Suprachoroidal delivery in a rabbit ex vivo eye model: influence of drug properties, regional differences in delivery, and comparison with intravitreal and intracameral routes

Abstract

Purpose: First, to determine the influence of drug lipophilicity (using eight beta-blockers) and molecular weight (using 4 kDa and 40 kDa fluoroscein isothiocyanate [FITC]-dextrans) on suprachoroidal delivery to the posterior segment of the eye by using a rabbit ex vivo eye model. Second, to determine whether drug distribution differs between the dosed and undosed side of the eye following suprachoroidal delivery. Third, to compare the suprachoroidal delivery of sodium fluorescein (NaF) with the intracameral and intravitreal routes by using noninvasive fluorophotometry.

Methods: Using a small hypodermic 26G needle (3/8") with a short bevel (250 µm), location of the suprachoroidal injection in an ex vivo New Zealand white rabbit eye model was confirmed with India ink. Ocular tissue distribution of NaF (25 µl of 1.5 µg/ml) at 37 °C was monitored noninvasively using the Fluorotron Master(TM) at 0, 1, and 3 h following suprachoroidal, intravitreal, or intracameral injections in ex vivo rabbit eyes. For assessing the influence of lipophilicity and molecular size, 25 µl of a mixture of eight beta-blockers (250 µg/ml each) or FITC-dextran (4 kDa and 40 kDa, 30 mg/ml) was injected into the suprachoroidal space of excised rabbit eyes and incubated at 37 °C. Eyes were incubated for 1 and 3 h, and frozen at the end of incubation. Ocular tissues were isolated in frozen condition. Beta-blocker and FITC-dextran levels in excised ocular tissue were measured by liquid chromatography-tandem mass spectrometry and spectrofluorometry, respectively.

Results: Histological sections of India ink-injected albino rabbit eye showed the localization of dye as a black line in the suprachoroidal space. Suprachoroidal injection of NaF showed signal localization to the choroid and retina at 1 and 3 h post injection when compared with intravitreal and intracameral injections. Drug delivery to the vitreous after suprachoroidal injection decreased with an increase in solute lipophilicity and molecular weight. With an increase in drug lipophilicity, drug levels in the choroid-retinal pigment epithelium (RPE) and retina generally increased with some exceptions. Beta-blockers and FITC-dextrans were localized more to the dosed side when compared to the opposite side of the sclera, choroid-RPE, retina, and vitreous. These differences were greater for FITC-dextrans as compared to the beta-blockers.

Conclusions: The suprachoroidal route of injection allows localized delivery to the choroid-RPE and retina for small as well as large molecules. Suprachoroidal drug delivery to the vitreous declines with an increase in drug lipophilicity and molecular weight. Drug delivery differs between the dosed and opposite sides following suprachoroidal injection, at least up to 3 h.

Figure 1

Suprachoroidal injection of India ink showed a thin black line between sclera and choroid-RPE in albino rabbit eye. Eyes were injected with 25 µl of 5% India ink dispersion in the suprachoroidal space. Eyes were fixed in Davidson’s fixative for 48 h immediately after injection and embedded in paraffin blocks, and 5-µm sections were obtained. Hematoxylin and eosin (H&E) stained sections were examined. A: Representative histological images of albino rabbit eye are shown. B: Representative histological images of pigmented rabbit eye are shown. C: Representative histological images of albino rabbit eye after suprachoroidal injection of India ink are shown. D: Collaged light microscopy image of H&E stained section of albino rabbit eye after suprachoroidal injection of India ink is shown.

Figure 2

Fluorophotometry scans show sodium fluorescein levels at various depths of the eye along the visual axis. Representative fluorophotometry scans attained using Fluorotron Master™ in albino rabbit eyes after intracameral, intravitreal, and suprachoroidal injections of sodium fluorescein are shown. Scans were collected using Fluorotron Master at 0, 1, and 3 h post injection. Peaks for different regions were assigned from left to right at 20-30, 55-80, and 105-115 data points corresponding to choroid–retina, vitreous, and aqueous humor regions, respectively.

Figure 3

Total retinal and total vitreal delivery after suprachoroidal injection decreases with an increase in solute molecular weight. Ocular tissue distribution of fluorescein isothiocyanate (FITC)-dextran in ex vivo albino rabbit eyes at (A) 1 h and (B) 3 h after suprachoroidal is shown. Data is represented as mean ± standard deviation for n=4. * means the value is significantly different from FITC-dextran 40 kDa at p≤0.05.

Figure 4

Suprachoroidal injection of fluoroscein isothiocyanate-dextrans (FITC-dextrans) showed regional differences in distribution, with drug levels being several fold higher on the injected side than the opposite side. Differences in regional distribution decreased with an increase in incubation time. Ocular tissue distribution of (A) FITC-dextran 4 kDa at 1 h, (B) FITC-dextran 4 kDa at 3 h, (C) FITC-dextran 40 kDa at 1 h, and (D) FITC-dextran 40 kDa at 3 h in ex vivo albino rabbit eyes after suprachoroidal injection is shown. Data is represented as mean ± standard deviation for n=4. *means the value is significantly different from opposite side tissues at p≤0.05.

Figure 5

Fluoroscein isothiocyanate-dextran entered the incubation medium after suprachoroidal injection. Figure shows the cumulative percentage entry of fluoroscein isothiocyanate (FITC)-dextran from the suprachoroidal space of ex vivo rabbit eyes into the incubation medium. Data represent mean±standard deviation for n=4.

Figure 6

Total vitreal delivery decreases, whereas choroid–RPE and retinal delivery increases with increase in solute lipophilicity after suprachoroidal injection. Effect of beta-blocker lipophilicity on (A) choroid–RPE, (C) retina, and (E) vitreous delivery in ex vivo albino rabbit eyes after suprachoroidal injection is shown. Beta-blocker lipophilicity showed a direct linear correlation with (B) choroid–RPE and (D) retinal levels and an inverse correlation with (F) vitreal levels. Data represent mean±standard deviation for n=4. * means the value is significantly different from atenolol, sotalol, nadolol, and pindolol at p≤0.05

Figure 7

Suprachoroidal injection of beta-blockers showed regional differences in distribution, with drug levels being higher on the injected side tissues than the opposite side. Differences in regional distribution decrease with an increase in incubation time. Data represent mean±standard deviation for n=4. * means the value is significantly different from injected side tissues at p≤0.05.