Inhibition of Corneal Metabolism of Deslorelin by EDTA and ZnCl2

Abstract

It was the aim of this study to determine whether deslorelin is degraded by the rabbit corneal tissue and to further delineate the mechanisms. Deslorelin was incubated with intact cornea either alone or in the presence of 0.1 mM ouabain, 0.1% 2,4-dinitrophenol, 0.1 mM phosphoramidon, 0.1 mM N-tosyl-L-phenylalanine chloromethylketone (TPCK), 0.1-2% EDTA, 0.1-1% ZnCl2, 0.1% dithiothreitol (DTT), or 0.1% N-ethylmaleimide (NEM) at 37°C. In addition, deslorelin alone was incubated with cornea at 4°C. Following α 90-min incubation, the supernatants were analyzed using a reversed-phase HPLC. Metabolite peaks observed in controls at 37°C were not detected in the low-temperature study, suggesting inhibition of metabolism at low temperature. Intact drug remaining in the supernatant was not altered by ouabain and dinitrophenol, suggesting that energy-dependent corneal uptake is not likely for deslorelin. Phosphoramidon and TPCK failed to alter deslorelin levels, indicating that phosphoramidon and TPCK-sensitive endopeptidases did not contribute to the observed metabolism. DTT and NEM also failed to affect deslorelin levels. However, 2% EDTA and 1% ZnCl2 significantly elevated the intact deslorelin levels by 44 and 60%, respectively, and the metabolite peaks almost completely disappeared. These observations are consistent with the corneal metabolism of deslorelin by either metallo-peptidases or metal-dependent peptidases.