Jwala

Recent Perspectives in Ocular Drug Delivery

Anatomy and physiology of the eye makes it a highly protected organ. Designing an effective therapy for ocular diseases, especially for the posterior segment, has been considered as a formidable task. Limitations of topical and intravitreal route of administration have challenged scientists to find alternative mode of administration like periocular routes. Transporter targeted drug delivery has generated a great deal of interest in the field because of its potential to overcome many barriers associated with current therapy. Application of nanotechnology has been very promising in the treatment of a gamut of diseases. In this review, we have briefly discussed several ocular drug delivery systems such as microemulsions, nanosuspensions, nanoparticles, liposomes, niosomes, dendrimers, implants, and hydrogels. Potential for ocular gene therapy has also been described in this article. In near future, a great deal of attention will be paid to develop non-invasive sustained drug release for both anterior and posterior segment eye disorders. A better understanding of nature of ocular diseases, barriers and factors affecting in vivo performance, would greatly drive the development of new delivery systems. Current momentum in the invention of new drug delivery systems hold a promise towards much improved therapies for the treatment of vision threatening disorders.

Development and validation of a fast and sensitive bioanalytical method for the quantitative determination of glucocorticoids--quantitative measurement of dexamethasone in rabbit ocular matrices by liquid chromatography tandem mass spectrometry.

A sensitive, selective, accurate and robust LC-MS/MS method was developed and validated for the quantitative determination of glucocorticoids in rabbit ocular tissues. Samples were processed by a simple liquid-liquid extraction procedure. Chromatographic separation was performed on Phenomenex reversed phase C18 gemini column (50mmx4.6mm i.d.,) with an isocratic mobile phase composed of 30% of acetonitrile in water containing 0.1% of formic acid, at a flow rate 0.2mL/min. Dexamethasone (DEX), prednisolone (PD) and hydrocortisone (HD) were detected with proton adducts at m/z 393.20-->355.30, 361.30-->147.20 and 363.20-->121.0 in multiple reaction monitoring (MRM) positive mode respectively. Finally, 50microL of 0.1% novel DEX mixed micellar formulation was topically administered to a rabbit eye and concentrations were measured. The method was validated over a linear concentration range of 2.7-617.6ng/mL. Lower limit of quantitation (LLOQ) of DEX and PD was measured in the concentration range of 2.7 and 11.0ng/mL respectively. The resulting method demonstrated intra and inter-day precision within 13.3% and 11.1% and accuracy within 19.3% and 12.5% for DEX and PD, respectively. Both analytes were found to be stable throughout freeze-thaw cycles and during bench top and postoperative stability studies (r(2)>0.999). DEX concentrations in various ocular tissue samples i.e., aqueous humor, cornea, iris ciliary body, sclera and retina choroid were found to be 344.0, 1050.07, 529.6, 103.9 and 48.5ng/mg protein respectively. Absorption of DEX after topical administration from a novel aqueous mixed micellar formulation achieved therapeutic concentration levels in posterior segment of the rabbit eye.

Novel nanoparticulate gel formulations of steroids for the treatment of macular edema.

PURPOSE This article describes the development and characterization of PLGA nanoparticles of dexamethasone (DEX), hydrocortisone acetate (HA), and prednisolone acetate (PA) suspended in thermosensitive gels indicated for the treatment of macular edema (ME). METHODS Nanoparticles were prepared by oil-in-water (O/W) emulsion and dialysis methods using PLGA 50:50 and PLGA 65:35. These particles were characterized for entrapment efficiency, size distribution, surface morphology, crystallinity, and in vitro release. Further, ex vivo permeation studies of DEX in suspension and nanoparticulate formulations were carried out across the rabbit sclera. RESULTS Entrapment efficiencies of DEX, HA, and PA were found to be lower with the dialysis method. O/W emulsion/solvent evaporation technique resulted in higher entrapment efficiencies, that is, 77.3%, 91.3%, 92.3% for DEX, HA, and PA, respectively. Release from nanoparticles suspended in thermosensitive gels followed zero-order kinetics with no apparent burst effect. Ex vivo permeability studies further confirmed sustained release of DEX from nanoparticles suspended in thermosensitive gels. CONCLUSIONS These novel nanoparticulate systems containing particles suspended in thermosensitive gels may provide sustained retina/choroid delivery of steroids following episcleral administration.

