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S. Katragadda, R.S. Talluri, A.K. Mitra; Simultaneous Modulation of Transport and Metabolism of Prodrugs Across Rabbit Cornea: An Approach Involving Enzyme Inhibitors Targeting Hydrolyzing Enzymes . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5095.
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© ARVO (1962-2015); The Authors (2016-present)
Prodrugs are designed to be inactive until in vivo activation to the parent drug, and hence reliable in vivo activation of the prodrug is considered critical for their pharmacological activity. The aim of this study is to identify the class of enzymes responsible for the hydrolysis of amino acid and dipeptide prodrugs of acyclovir across rabbit cornea and to modulate the simultaneous transport and metabolism of amino acid and dipeptide prodrugs in presence of various enzyme inhibitors across rabbit cornea.
L–valine ester of acyclovir, val–acyclovir (VACV) was a generous gift from GlaxoSmithKline (Research Triangle Park, NC, USA) and L–glycine–valine ester of acyclovir, gly–val–acyclovir (GVACV) was synthesized in our laboratory. Corneal homogenate hydrolysis studies of VACV and GVACV were conducted in presence of various enzyme inhibitors. [3H] Glycylsarcosine (GS) was chosen as a model peptide transporter (PEPT) substrate. The uptake studies were performed on rPCEC (Rabbit Primary Corneal Epithelial Culture) using 12–well plates. Transport studies were conducted with isolated rabbit corneas at 34°C.
Complete abolition of VACV hydrolysis rate was observed in presence of caroxyl esterase enzyme inhibitors, PCMB (p–Chloromercuribenzoic acid) and Pefabloc SC and similar trend was observed for GVACV in presence of bestatin (aminopeptidase inhibitor). The IC50 value of PCMB and bestatin for VACV and GVACV were found to be 3.81 ± 0.94 and 0.34 ± 0.08 µM respectively. Choline esterase inhibitors like eserine, tetraethyl pyrophosphate (TEPP) and diisopropyl fluorophosphate (DFP) were also showed significant inhibition in VACV hydrolytic rate. Surprisingly, EDTA (chelating agent) showed significant inhibition for GVACV hydrolytic rate in corneal homogenate. Transport of VACV and GVACV across rabbit cornea showed decreased metabolic rate and modulation in transport in presence of PCMB and bestain respectively.
The principle enzyme classes responsible for the hydrolysis of VACV and GVACV were proven as carboxylesterases and aminopeptidases respectively. Enzyme inhibitors (PCMB and bestatin) modulated the transport and metabolism of prodrugs simultaneously across rabbit cornea even though their affinity towards prodrugs was distinct. The affinity of these prodrugs towards peptide transporter was also not influenced by enzyme inhibitors. In conclusion, utility of enzyme inhibitors to modulate transport and metabolism of prodrugs seems to be promising.
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