Bovine and porcine transscleral solute transport: influence of lipophilicity and the Choroid-Bruch's layer

Abstract

Purpose: To determine the influence of the choroid-Bruch's layer and solute lipophilicity on in vitro transscleral drug permeability in bovine and porcine eyes.

Methods: The in vitro permeability of two VEGF inhibitory drugs, budesonide and celecoxib, which are lipophilic and neutral at physiologic pH, and of three marker solutes, 3H-mannitol (hydrophilic, neutral), sodium fluorescein (hydrophilic, anionic), and rhodamine 6G (lipophilic, cationic), were determined across freshly excised scleras, with or without the underlying choroid-Bruch's layer. Select studies were performed using porcine sclera with and without choroid-Bruch's layer. Neural retina was removed by exposure of the eyecup to isotonic buffer and wherever required, the retinal pigment epithelial (RPE) layer of the preparation was disrupted and removed by exposure to hypertonic buffer. Because of the poor solubility of celecoxib and budesonide, permeability studies were conducted with 5% wt/vol of hydroxypropyl-beta-cyclodextrin (HPbetaCD). For other solutes, permeability studies were conducted, with and without HPbetaCD. Partitioning of the solutes into bovine sclera and choroid-Bruch's layer was also determined.

Results: The calculated log (distribution coefficient) values were -2.89, -0.68, 2.18, 3.12, and 4.02 for mannitol, sodium fluorescein, budesonide, celecoxib, and rhodamine 6G, respectively. Removal of RPE was confirmed by transmission electron microscopy and differences in the transport of mannitol. The order of the permeability coefficients (Papp) across sclera and sclera-choroid-Bruch's layers in bovine and porcine models was 3H-mannitol > fluorescein > budesonide > celecoxib > rhodamine 6G, with HPbetaCD, and 3H-mannitol > fluorescein > rhodamine 6G, without HPbetaCD. The presence of choroid-Bruch's layer reduced the bovine scleral permeability by 2-, 8-, 16-, 36-, and 50-fold and porcine tissue permeability by 2-, 7-, 15-, 33-, and 40-fold, respectively, for mannitol, sodium fluorescein, budesonide, celecoxib, and rhodamine 6G. The partition coefficients measured in bovine tissues correlated positively with the log (distribution coefficient) and exhibited a trend opposite that of transport. The partition coefficient ratio of bovine choroid-Bruch's layer to sclera was approximately 1, 1.5, 1.7, 2, and 3.5, respectively, for the solutes, as listed earlier.

Conclusions: The choroid-Bruch's layer is a more significant barrier to drug transport than is sclera. It hinders the transport of lipophilic solutes, especially a cationic solute, more than hydrophilic solutes and in a more dramatic way than does sclera. The reduction in transport across this layer directly correlates with solute binding to the tissue. Understanding the permeability properties of sclera and underlying layers would be beneficial in designing better drugs for transscleral delivery.

Figure 1

TEM image of the bovine eyecup after assay buffer treatment and (A) before and (B) after hypertonic treatment. TEM image of the porcine eyecup (C) before any treatment and (D) after assay buffer and hypertonic treatment. Hypertonic treatment removed the RPE layer in both the bovine and porcine tissues. Bar, 2 μm.

Figure 2

Cumulative percentage transport of various solutes across the sclera and the sclera–choroid–Bruch’s layer in bovine (left) and porcine (right) models. All donor solutions contained 5% HPβCD, which was required for solubilizing some of the solutes used. (▲) Sclera; (■) sclera-choroid–Bruch’s layer. Data are expressed as the mean ± SD (n = 6).

Figure 3

Cumulative percentage transport of mannitol, sodium fluorescein, and R6G, with (●) and without (○) 5% HPβCD across bovine sclera and bovine sclera–choroid–Bruch’s layer. Data are expressed as the mean ± SD (n = 6).

Figure 4

Correlation of solute permeability coefficients across (A) bovine and (B) porcine tissues with log (distribution coefficients) data. Results are expressed as the mean (n = 6).

Figure 5

Correlation of solute permeability coefficients between porcine and bovine models for (A) sclera, (B) sclera-choroid–Bruch’s layer, and (C) choroid–Bruch’s layer. Permeabilities of (1) mannitol, (2) sodium fluorescein, (3) budesonide, (4) celecoxib, (5) and R6G were compared. Data are expressed as the mean ± SD (n = 6). Dashed line: the trend line for 1:1 relationship.

Figure 6

Influence of R6G on the transport of ³H-water (A, B) and ³H-mannitol (C, D) across bovine sclera (A, C) and sclera–choroid–Bruch’s layer (B, D). Data are expressed as the mean ± SD (n = 6). (△) 0 μg/mL; (○) 100 μg/mL; (□) 200 μg/mL of R6G. Only 0 and 200 μg/mL of R6G were assessed for water transport.