The main goal of this proposal is to test the hypotheses that the lipophilicity of a topically applied prodrug significantly influences its topical and systemic activities and that chronic prodrug administration may alter esterase activities in the eye. Timolol, the most widely used topical anti-glaucoma drug, has been chosen as a model drug for prodrug derivatization because of a need to reduce its relatively high incidence of systemic side effects. There are 5 specific aims in this proposed research: (1) Using 5 ester prodrugs of timolol, to demonstrate that prodrug hydrolysis is required for expression of its beta adrenergic blockade activity, the degree of which may be controlled further by its diffusion rate across the cornea, (2) To demonstrate that the rate and duration of timolol release in the eye can be controlled further by using combinations of its prodrugs, (3) To demonstrate that the extent of corneal transport and hydrolysis of timolol prodrugs vary non-linearly with the applied dose due to esterase saturation, (4) Based on these results, to select a timolol prodrug for evaluation of its time course of ocular disposition and systemic absorption following single and multiple doses, and (5) To evaluate the changes in ocular esterase activity and to determine the basis for these changes upon chronic application of a timolol prodrug. An ancillary aim is to confirm that the ocular tissues of rabbits and human subjects differ in the relative proportions of acetyl- and butyrylcholinesterase, resulting in different esterase activities. These experiments will be performed using live rabbits as well as tissues from enucleated human (eye bank) and pigmented rabbit eyes. The methodology includes dihydroalprenolol displacement and inhibition of cAMP production assays to determine beta adrenergic blockage, the pH-stat method to evaluate prodrug hydrolysis, and the use of lucite-block perfusion chambers to evaluate corneal transport of prodrugs in vitro. HPLC will be used to assay for timolol and its prodrugs. The importance of this research is that it demonstrates the generality of the prodrug approach to enhance the therapeutic indices of other topical beta blockers. Furthermore, this research will lead to studies to evaluate the long-term effects of prodrugs on the physiological role of esterases and to investigate the effect of age, disease states, and nutritional status on esterase activity and relative proportions in the eye.
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