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Ilva D. Rupenthal, Simon Raesch, Bo Li, Ulrich F. Schaefer, Claus-Michael Lehr, Julie C. Lim; Ex Vivo Permeation of Cystine across Bovine Corneas - A Step towards the Development of an Antioxidant Eye Drop for Cataract Prevention. Invest. Ophthalmol. Vis. Sci. 2012;53(14):489.
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Age-related nuclear (ARN) cataract is the leading cause of blindness worldwide and is estimated to affect more than 40 million people by 2020. Even though surgical removal of the clouded lens is highly effective, the current demand exceeds the supply. Since ARN cataract is associated with a depletion of antioxidants, mainly glutathione, in the lens core, our research has concentrated on restoring antioxidant levels in this region in order to prevent or delay the onset of cataract formation. This study investigated the permeation of cystine, the limiting factor for glutathione synthesis, across bovine corneas.
Freshly excised bovine corneas were placed between donor and acceptor chambers of standard Franz diffusion cells. A 0.15 mg/ml cystine solution (1 ml) was placed in the donor compartment, while modified Ringer solution, kept at 37°C, served as acceptor medium. Samples were taken at predetermined time points over 24 h and cystine concentrations were analyzed by derivatization with monobromobimane and subsequent fluorescence HPLC analysis. In addition to a simple cystine solution, various penetration enhancers were tested for their ability to improve the cystine permeation across bovine corneas.
The established HPLC method allowed cystine quantification to a lower limit of <0.1 nmol/ml with high linearity (R2=0.9994) and a reduced sample run time (17 min). Over the 24 h period 45 nmol of cystine permeated across the bovine cornea using the simple solution as donor. This amount was almost tripled (118 nmol over 24 h) when incorporating 0.5% EDTA, a tight junction opener, while 0.01% BAC only exhibited a slight improvement. Immunolabelling of frozen tissue sections revealed the presence of the cystine/glutamate (Xc-) exchanger in the corneal epithelium, which may be responsible for partial active transport of the antioxidant across the tissue.
Franz diffusion cell experiments combined with HPLC analysis offer an accurate way to study the permeation of various cystine eye drops across bovine corneas, with the addition of 0.5% EDTA showing the most promising results so far. Further penetration enhancers in combination with other delivery approaches, such as in situ gelling eye drops, will further be evaluated before testing the most promising formulations in vivo.
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