Recent Patents and Emerging Therapeutics in the Treatment of Allergic Conjunctivitis

Ocular allergy is an inflammatory response of the conjunctival mucosa that also affects the cornea and eyelids. Allergic conjunctivitis includes seasonal allergic conjunctivitis (SAC), perennial allergic conjunctivitis (PAC), vernal keratoconjunctivitis (VKC), atopic keratoconjunctivitis (AKC) and giant papillary conjunctivitis (GPC). In general, allergic conditions involve mast cell degranulation that leads to release of inflammatory mediators and activation of enzymatic cascades generating pro-inflammatory mediators. In chronic ocular inflammatory disorders associated with mast cell activation such as VKC and AKC constant inflammatory response is observed due to predominance of inflammatory mediators such as eosinophils and Th2-generated cytokines. Antihistamines, mast-cell stabilizers, nonsteroidal anti-inflammatory agents, corticosteroids and immunomodulatory agents are commonly indicated for the treatment of acute and chronic allergic conjunctivitis. In recent years newer drug molecules have been introduced in the treatment of allergic conjunctivitis. This article reviews recent patents and emerging therapeutics in the treatment of allergic conjunctivitis.

Ocular Sustained Release Nanoparticles Containing Stereoisomeric Dipeptide Prodrugs of Acyclovir

PURPOSE The objective of this study was to develop and characterize polymeric nanoparticles of appropriate stereoisomeric dipeptide prodrugs of acyclovir (L-valine-L-valine-ACV, L-valine-D-valine-ACV, D-valine-L-valine-ACV, and D-valine-D-valine-ACV) for the treatment of ocular herpes keratitis. METHODS Stereoisomeric dipeptide prodrugs of acyclovir (ACV) were screened for bioreversion in various ocular tissues, cell proliferation, and uptake across the rabbit primary corneal epithelial cell line. Docking studies were carried out to examine the affinity of prodrugs to the peptide transporter protein. Prodrugs with optimum characteristics were selected for the preparation of nanoparticles using various grades of poly (lactic-co-glycolic acid) (PLGA). Nanoparticles were characterized for the entrapment efficiency, surface morphology, size distribution, and in vitro release. Further, the effect of thermosensitive gels on the release of prodrugs from nanoparticles was also studied. RESULTS L-valine-L-valine-ACV and L-valine-D-valine-ACV were considered to be optimum in terms of enzymatic stability, uptake, and cytotoxicity. Docking results indicated that L-valine in the terminal position increases the affinity of the prodrugs to the peptide transporter protein. Entrapment efficiency values of L-valine-L-valine-ACV and L-valine-D-valine-ACV were found to be optimal with PLGA 75:25 and PLGA 65:35 polymers, respectively. In vitro release of prodrugs from nanoparticles exhibited a biphasic release behavior with initial burst phase followed by sustained release. Dispersion of nanoparticles in thermosensitive gels completely eliminated the burst release phase. CONCLUSION Novel nanoparticulate systems of dipeptide prodrugs of ACV suspended in thermosensitive gels may provide sustained delivery after topical administration.

In vitro evaluation of a targeted and sustained release system for retinoblastoma cells using Doxorubicin as a model drug.

PURPOSE The objective of this study was to develop a novel folate receptor-targeted drug delivery system for retinoblastoma cells using doxorubicin (DOX) as a model drug. METHODS Biodegradable DOX-loaded poly(d,l-lactide-co-glycolide)-poly(ethylene glycol)-folate (PLGA-PEG-FOL) micelles (DOXM) were prepared with various solvents (dimethylsulfoxide, acetone, and dimethylformamide). The effects of solvents on entrapment efficiency, particle size, and polydispersity were examined. The effects of thermosensitive gel structure on the release of DOX from the DOXM were also studied. Qualitative and quantitative uptake studies of DOX and DOXM were carried out in Y-79 cell line. Cytotoxicity studies of DOXM were performed on Y-79 cells. RESULTS Based on size, polydispersity, and entrapment efficiency, dimethylformamide was found to be the most suitable solvent for the preparation of DOXM. Dispersion of DOXM in PLGA-PEG-PLGA gel sustained drug release for a period of 2 weeks. Uptake of DOX was ∼4 times higher with DOXM than DOX in Y-79 cells overexpressing folate receptors. This was further confirmed from the quantitative uptake studies. DOXM exhibited higher cytotoxicity in Y-79 cells when compared with pure DOX. CONCLUSION These polymeric micellar systems suspended in thermosensitive gels may provide sustained and targeted delivery of anticancer agents to retinoblastoma cells following intravitreal administration.

