Melanin-Binding-Based Discovery of Topically Instilled Carbonic Anhydrase Inhibitors for Targeted Delivery and Prolonged Action in the Eye

Abstract

Glaucoma is a vision-threatening disease that is currently treated with intraocular-pressure-reducing eyedrops that are instilled once or multiple times daily. Unfortunately, the treatment is associated with low patient adherence and suboptimal treatment outcomes. We developed carbonic anhydrase II inhibitors (CAI-II) for a prolonged reduction of intraocular pressure (IOP). The long action is based on the melanin binding of the drugs that prolongs ocular drug retention and response. Overall, 63 new CAI-II compounds were synthesized and tested for melanin binding in vitro. Carbonic anhydrase affinity and IOP reduction of selected compounds were tested in rabbits. Prolonged reduction of IOP in pigmented rabbits was associated with increasing melanin binding of the compound. Installation of a single eye drop of a high melanin binder carbonic anhydrase inhibitor (CAI) resulted in ≈2 weeks' decrease of IOP, whereas the effect lasted less than 8 h in albino rabbits. Duration of the IOP response correlated with melanin binding of the compounds. Ocular pharmacokinetics of a high melanin binder compound was studied after eye drop instillation to the rat eyes. The CAI showed prolonged drug retention in the pigmented iris-ciliary body but was rapidly eliminated from the albino rat eyes. The melanin-bound drug depot maintained effective free concentrations of CAI in the ciliary body for several days after application of a single eye drop. In conclusion, melanin binding is a useful tool in the discovery of long-acting ocular drugs.

Keywords: carbonic anhydrase inhibitor; drug delivery; drug discovery; eye drops; glaucoma; intraocular pressure; melanin binding; sustained delivery.

Figure 1

IOP-lowering effect of the CAIs in albino (A) and pigmented (B) rabbits (n = 5 animals/group). Relative mean (±S.D.) changes of pressure compared to pretreatment levels are shown. PBS = phosphate buffered saline, DRZ = dorzolamide, A22 = low melanin binding CAI, A12 = intermediate melanin binding CAI, and A01 = high melanin binding CAI. (A) In albino eyes, AUC_0-last values of IOP responses were statistically tested with Friedman Repeated Measurement Analysis of Variance on Ranks. The statistical test against placebo (paired t-test with each molecule separately) shows statistically significant difference in AUC_0-last values of A01 (P = 0.005, t = −5.569 with 4 degrees of freedom), and dorzolamide (P = 0.043, t = −2.923 with 4 degrees of freedom), but not for A12 (P = 0.052, t = −2.742 with 4 degrees of freedom) and A22 (P = 0.074 t = −2.406 with 4 degrees of freedom). (B) In pigmented eyes, the AUC_0-last of IOP response of A01 was significantly greater (P < 0.001, t = 7.509) than that of dorzolamide (one-way repeated measures analysis of variance with Bonferroni t-test; F = 30.755, power of the performed test with alpha 0.050:1.000). Significant difference was not seen between dorzolamide and A12 (P = 1.0, t = 0.388) or between dorzolamide and A22 (P = 1.0, t = 0.571).

Figure 2

Total A01 concentrations (ng/mg) (purple) in albino (A) and pigmented (B) iris-ciliary body tissues after instillation of a single topical eye drop (V = 7 μL; A01 concentration = 10 mg/mL) to rats. Unbound (blue) and bound A01 concentrations (red) are also shown. The data show mean ± SD (n = 4) values. There is no statistically significant difference in C_max values of total drug in albino (t_{max(total drug)} = 2 h) and pigmented (t_{max(total drug)} = 24 h) eyes (two-tailed P-value = 0.398, t = 0.910 with 6 degrees of freedom, tested with Student’s t-test), whereas the difference in bound drug concentrations (C_{max(bound drug)}) between albino (t_max = 2 h) and pigmented (t_max = 24 h) eyes was significant (two-tailed P-value = 0.0172, t = −3.264 with 6 degrees of freedom, tested with Student’s t-test).

Figure 3

Bound fraction of high melanin binder CAI (A01) in albino and pigmented rat iris-ciliary body after single eye drop administration. Means ± SD (n = 4) are presented.

Figure 4

AUC of the IOP-lowering effect in pigmented rabbits presented in logarithmic scale and melanin binding affinity (K_d) of the CA-II inhibitors with different melanin-binding properties (A01 = CAI with high melanin binding, A12 = CAI with intermediate melanin binding, DRZ = dorzolamide, and A22 = CAI with low melanin binding). The filled symbols show the response AUC results in pigmented rabbits and the open symbols represent the results in albino rabbits.