cassette of eight beta-blockers with varying lipophilicities solubilized in assay buffer (pH 7.4) was used for eye drop administration in ex vivo studies and donor solutions in isolated corneal transport studies. The cassette included the following beta-blockers: sotalol, atenolol, nadolol, pindolol, metoprolol, timolol, betaxolol, and propranolol. In both studies, samples were collected up to 6 hours. The penetration of beta-blockers after topical application as a multiple-dose cassette eye drop was evaluated by microdialysis in the anterior chamber of an ex vivo bovine eye model (37 oC) using linear microdialysis probe (10 mm). In ex vivo studies, as an initial approach, we assessed penetration from multiple 50 µl drug drops applied every 30 minutes throughout the study, with 15 min intermittent blank buffer drops. In-vitro transport of beta-blocker cassette across isolated bovine corneas was also assessed. An LC/MS/MS method using Q-trap LC-MS/MS (positive ionization mode) was developed and validated for high-throughput cassette analysis of beta-blockers
The LCMS method for beta-blockers has short run-time (13 min) and is highly specific for the beta-blockers with good sensitivity (LOD: 10 ng/ml), accuracy (85-110 %), and reproducibility (RSD < 10 %). The beta-blocker penetration (µg/min) in the ex vivo model correlated well with their permeability (Papp, cm/sec) across isolated cornea (R2 = 0.92), with the delivery generally increasing with an increase in lipophilicity. Further, isolated bovine corneal permeabilities, although lower, correlated well with previously reported rabbit corneal permeabilities (R2 = 0.96) for five beta-blockers in common, after excluding propranolol, which exhibited much lower permeability across bovine cornea.
Cassette dosing in an ex vivo model that reflects normal corneal architecture is a useful method for corneal drug penetration studies. Such an approach, by increasing throughput, can potentially minimize animal usage and cost.
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