Differential Expression of Folate Receptor-Alpha, Sodium-Dependent Multivitamin Transporter, and Amino Acid Transporter (B (0, +)) in Human Retinoblastoma (Y-79) and Retinal Pigment Epithelial (ARPE-19) Cell Lines

PURPOSE The overall objective of this study was to investigate the differential expression of folate receptor-alpha (FR-α), sodium-dependent multivitamin transporter (SMVT), and amino acid transporter [B ((0, +))] in retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cells. METHODS Polymerase chain reaction (PCR) analysis was performed to confirm the existence of FR-α, SMVT, and B ((0, +)) in Y-79 and ARPE-19 cell lines. Quantitative real-time PCR was also performed to determine the relative expression of FR-α, SMVT, and B ((0, +)) at mRNA level in these cell lines. Quantitative uptake of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was studied in Y-79 and ARPE-19 cells. Further, saturation kinetics of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was performed in the presence of various concentrations of respective cold substrates to determine the kinetic parameters (K(m) and V(max)) in Y-79 and ARPE-19 cells. RESULTS PCR analysis had confirmed the existence of FR-α, SMVT, and B ((0, +)) in Y-79 and ARPE-19 cells. Quantitative real-time PCR analysis had shown significantly higher expression of FR-α, SMVT, and B ((0, +)) mRNA levels in Y-79 cells compared with ARPE-19 cells. Quantitative uptake of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was found to be significantly higher in Y-79 cells relative to ARPE-19 cells. [(3)H] Folic acid uptake process followed saturation kinetics with apparent K(m) of 8.29 nM and V(max) of 393.47 fmol/min/mg protein in Y-79 cells and K(m) of 80.55 nM and V(max) of 491.86 fmol/min/mg protein in ARPE-19 cells. [(3)H] Biotin uptake process also displayed saturation kinetics with K(m) of 8.53 μM and V(max) of 14.12 pmol/min/mg protein in Y-79 cells and K(m) of 138.25 μM and V(max) of 38.85 pmol/min/mg protein in ARPE-19 cells. [(14)C] Arginine uptake process followed saturation kinetics with K(m) of 16.77 μM and V(max) of 348.27 pmol/min/mg protein in Y-79 cells and K(m) of 52.03 μM and V(max) of 379.21 pmol/min/mg protein in ARPE-19 cells. CONCLUSIONS This work demonstrated for the first time the higher expression and affinity of FR-α, SMVT, and B ((0, +)) mRNA levels in retinoblastoma (Y-79) cells compared with retinal pigment epithelial (ARPE-19) cells.

Functional Characterization of Folate Transport Proteins in Staten’s Seruminstitut Rabbit Corneal Epithelial Cell Line

Purpose:The overall objective of this study was to investigate and characterize the expression of folate transport proteins in Staten’s Seruminstitut rabbit corneal (SIRC) epithelial cell line. Methods: [3H]Folic acid uptake was studied with respect to time, pH, temperature, sodium, and chloride ion dependency. Inhibition studies were conducted with structural analogs, vitamins, and metabolic inhibitors. [3H]Folic acid uptake was also determined with varying concentrations of cold folic acid. Uptake kinetics was studied in the presence of various modulators of intracellular regulatory pathways, protein kinases A and C (PKA and PKC), protein tyrosine kinase (PTK), and calcium-calmodulin modulators. Ex vivo corneal permeability studies were carried out with [3H]folic acid in the presence and absence of 1 mM cold folic acid. Results: Linear increase in [3H]folic acid uptake was observed over 30 min. The process followed saturation kinetics with apparent Km of 14.2 ± 0.2 nM, Vmax of (1.5 ± 0.1)*10−5 micro.moles/min/mg protein, and Kd of (2.1 ± 0.2)*10−6 min−1. The uptake process was found to be dependent on pH, sodium ions, chloride ions, temperature, and energy. Uptake was inhibited in the presence of structural analogs (cold folic acid, methyltetrahydro folate, and methotrexate), but structurally unrelated vitamins did not show any effect. Membrane transport inhibitors SITS, DIDS, probenecid and endocytic inhibitor, colchicine significantly inhibited the [3H]folic acid uptake indicating the involvement of receptor/transporter mediated process. PKA, PTK, and Ca2+/calmodulin pathways significantly regulate the process. RT-PCR and Western blot analysis confirmed the presence of folate receptor-α (FR-alpha) and proton-coupled folate transporter (PCFT). Permeability of [3H]folic acid across the rabbit cornea was (1.48 ± 0.13)*10−05 cm/sec, and in the presence of cold folic acid it was (1.08 ± 0.10)*10−05 cm/sec. Conclusions: This work demonstrated the functional and molecular presence of FR-alpha and PCFT in SIRC epithelial cell line.