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A. Trendelenburg, C. Keller, M. Haslebacher, R. Markstein, G.N. Lambrou; Comparison of Melanin Binding for Antiglaucomatous Drugs . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4440.
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Purpose: It has been shown that the effect of antiglaucomatous drugs markedly differ between species and strains. This is believed to be partly due to binding of these drugs to melanin. Timolol, for example, binds highly to melanin from which it is then slowly released. Consequently, single administration of timolol reduces introcular pressure (IOP) in albino, but not in pigmented rabbits, whereas prolonged effects are found after repeated dosing (Nagata et al., Jpn. J. Ophthalmol. 37, 32-38, 1993). Thus, data on melanin binding may be important for early drug selection. The purpose of the present study was (i) to develop a suitable melanin binding assay, and (ii) to compare melanin binding for two structurally similar IOP-lowering compounds, namely GLC 756 and GLA 221 using timolol as a positive control. Methods: Melanin from Sepia officinalis was suspended in phosphate buffer. For background determination an aliquot was removed and then replaced by drug solutions (1, 10 or 100 µM) containing 1-10% of the respective tritiated derivative, 3H-timolol, 3H-GLC 756 or 3H-GLA 221. Binding was determined by taking aliquots after 1, 5, 10, 15 and 30 min. Aliquots were centrifuged and the radioactive content of the supernatants was measured. Results were expressed as percentage of bound drug. Results and discussion: All three compounds rapidly bound to melanin at all tested concentrations. The amount of bound drug increased only slightly with increasing incubation time and maximum binding was reached after approximately 15 min. For timolol, maximum binding decreased with increasing timolol concentrations reaching 93 % (1 µM), 90 % (10 µM) and 81 % (100 µM). The data obtained for timolol 100 µM agreed well with data from literature. Concentration dependence of timolol binding to melanin has not been previoulsy studied. For both GLC 756 and GLA 221, the maximum binding was similar at all concentrations tested. However, differences in maximum binding were observed: 82-85% for GLC 756 and 98% for GLA 221. Thus, differences have to be expected concerning onset and duration of ocular effects. Moreover, since both compounds bind strongly to melanin (>82%) prolonged effects seem likely. Conclusion: Melanin binding can be measured by means of 3H-labelled compounds. The differences in melanin binding observed for the two structurally similar IOP-lowering drugs, GLC756 and GLA221 may influence the onset and duration of their ocular effects. Thus, data on melanin binding may be indeed helpful for early selection of compounds for drug development.
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