In Vivo Ocular Fluorophotometry: Delivery of Fluoresceinated Dextrans via Transscleral Diffusion in Rabbits

The rapidly changing field of ocular pharmacology requires an equally dynamic approach to ocular drug delivery. In recent years, new developments in ocular therapeutics have favored protein-based agents in the treatment of posterior segment disease, suggesting a shift toward future drugs engineered to treat specific conditions.1 If the therapeutic effect of these high-molecular-weight agents is to be maximized, successful delivery to their target tissues is essential. Intravitreal injection is currently the most common route of delivery of proteins into the posterior segment. Ranibizumab (Lucentis; Genentech, South San Francisco, CA) is a commonly prescribed pharmacologic treatment for age-related macular degeneration, and although clinical trials have demonstrated clinical efficacy with visual improvement, recommended dose frequency is one 0.5-mg (50 μL) intravitreal injection per month for at least 9 months.2,3 Each intravitreal injection carries a small risk of adverse effects (including endophthalmitis, retinal detachment, and hemorrhage); although the incidence of complications is low, their vision-threatening nature has spurred the search for a safer alternative.4,5 One proposed alternative to repeated intravitreal injections has been to decrease the injection frequency via an implantable drug delivery device. These devices, however, are currently available only for delivery of small-molecule medications, and the procedures necessary to place them inside the globe are themselves not without risk.6–8 Furthermore, once a drug has entered the vitreous cavity, its exact distribution within the vitreous humor is not completely understood. Changes related to natural vitreous aging (known as syneresis), vitreoretinal surgery, and multiple injection procedures may affect a drug's distribution characteristics and kinetics. Recent in vivo findings suggest that the half-life of VEGF is significantly shortened in vitrectomized rabbit eyes9; a similar change in drug pharmacokinetics is likely in human eyes after vitrectomy. A delivery technique that does not rely on the vitreous for distribution could therefore achieve more predictable drug levels at the retina, RPE, and choroid. Transscleral drug delivery has been proposed as a less invasive alternative to vitreous-based delivery, seeking to minimize the trauma and risk associated with penetration of the sclera and vitreous. In vitro studies have established that the sclera is permeable to compounds such as fluorescein, dextran (up to 70-kDa), steroids, and, most recently, immunoglobulins.10,11 These and other studies have described the sclera's ability to absorb and retain compounds, suggesting a potential for sustained delivery without the need for highly specialized formulations or implants. When examining the sclera in vivo, however, it becomes clear that its ability to absorb, retain, and release drugs is heavily influenced by normal physiologic factors not reproducible in vitro. These factors can be classified as static (barrier functions of the choroid, Bruch's membrane, and RPE) or dynamic (loss of drug from the eye via bulk flow or blood, lymph or aqueous humor flow), and their combined effects prevent the ocular penetration of drugs that would easily move across isolated sclera.12,13 An overview of these barriers, as well as plausible strategies for overcoming them, can be found in a recent review by Edelhauser et al.5 In addition, injection-related factors such as the volume of drug solution injected into the sub-Tenon space can have a profound effect on tissue drug levels.14 Ocular fluorophotometry was developed during the 1980s to identify preclinical changes in the blood–retinal barrier of diabetic patients. In contrast to bench-top spectrofluorometry, both excitation and detection mechanisms share a single, mobile optical element, which allows for continuous fluorescence measurements along a straight line extending from the center of the cornea to the center of the posterior pole. Although fluorescein angiography became the preferred modality for assessing the retinal vasculature, fluorophotometry proved useful in the characterization of tear turnover, aqueous turnover, and early cataract formation.15,16 More recently, fluorophotometry has been used to evaluate the transscleral delivery of small molecules in a rabbit model.14,17 Traditional in vivo pharmacokinetic experiments have involved killing several animals at predetermined time points and determining drug levels in the tissue. This technique requires the use of a large number of animals to characterize the change in drug levels over time. It is also susceptible to the changes in drug location and concentration that most likely occur during tissue harvesting and subsequent manipulation. Fluorophotometry offers a noninvasive alternative for monitoring intraocular drug concentrations in vivo, providing a longitudinal view of drug behavior within a single animal. In rabbit studies, fluorophotometry scans can be obtained without the need for anesthesia or active restraint. The eye is not manipulated during the measurement, thus allowing normal physiologic conditions (blood and aqueous flow) to remain unaltered. Fluorescein and fluorescein-conjugated compounds are a logical choice for in vivo fluorophotometry studies because of their intense fluorescence, favorable safety profile, and stable molecular bonding.18,19 We selected fluoresceinated dextrans in particular for this study because of their high solubility and stability in aqueous solution, as well as customizable molecular weight. With the use of fluoresceinated dextrans of various molecular weights, the effect of molecular size and weight on transscleral diffusion can be characterized while keeping other molecular parameters constant. The purpose of this study was to deliver fluoresceinated dextrans, with molecular mass up to 70 kDa, to the posterior retina and choroid using sub-Tenon injections, and to characterize the intraocular movement of the dextrans using in vivo ocular fluorophotometry.