Sriram Gunda

Novel approaches to retinal drug delivery

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. Keywords:

Enhanced Corneal Absorption of Erythromycin by Modulating P-Glycoprotein and MRP Mediated Efflux with Corticosteroids

PurposeThe 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.MethodsCultured 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.ResultsBi-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.ConclusionMRP2 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.

Ocular pharmacokinetics of acyclovir amino acid ester prodrugs in the anterior chamber: evaluation of their utility in treating ocular HSV infections.

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 Stability of L-alanine-ACV (AACV), L-serine-ACV (SACV), L-isoleucine-ACV (IACV), gamma-glutamate-ACV (EACV) and 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 (lambda(z)). SACV and VACV exhibited approximately two-fold increase in area under the curve (AUC) relative to ACV (p<0.05). Clast (concentration at the last time point) of SACV was observed to be 8+/-2.6microM 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 (ka) 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 (Clast) seems to be a promising candidate for the treatment of ocular HSV infections.

Nanomicellar Topical Aqueous Drop Formulation of Rapamycin for Back-of-the-Eye Delivery

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 (1H 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 1H 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.

Identification and functional expression of a carrier-mediated riboflavin transport system on rabbit corneal epithelium.

Purpose: To investigate the functional expression of a carrier-mediated transport mechanism for riboflavin (vitamin B2) across cultured rabbit primary corneal epithelial cells (rPCECs) and intact rabbit cornea. The secondary objective was to understand the physiological significance behind the presence of such a transport system for riboflavin on the apical side of the corneal epithelium. Methods: rPCECs and freshly excised rabbit corneas were selected as in vitro and ex vivo models, respectively. Transport and uptake characteristics of [3H]riboflavin were determined at various time points, concentrations, temperatures, and pH. Substrate specificity, energy, and ion dependence studies were carried out to characterize the translocation mechanism. Rabbit tear analysis was done with liquid chromatography/tandem mass spectrometry (LC-MS/MS) to understand the physiological relevance of this transporter. Results: The uptake process in rPCECs was found to be concentration dependent and saturable at higher concentrations. The process was also independent of pH, Na+, and Cl− but dependent on energy and temperature. Unlabeled riboflavin and its structural analogues caused significant inhibition, whereas unrelated vitamins did not interfere with the process. Transport of [3H]riboflavin across rabbit cornea was also saturable at higher concentration and energy dependent but Na+ independent. Substrate specificity studies across intact rabbit cornea produced results similar to the uptake studies in cultured rPCECs. LC-MS/MS analysis of rabbit tears showed the presence of riboflavin. Conclusions: Results suggest the presence of a specialized, high-affinity transport mechanism for riboflavin that is expressed on the apical side of rabbit corneal epithelium and may in turn be responsible for influx of riboflavin from tears to cornea.

Bioanalytical method validation of rapamycin in ocular matrix by QTRAP LC-MS/MS: application to rabbit anterior tissue distribution by topical administration of rapamycin nanomicellar formulation.

A novel, fast and sensitive 3200 QTRAP LC-MS/MS method was validated for rapamycin analysis in the rabbit eye following 0.2% administration of nanomicellar eye drop formulation. The LC-MS/MS technique was developed with electrospray ionization (ESI) in positive mode. Rapamycin was extracted from individual eye tissues and fluids by a simple protein precipitation method. Samples were reconstituted in 200μL of 80% of acetonitrile in water containing 0.05% formic acid. Twenty microliter of the sample was injected on LC-MS/MS. Chromatographic separations was achieved on reversed phase C 8 Xterra column, 50mm×4.6mm, 5μm. Multiple reactions monitoring (MRM) transition m/z 936.6/409.3 for rapamycin and 734.4/576.5 for erythromycin were employed as internal standard. The calibration curves were linear r(2)>0.9998 over the concentration range from 2.3ng/mL to 1000.0ng/mL. Rapamycin was found to be stable in ocular tissue homogenates for 6weeks at a refrigerated -80°C and -20°C temperatures. Rapamycin concentration was found to be 2260.7±507.1 (mean±S.D.)ng/g tissue and 585.5±80.1 (mean±S.D.)ng/g tissue in the cornea and iris ciliary muscle, respectively. This method has two advantages. First, a volatile base was used in the extraction procedure, which is easy to evaporate and generate consistent results. Second, the sodium adduct is employed that was stable in non-ammoniated mobile phase. The method demonstrates that absorption of rapamycin by a topical application of 0.2% rapamycin nanomicellar formulation generates therapeutically effective concentrations in the anterior segment of the eye.