Research into treatment modalities affecting vision is rapidly progressing due to the high incidence of diseases such as diabetic macular edema, proliferative vitreoretinopathy, wet and dry age-related macular degeneration and cytomegalovirus retinitis. The unique anatomy and physiology of eye offers many challenges to developing effective retinal drug delivery systems. Historically, drugs have been administered to the eye as liquid drops instilled in the cul-de-sac. However retinal drug delivery is a challenging area. The transport of molecules between the vitreous/retina and systemic circulation is restricted by the blood-retinal barrier, which is made up of retinal pigment epithelium and endothelial cells of the retinal blood vessels. An increase in the understanding of drug absorption mechanisms into the retina from local and systemic administration has led to the development of various drug delivery systems, such as biodegradable and non-biodegradable implants, microspheres, nanoparticles and liposomes, gels and transporter-targeted prodrugs. Such diversity in approaches is an indication that there is still a need for an optimized noninvasive or minimally invasive drug delivery system to the eye. A number of large molecular weight compounds (i.e., oligonucleotides, RNA aptamers, peptides and monoclonal antibodies) have been and continue to be introduced as new therapeutic entities. However, for high molecular weight polar compounds the mechanism of epithelial transport is primarily through the tight junctions in the retinal pigment epithelium, as these agents undergo limited transcellular diffusion. Delivery and administration of these new drugs in a safe and effective manner is still a major challenge facing pharmaceutical scientists. In this review article, the authors discuss various drug delivery strategies, devices and challenges associated with drug delivery to the retina.

Efflux pump like P-glycoprotein (P-gp) is known to be a major barrier to drug delivery. Functional P-glycoprotein has been recently identified in cornea and corneal cell lines. Thus, it is probable that P-glycoprotein may restrict in vivo ocular drug absorption, resulting in low ocular bioavailability. Experiments were designed using New Zealand albino (New Zealand White) rabbits to assess inhibitors of P-gp efflux to increase drug absorption. Anesthetized rabbits were given constant topical infusions of [14 C]erythromycin in the presence and absence of inhibitors. Testosterone, verapamil, quinidine, and cyclosporine A were selected as P-gp inhibitors. Transport experiments were conducted in Madin-Darby canine kidney cells transfected with the human mdr1 gene (MDCK-MDR1). Erythromycin exhibited significant efflux out of MDCK-MDR1 cells, suggesting that erythromycin is a good substrate for P-gp. Ocular pharmacokinetic studies were conducted using a topical single-dose infusion method. Maximum inhibition of P-gp mediated efflux was observed with 500 M testosterone. Area under the curve (AUC) 0-ϱ of erythromycin with 500 M testosterone was almost 4 times higher than AUC 0-ϱ without any inhibitor. Rate of elimination (k 10 ) for erythromycin and those with inhibitors was found to be similar (141 Ϯ 23 min), suggesting that elimination pathways were not altered. All the inhibitors were found to be nontoxic. Verapamil also inhibited the efflux pump with moderate change in AUC 0-ϱ and C max compared with control. Thus, P-gp is found to be active in vivo, and it restricts topical erythromycin absorption across the cornea, which can be inhibited by known P-gp inhibitors. Therefore, ocular bioavailability of P-gp substrates can be significantly enhanced by proper selection of P-gp inhibitors.

