human donor eyes analyzed, mean scleral surface area was measured as 16.3 ± 1.8 cm2 (N = 17).

Drug delivery through the sclera is dependent upon the thickness of the tissue that the solute must traverse as well as the surface area available to the compound for diffusion. The sclera is relatively thick near the limbus. It thins at the equator and becomes significantly thicker near the optic nerve. If transscleral drug delivery could be directed to near the equator, 12–17 mm posterior to the limbus, transscleral flux of the applied solute could be maximized. Because of its large surface area the tissue, thus, provides a significantly larger avenue for drug diffusion to the inside of the eye compared to the 1 cm2 surface area of the cornea. Also, if regional differences in scleral thickness could be taken advantage of by localized delivery, for example by regional application of sustained-release delivery systems, regional scleral delivery could be further optimized.

7.4  Scleral Permeability: Initial Studies

Initial in vitro permeability studies, largely from our laboratory, have shown the sclera to be permeable to a wide molecular weight range of solutes. Scleral solute permeability is, in fact, comparable to that of the corneal stroma; and passive solute diffusion through an aqueous pathway is the primary mechanism of drug permeation across the tissue. The sclera by virtue of its large surface area, accessibility, and relatively high permeability may indeed provide a useful vector for delivering drugs to tissues in the posterior of the eye.

These studies do demonstrate a clear inverse relationship between permeability and molecular weight, with an abrupt decline in permeability at the larger molecular weights. The data for human tissue are comparable to those reported by Maurice and Polgar (1977) for bovine tissue, if one considers the differences in thickness comparing the tissues of the two species. These studies do demonstrate a clear inverse relationship between permeability and molecular weight, with an abrupt decline in permeability at the larger molecular weights, Table 7.1. A number of permeability studies, using essentially comparable methods, have shown the sclera to be permeable to a wide range of solutes and that permeability is best correlated with molecular radius (Prausnitz and Noonan 1998).

Subsequent in vitro studies in our laboratory have demonstrated that the sclera, in vitro, remains permeable to a range of lower molecular weight solutes in the presence of a transscleral pressure as high as 60 mmHg (Prausnitz and Noonan 1998).

Solute permeability of the human sclera does not appear to be correlated with age. In a series of experiments, the permeability to inulin (MW: 5,000) was determined in a series of donor ages (9 days to 87 years of age). For the 22 donor globes studied. No significant correlation was found between scleral permeability to inulin and donor age (Olsen et al. 1995).

The results of the in vitro permeability studies indicate that scleral permeability is comparable to that of the corneal stroma (Prausnitz and Noonan 1998). As in the

the tissue, by increasing scleral hydration, or by transient modification of the scleral extracellular matrix. This approach to enhancing scleral permeability might be achieved by chemical, electrical, or ultrasonic approaches, for example.

Lateral diffusion, parallel to the scleral surface, could affect drug distribution and delivery following periocular delivery. The nonisotropic architecture of collagen lamellae and other features of the scleral microanatomy suggest that lateral diffusion may behave differently than transscleral diffusion. To investigate lateral diffusion within the human sclera, rates of diffusion of sulforhodamine, a model of a hydrophilic drug, were measured in strips of human donor sclera for period up to 1 week (Jiang et al. 2006). Measureable amounts of drug were detected at distances of 5 and 10 mm from the drug delivery reservoir at 4 h and 3 days, respectively. Calculations of lateral diffusivity showed that a point source of sulforhodamine would require 6 weeks to diffuse throughout all the sclera in a human eye. Lateral diffusion within the sclera is thus a slow process that localizes drug distribution to the scale of millimeters for hours to days. Lateral diffusion over larger surface areas could occur over longer periods of time – for example during extended release drug delivery from an implant.

7.5  Sustained-Release Delivery In Vitro

Drug delivery across the sclera is governed, in part, by the transient diffusion of a solute across the tissue that typically occurs over a time course of minutes, unless some type of sustained-release formulation or device is used. By comparison, experimental studies aimed at determining scleral permeability typically derive scleral permeability for a particular solute from steady-state flux data. In the absence of some type of sustained-release system, drug–sclera contact times would be too brief to permit the attainment of steady-state flux. Consequently, the in vitro flux measurements would be expected to overestimate transscleral drug delivery (Prausnitz et al. 1998). Experimental measurements for carboxyfluorescein confirm that the lag time for solute diffusion across the sclera is similar to or actually longer than the drug–sclera contact time during conventional drug administration. The scleral lag time for carboxyfluorescein diffusion, for example, is greater than 20 min. This solute is comparable in size and diffusivity to many conventional drugs. This lag time is significantly longer than the few minutes which eye drops remain on the eye’s surface before being washed away by tear fluid. The scleral lag time for carboxyfluorescein is also similar to the residence time at the scleral surface for a drug introduced by peribulbar injection. Consequently, flux across the sclera would not achieve steady state for most drug delivery applications.

Although carboxyfluorescein provides a good model solute for many drugs, other compounds will bind to the sclera to different extents and at different rates and their scleral lag times could be significantly different. Also, larger sized solutes including macromolecules will diffuse across the tissue much more slowly. Their scleral lag times would be significantly greater.

optimized through the use of sustained-release delivery systems. With a basic understanding of the permeability characteristics of the sclera to a range of solutes provided by in vitro studies, the application of these data must ultimately be extended to the real world delivery of drugs and solutes to the posterior tissues of the eye in vivo.

