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A. Kriatchko, G. Zhan, N. Cheruvu, S.P. Ayalasomayajula, C.B. Camras, R.V. Patil, U.B. Kompella; In Vitro Transport and Hydrolysis of Bimatoprost in Bovine Cornea . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4422.
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Purpose:Bimatoprost is a prostaglandin analog that is proposed to act via a putative prostamide receptor. However, bimatoprost can be hydrolyzed to a free acid, which is much more potent than the parent drug in activating the prostaglandin FP receptor. The objective of this study was to assess the in vitro transport and hydrolysis of bimatoprost in bovine cornea. Methods:The in vitro transport of bimatoprost (1.5 ml of 0.03% solution on donor side) across freshly isolated bovine cornea mounted in modified Ussing chambers was assessed over a period of 3 hours. Bimatoprost (100 µl of 0.03% solution) was also incubated with 100 mg of excised intact cornea, and the formation of the free acid in the incubation mixture at the end of 90 min was assessed. Both bimatoprost and its free acid were analyzed using a HPLC assay. Results:During the 3 hour transport study, the permeability of bimatoprost was negligible, with the highest concentrations detected on the receiver side being 1.2 ± 1.7 µg/ml (n = 6) (~0.4%). The highest concentrations of the free acid detected in the receiver chamber were 0.1 ± 0.1 µg/ml (n = 6) (0.03%). In vitro incubation with isolated tissue resulted in conversion of the bimatoprost to its free acid, which accounted for 2.2 ± 0.6 % (n = 4) of the initial mass of bimatoprost. Co-incubation of bimatoprost with 5% protease inhibitor cocktail (Sigma Chemical Co.), 50 µg/ml bestatin (amino peptidase inhibitor), and 2% EDTA (metalloenzyme inhibitor) resulted in a free acid formation of 1.3 ± 0.3% (p = 0.07), 1.64 ± 0.5% (p = 0.3), and 0% (p = 0.004), respectively. Thus, a protease inhibitor cocktail partly inhibited the hydrolysis and a metalloenzyme inhibitor completely abolished the hydrolysis. Conclusions:Consistent with earlier reports indicating the conversion of bimatoprost to its free acid in human and rabbit tissues, we observed the conversion of bimatoprost to free acid in the bovine cornea at a rate of 1.5%/hr/100 mg tissue. It appears that metalloenzymes, likely metallopeptidases, are responsible for the conversion of bimatoprost to its free acid in the bovine cornea.
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