Purpose The objectives were (i) to test in vivo functional activity of MRP2 on rabbit corneal epithelium and (ii) to evaluate modulation of P-gp and MRP2 mediated efflux of erythromycin when co-administered with corticosteroids. Methods Cultured rabbit primary corneal epithelial cells (rPCECs) was employed as an in vitro model for rabbit cornea. Cellular accumulation and bi-directional transport studies were conducted across Madin-Darby Canine Kidney (MDCK) cells overexpressing MDR1 and MRP2 proteins to delineate transporter specific interaction of steroids. Ocular pharmacokinetic studies were conducted in rabbits following a single-dose infusion of erythromycin in the presence of specific inhibitors and steroids. Results Bi-directional transport of erythromycin across MDCK-MDR1 and MDCK-MRP2 cells showed significant difference between BL-AP and AP-BL permeability, suggesting that erythromycin is a substrate for P-gp and MRP2. Cellular accumulation of erythromycin in rPCEC was inhibited by steroids in a dose dependent manner. MK571, a specific MRP inhibitor, modulated the aqueous humor concentration of erythromycin in vivo. Even, steroids inhibited P-gp and MRP2 mediated efflux with maximum increase in ka, AUC0−∞, Cmax and Clast values of erythromycin, observed with 6α-methyl prednisolone. Conclusion MRP2 is functionally active along with P-gp in effluxing drug molecules out of corneal epithelium. Steroids were able to significantly inhibit both P-gp and MRP2 mediated efflux of erythromycin.

Abstract. The objective of this study was to develop a clear, aqueous rapamycin-loaded mixed nanomicellar formulations (MNFs) for the back-of-the-eye delivery. MNF of rapamycin (0.2%) was prepared with vitamin E tocopherol polyethylene glycol succinate (TPGS) (Vit E TPGS) and octoxynol-40 (Oc-40) as polymeric matrix. MNF was characterized by various parameters such as size, charge, shape, and viscosity. Proton nuclear magnetic resonance ( 1 H NMR) was used to identify unentrapped rapamycin in MNF. Cytotoxicity was evaluated in human retinal pigment epithelial (D407) and rabbit primary corneal epithelial cells (rPCECs). In vivo posterior ocular rapamycin distribution studies were conducted in male New Zealand white rabbits. The optimized MNF has excellent rapamycin entrapment and loading efficiency. The average size of MNF was 10.98±0.089 and 10.84±0.11 nm for blank and rapamycin-loaded MNF, respectively. TEM analysis revealed that nanomicelles are spherical in shape. Absence of free rapamycin in the MNF was confirmed by 1 H NMR studies. Neither placebo nor rapamycin-loaded MNF produced cytotoxicity on D407 and rPCECs indicating formulations are tolerable. In vivo studies demonstrated a very high rapamycin concentration in retina-choroid (362.35±56.17 ng/g tissue). No drug was identified in the vitreous humor indicating the sequestration of rapamycin in lipoidal retinal tissues. In summary, a clear, aqueous MNF comprising of Vit E TPGS and Oc-40 loaded with rapamycin was successfully developed. Back-of-the-eye tissue distribution studies demonstrated a very high rapamycin levels in retina-choroid (place of drug action) with a negligible drug partitioning into vitreous humor.

The purpose of this study was to identify and functionally characterize an active transport system for L-arginine on rabbit corneal epithelium and human cornea and study its interaction with the amino acid ester prodrugs of acyclovir (Anand, B. S.; Mitra, A. K. Pharm. Res. 2002, 19, 1194-1202). Transport characteristics of [3H]-L-arginine across freshly excised rabbit corneas were determined at various concentrations, in the absence of sodium and chloride ions. Inhibition studies were conducted in the presence of other amino acids, ouabain, and amino acid ester prodrugs of acyclovir (glycine-ACV, phenylalanine-ACV and gamma-glutamate-ACV). Reverse transcription-polymerase chain reaction (RT-PCR) for amino acid transporter B(0,+) was carried out on total RNA isolated from rabbit cornea, rabbit corneal epithelium, and human cornea. Transport of L-Arg across rabbit cornea was saturable (Km = 306 +/- 72 microM and Vmax = 0.12 +/- 0.01 nmol min(-1) cm(-2)) and was Na+, Cl-, and energy dependent. Transport was inhibited by neutral and cationic amino acids and a B(0,+) system specific inhibitor, BCH (Sloan, J. L.; Mager, S. J. Biol. Chem. 1999, 274, 23740-23745), but not by anionic amino acids. Amino acid prodrugs of ACV (Glu-ACV and Phe-ACV) also inhibited transport of [3H]-L-Arg across rabbit cornea. Amino acid transporter B(0,+) was identified by RT-PCR and its identity confirmed by subcloning and sequencing in rabbit cornea, rabbit corneal epithelium, and human cornea. A Na+-, Cl(-)-, and energy-dependent carrier for L-Arg, B(0,+), was identified on rabbit corneal epithelium and human cornea. Glu-ACV and Phe-ACV appear to be substrates for this transporter. The presence of such transporters on the corneal epithelium may provide new opportunities for transporter-targeted prodrug design for enhanced corneal absorption.