Retinoblastoma is the most common primary intraocular malignancy of childhood. While focal treatment is effective for smaller single tumors, systemic or local chemotherapy with vincristin, etoposide and carboplatin has become the treatment of choice for larger tumors and vitreous seeds. Because of significant adverse effects associated with systemic treatment, localized periocular delivery of carboplatin might be an option that could maintain local treatment while avoiding the adverse events that accompany systemic administration of the drug. In a series of experiments (Simpson et al. 2002), Dutch Belted rabbits were injected subconjunctivally with carboplatin in either fibrin sealant or in a balanced salt solution. Eyes were enucleated at various times following injection through 2 weeks, and levels of carboplatin were measured in various ocular tissues. The results of these studies demonstrated that fibrin sealant provided a more controlled and localized release of carboplatin, and provided sustained delivery of carboplatin to ocular tissues for up to 2 weeks. Compared to intravitreal injection, subconjunctival administration of 25.1 mg/ml in rabbits was observed to be well tolerated with no retinal toxicity as determined by electroretinogram (Pardue et al. 2004). Using a transgenic murine retinoblastoma model, subconjunctival carboplatin in fibrin sealant was shown to be effective in inducing complete or near-complete intraocular tumor regression in 10 of 11 eyes with no histological evidence of toxicity (Van Quill et al. 2005). In an additional study, the effects of subconjunctival topotecan (TPT) in fibrin sealant were tested in a transgenic murine retinoblastoma model (Tsui et al. 2008). For these experiments the therapeutic effects of a single subconjunctival injection of TPT in fibrin sealant was used. Treatment resulted in a bilateral reduction of tumor burden without a significant difference between treated and untreated eyes. These results suggest that drug was delivered to both eyes predominantly through the hematogenous route. Periocular administration resulted in low systemic plasma drug levels, suggesting that this approach could provide therapeutic benefits comparable to those of intravenous administration while reducing potential systemic toxicities. Taken together, the results of these studies suggest that chemotherapy drugs including carboplatin, cisplatin, and topotecan in fibrin sealant delivered subconjunctivally provide sustainedrelease delivery in vivo and could have clinical use in the treatment of intraocular retinoblastoma.

Traditional methods of evaluating ocular pharmacokinetics are invasive and involve either on-time sampling of the aqueous/vitreous in humans during intraocular surgery or euthanizing animals a various time points, followed by enucleation, dissection, and isolation of the intraocular tissues (including vitreous, iris/ciliary body, lens, neuroretina, RPE, choroid). Ocular fluorophotometry is a noninvasive technique that does not require anesthesia; does not disturb ocular structures; and determines the concentration of fluorescein-labeled compound in the aqueous, vitreous, and retina on a real-time basis at different time points. In albino rabbits, the

technique also permits measurement of fluorescein in the contralateral choroidal circulation as an excellent real-time measure of the concentration in the systemic circulation.

Sodium fluorescein (NaF) has been an invaluable diagnostic tool for retinochoroidal disease. It has been useful in the investigation of the transscleral delivery of drugs administered by the periocular route. Drugs labeled with NaF permit accurate detection and measurement of drug concentration using fluorophotometry both in vitro and in vivo. Even though NaF has proven useful in drug delivery research its use is limited to some extent by photobleaching and pH sensitivity. More stable alternative fluorescent agents such as Oregon Green (OG, Molecular Probes, Eugene, OR) have recently been developed, providing a fluorescent agent without the photobleaching and pH sensitivity limitations of NaF. Its higher level of fluorescence provides an additional advantage. Since Oregon Green has nearly the same structure, molecular weight and emission and excitation characteristics as NaF, efficient utilization of existing equipment and research protocols is possible.

To investigate its potential application in drug delivery research, we evaluated the transscleral permeability and pharmacokinetics of OG compared to NaF using both in vitro and in vivo experimental models (Lee et al. 2008a, b). The results of these initial studies demonstrated that although the sclera had a lower permeability to OG, this fluorescent agent was able to diffuse across the sclera. In vivo, following subtenon injection, Oregon Green does penetrate the sclera and cross the bloodretinal barrier. Vitreous and anterior segment concentrations of OG were directly influenced by the retina/choroid concentration. These initial experiments thus demonstrate the pharmacokinetic differences between OG and NaF after subtenon injection and provide an important starting point for future interpretation of transscleral drug delivery studies utilizing OG.

In vivo ocular fluorophotometry following periocular injection has been used to study the intraocular pharmacokinetics of NaF (Ghate et al. 2007), Oregon Green 488 (Lee et al. 2008a, b), and Oregon Green labeled triamcinolone (Lee et al. 2008b). Results of these studies have shown that these agents are capable of diffusing across the sclera in vivo from a subtenon depot. In experiments with Oregon Green labeled triamcinolone (OGTA), peak OGTA concentrations in the retina/ choroid were achieved at 3 h after injection. This level was maintained for 3 h and then was observed to decrease to a baseline at 7 h after injection. These studies demonstrate that OGTA can diffuse through the sclera and accumulate in the vitreous in rabbit eyes after a subtenon injection. Although the dose was low (1 mg), the vitreous concentrations were still measurable by ocular fluorophotometry. Once the OGTA has diffused into the choroid, it has to cross the blood-retinal barrier to the vitreous. The OGTA levels in the mid vitreous and anterior segment were observed to peak at 3–4 h after subtenon injection in live rabbits, immediately after the retina/choroid peaks were observed to occur. This finding suggests that the vitreous and anterior segment concentrations of OGTA are closely and quickly affected by the retina/choroid circulation.

Conjunctival, lymphatic, and choroidal vessels provide barriers to drug delivery. Euthanization stops the conjunctival and choroidal circulation and enhances