Episcleral clearance of sodium fluorescein from a bioerodible sub-tenon's implant in the rat

Abstract

We quantified episcleral drug clearance of sodium fluorescein (NaFl) in rats to examine the hypothesis that there is rapid clearance of episcleral water soluble drugs, and that this rapid clearance may limit the amount of drug that is able to reach the posterior segment from an episcleral location. 2 mm implants containing either 12 or 22 microg of NaFl were manufactured and in vitro release rates were determined. Implants were placed in the sub-Tenon's space and the amount of drug remaining in the conjunctiva/sclera/choroid complex (CSCC) at various time points was quantified following tissue solubilization and fluorescence quantification using a spectrofluorometer. Kinetics of NaFl clearance was determined in live animals, following euthanasia and in animals in which choroidal non-perfusion had been achieved with indocyanine green-enhanced 810 nm diode laser thrombosis of the choroidal vasculature. Choroidal non-perfusion in these laser-treated rats was verified with Concavalin-A staining of choroidal flatmounts. In vitro, >99% of drug was released by 25 min for the low dose implants, and by 60 min for the high dose implants. In vivo, both implant doses were >99% cleared from the episcleral tissue by 3 h. By 7 h, an average of only 0.14 +/- 0.131 ng of NaFl per mg of wet tissue weight (mean +/- SD) remained in the CSCC with the low dose implant, and 0.29 +/- 0.428 ng of NaFl per mg of wet tissue weight remained in animals with the high dose implant. By comparison, in euthanized animals at 7 h following sub-Tenon's implantation, 432.0 +/- 181.40 ng of NaFl per mg of wet tissue weight was in the episcleral tissue of animals with the low dose implant, and of 787.8 +/- 409.89 ng of NaFl per mg of wet tissue weight remained in the animals with the high dose implant. In live animals with selective thrombosis of the choroidal vasculature, the difference in the amount of drug remaining in the episcleral tissue as compared to control live animals was not significant at all time points for both implant doses. In conclusion, there is rapid clearance of episcleral NaFl delivered from a bioerodible sub-tenon's implant. The clearance mechanisms are dramatically reduced following euthanasia, suggesting that elimination is occurring via active physiologic mechanisms, rather than by passive diffusion clearance (CL(diff)) (Pfister et al., 2003). Interestingly, the choroid does not appear to play a prominent role as clearance of episcleral NaFl was not affected by elimination of choroidal blood flow. Further work is needed to delineate the pathways of episcleral drug clearance.

Keywords: animal model; drug delivery; episcleral lymphatics; sodium fluorescein.

Figure 1

(A) Sodium fluorescein (NaFl) implant. Bars measure 1 mm each. (B) Insertion of implant into sub-Tenon’s space in the superotemporal quadrant of a rat eye.

Figure 2

In vitro release of NaFl (µg) from both the low dose implants (A) and high dose implants (B) over time. The error bars represent the standard deviation.

Figure 3

Standard curve showing the relationship between the concentration of NaFl in the dissected conjunctiva/sclera/choroid complex (CSCC) and the amount of fluorescence, expressed as relative fluorescence units (RFU).

Figure 4

Clearance of NaFl from episcleral implants, both low dose (A) and high dose (B), over 7 hrs in live animals. The amount of NaFl is expressed as ng per wet tissue weight (mg) of CSCC. The error bars represent the standard deviation.

Figure 5

Clearance of NaFl from episcleral implants, both low dose (A) and high dose (B), over 7 hrs in live and dead animals. The amount of NaFl is expressed as ng per wet tissue weight (mg) of CSCC. The error bars represent the standard deviation.

Figure 6

Flatmount of a choroid of an untreated control eye (A), showed normal perfusion of the choroidal vessels, while the choroid of the laser-treated eye (B) showed >90% non-perfusion of the vasculature. Flatmounts are representative of a sample of n = 5.

Figure 7

Clearance of NaFl from episcleral implants, both low dose (A) and high dose (B) in untreated control eyes versus laser-treated eyes. The amount of NaFl is expressed as ng per wet tissue weight (mg) of episcleral tissue. The error bars represent the standard deviation.