Digoxin derivatives with selectivity for the α2β3 isoform of Na,K-ATPase potently reduce intraocular pressure

Abstract

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1β1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2β3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2β3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO4 oxidation of the third digitoxose and reductive amination with various R-NH2 substituents and identified derivatives with significant selectivity for human α2β1 over α1β1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the β1 subunit, Gln84, suggesting a role for β in digoxin binding. Gln84 in β1 is replaced by Val88 in β3. Assuming that alkyl substituents might interact with β3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2β3 over α1β1 (22-33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2β3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.

Keywords: Na/K-ATPase; digoxin derivatives; intraocular pressure; α2β3 isoform.

Fig. 1.

Model of human Na,K-ATPase α1β1 and α2β3 isoforms. The model depicts the human α1β1 complex and human α2β3 complex on the pig α1β1 structure with bound digoxin (4RET). α1 and α2 subunits, red; β1 subunit, gray; β3 subunit, blue; digoxin, green.

Fig. S1.

Structures, masses, and LogP values of digoxin and digitoxin derivatives. LogP values were calculated using the ALOGPs2.1 programme (www.vcclab.org) and represent the average of seven predictions ± SD. Reference values for Digoxin and Digitoxin are 1.26 and 1.85, respectively.

Fig. S2.

Inhibition curves for α1β1, α2β1, and α2β3 and structures of (A) DcB and (B) DMcP.

Fig. 2.

Inhibition of Na,K-ATPase activity of bovine NPE cells by DcB. Inhibition of Na,K-ATPase activity in NPE cells by DcB was fitted to the two sites model [V_CG/V₀ = Aα2 * K_i2/([CG] + K_i2) + Aα1 * K_i1/([CG] + K_i1), where A and K_i represent the amplitudes and K_i values for α2 and α1, respectively]. The dashed line shows the theoretical fit for the one site model [V_CG/V₀ = K_i/([CG] + K_i), with a K_i of 17.3 nM]. Each point represents the average value from four independent experiments ± SEM.

Fig. S3.

Density gradient separation of bovine NPE and PE cells and Western blots of α1 and α2 isoforms.

Fig. 3.

Effects of DMcP (A) and DcB (B) on 4AP-induced ocular hypertension. One drop of DMcP or DcB was added to both eyes 30 min before addition of 4AP. During this preincubation period, there was little or no change in IOP. IOP was measured at the indicated times after addition of 4AP. The values of IOP/IOP t = 0 represent the average for three rabbits (i.e., six eyes) ± SEM.

Fig. 4.

Effects of Digoxin (A), DMcP (B), and DcB (C) on basal IOP. One drop of 1 mM of digoxin, DMcP, or DcB was added to the RE and one drop of PBS to the LE as the control, at time 0. IOP was measured at the indicated times. The values of IOP/IOP t = 0 represent the average for three rabbits ± SEM.

Fig. 5.

Effect of DcB and Latanoprost, separately and combined, on basal IOP. RE: (A) DcB, 1 mM; (B) Latanoprost, 0.005%; (C) 1 mM DcB + Latanoprost 0.005%. LE: PBS, control. Within each figure, the inserted symbol Δ indicates the absolute difference in IOP (in mmHg) between time IOP t = 0 and the average of IOP between 3 and 8 h ± SEM. The P values refer to the difference between DcB versus Latanoprost (A) or DcB/Latanoprost versus Latanoprost (C). Points represent averages IOP/IOP t = 0 from five rabbits ± SEM.

Fig. S4.

Exterior view and histological sections of rabbit eye after DcB treatment. (A) whole eye; (B) cornea, iris, lens; (C) ciliary body; (D) cornea; and (E) retina.