The main objective of the proposal is to investigate various enzyme systems in eye tissues which play a key role in recently developed site-specific and site-activated drug delivery systems, a unique concept for treating eye diseases such as glaucoma, by metabolically targeting medication into the site of action, without systemic side effects. The presence and specificity of such enzymes offer perspectives for their use in developing specific treatments for other ophthalmic diseases. The specific enzymes studied in this proposal involve i) oxime isomerase, ii) oxime hydrolyse, and iii) ketone reductase enzymes. Experiments will be carried out to characterize and attempts will be made to isolate enzymes capable of hydrolyzing oximes thus making the corresponding carbonyl compounds available to further metabolic processes. Primary attention will be given to eye compartments (iris, ciliary body, aqueous humor, cornea) found to be directly involved in ocular biotransformation of topically administered drugs and comparisons will be made with tissues exhibiting high hydrolytic capacity (liver, blood, gastrointestinal tissues). The oxime hydrolase activity and substrate specificity will be estimated in vitro and in vivo, using various oximes related to beta-adrenergic antagonists some of which have already been shown to undergo sequential biotransformation-activation in the eye. Since oximes are subject to isomerization in biological media either by enzymatically or by acid-catalyzed reaction and the E- and Z-isomers tend to form an equilibrium mixture, the enzymatic basis of the interplay between oxime hydrolysis and isomerization will also be studied. Preliminary recent data demonstrated that there is indeed a fast enzymatic E-Z interconversion of these oximes, which appears to be sensitive to structural features. Experiments also will be carried out to characterize and attempts will be made to isolate and purify and carbonyl reductase enzyme(s) found in the eye which is responsible to the conversion of certain oxo-compounds to the corresponding alcohols in the presence of NAD(P)H coenzyme. Primary attention will be given to those eye compartments that are directly involved in the ocular biotransformation of topically administered drugs, i.e., to the iris and the ciliary body. Issues on activity, substrate specificity and stereo-specificity (where appropriate) will be approached by in vitro and in vivo experiments involving various carbonyl compounds related to the beta-adrenergic agents. Derivatives (oximes, methoximes), which may be hydrolytically activated prior to the subsequent reduction step, will also be included. These compounds have direct relevance for treating specific eye diseases such as glaucoma, and their activity is principally confined to one of the possible stereoisomeric forms. Correlation between the enzyme activity/stereoselectivity and the structure (along with derived physicochemical properties such as partition coefficient) of the substrates will be determined. The ultimate objective is to facilitate discovery of novel site-and stereospecific delivery of ophthalmic drugs via the chemical delivery system concept.
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