YVGCV and VVGCV exhibited superior corneal absorption and bioavailability, in comparison with GVGCV, VGCV, and GCV. Such facilitated absorption of prodrugs may be a result of a combination of transcellular passive diffusion and peptide transporter (PEPT1)-mediated transport across the corneal epithelium.

Purpose-To evaluate in vivo corneal absorption of the amino acid prodrugs of acyclovir (ACV) using a topical well model and microdialysis in rabbits. Methods-Stabilityand L-Valine-ACV (VACV) prodrugs was evaluated in various ocular tissues. Dose dependent toxicity of these prodrugs was also examined in rabbit primary corneal epithelial cell culture (rPCEC) using 96-well based cell proliferation assay. In vivo ocular bioavailability of these compounds was also evaluated with a combination of topical well infusion and aqueous humor microdialysis techniques.Results-Among the amino acid ester prodrugs, SACV was most stable in aqueous humor. Enzymatic degradation of EACV was the least compared to all other prodrugs. Cellular toxicity of all the prodrugs was significantly less compared to trifluorothymidine (TFT) at 5mM. Absorption rate constants of all the compounds were found to be lower than the elimination rate constants. All the prodrugs showed similar terminal elimination rate constants (λ z ). SACV and VACV exhibited approximately two fold increase in area under the curve (AUC) relative to ACV (p < 0.05). C last (concentration at the last time point) of SACV was observed to be 8 ± 2.6µM in aqueous humor which is two and three times higher than VACV and ACV, respectively.Conclusions-Amino acid ester prodrugs of ACV were absorbed through the cornea at varying rates (k a ) thereby leading to varying extents (AUC). The amino acid ester prodrug, SACV owing to its enhanced stability, comparable AUC, and high concentration at last time point (C last ) seems to be a promising candidate for the treatment of ocular HSV infections.

Abstract. The purpose of this study was to develop a clear aqueous mixed nanomicellar formulation (MNF) of dexamethasone utilizing both D-α-tocopherol polyethylene glycol-1000 succinate (Vit E TPGS) and . In this study, Vit E TPGS and Oc-40 are independent variables. Formulations were prepared following solvent evaporation method. A three level full-factorial design was applied to optimize the formulation based on entrapment efficiency, size, and polydispersity index (PDI). A specific blend of Vit E TPGS and Oc-40 at a particular wt% ratio (4.5:2.0) produced excellent drug entrapment, loading, small mixed nanomicellar size and narrow PDI. Solubility of DEX in MNF is improved by~6.3-fold relative to normal aqueous solubility. Critical micellar concentration (CMC) for blend of polymers (4.5:2.0) was found to be lower (0.012 wt%) than the individual polymers (Vit E TPGS (0.025 wt%) and Oc-40 (0.107 wt%)). No significant effect on mixed nanomicellar size and PDI with one-factor or multifactor interactions was observed. Qualitative 1 H NMR studies confirmed absence of free drug in the outer aqueous MNF medium. MNF appeared to be highly stable. Cytotoxicity studies on rabbit primary corneal epithelial cells did not indicate any toxicity suggesting MNF of dexamethasone is safe and suitable for human topical ocular drops after further in vivo evaluations.