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The present work aimed to evaluate the efficacy of topical tacrolimus (0.01%) loaded propylene glycol (PG) modified nano-vesicles (Proglycosomes Nano-vesicles, PNVs) for the treatment of experimental dry eye syndrome (DES) in rabbits. DES was induced by topical application of atropine (1.0%) and benzalkonium chloride (0.1%) aqueous solution. PNVs treatment (PNV group) was compared with tacrolimus solution 0.01% (TAC group) and untreated group and healthy group were used as controls. PNV treated animals showed improved clinical performance with marked increase in tear production and tear break-up time (TBUT). Further, PNVs also subside ocular inflammation as evident from absence of matrix metalloprotenaise-9 and normal ocular surface temperature (32.3 ± 0.34 °C). Additionally, PNVs have positive effect on ocular and epithelial damage observed through low ocular surface staining score and improved globlet cell density. The PNV treatment was found to more effectively compared to TAC solution and most of the parameters were close to those of healthy animals. In conclusion, tacrolimus PNV formulation (0.01%) could be a potential therapy for treatment of dry eye syndrome.

The study aimed to evaluate the intraocular pharmacokinetics and efficacy of aflibercept after subconjunctival injection in animal models for treating choroidal neovascularization (CNV) associated with Age-Related Macular Degeneration (AMD). New Zealand albino rabbits received aflibercept (2000 μg/50 μl) in one eye, and the other eye was used as control. At 7, 14, 21 and 28 days, the animals were sacrificed to dissect the ocular tissues, and serum was collected at 1hr, 3 h, 1, 7, 14, 21 and 28 days. The concentration of aflibercept in various ocular tissues and serum were measured using the immunoassay technique. The concentration maximum (Cmax) at the Retinal Pigment Epithelium (RPE)-choroid complex and retina in treated eyes was 261.55 and 33.83 ng/gm, respectively. The area under the curve (AUC0-last) for RPE-Choroid and retina were 2094.02 and 290.33 days. ng/gm respectively. The time maximum (Tmax) for the ocular tissues was reached on day 7. In the vitreous humour, a lower level of aflibercept was retrieved. The Cmax (1766.84 ng/ml) in the serum was reached on day 1, followed by a decline in the concentration till the end of the study period. In treated eyes, the levels of aflibercept in most of the ocular tissues were maintained for at least 21 days above the invitro IC50 concentration. The results of the efficacy study show that subconjunctival aflibercept could reach the therapeutic target to inhibit CNV. The subconjunctival aflibercept could be a less invasive route for treating CNV with AMD.

The purpose of this study was to develop, characterize and evaluate novel water soluble formulation for its topical and intrastromal application. Natamycin complexed with cyclodextrin was characterized by Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). The complexed powder was used to formulate 1% w/v aqueous natamycin formulation (Natasol 1%) for topical use. The developed formulation was subjected to stability testing at various conditions. Single and multi-dose trans-corneal permeation of Natasol 1% was evaluated in New Zealand Albino rabbits in comparison with marketed 5% natamycin suspension. Sterile unpreserved Natasol (0.01% w/v natamycin) formulation was also developed for intrastromal injection. Both formulations were evaluated for the ocular toxicity. FTIR and DSC studies revealed successful complexation of natamycin that further protected the degradation at various stability conditions. Single dose trans-corneal permeation studies revealed that Natasol 1% attained Cmax at one hr and maintain its intraocular concentration 5 and 2.5 times higher at 4th and 6th hr compared to 5% natamycin suspension. Multi-dose kinetics revealed the steady state pharmacokinetics after hourly and two hourly dosing schedules. Topical Natasol 1% and sterile unpreserved Natasol 0.01% intrastromal injection, did not show any ocular toxicity in the animals. To conclude, the newly developed topical 1% w/v natamycin formulation was found to be non-inferior to 5% natamycin topical suspension. It is expected to increase patient compliance for the treatment of fungal keratitis.

Uveitis is an important cause of vision loss worldwide due to its sight-threatening complications, especially cystoid macular edema, as well as choroidal neovascularization, macular ischemia, cataract, and glaucoma. Systemic corticosteroids are the mainstay of therapy for noninfectious posterior uveitis; however, various systemic side effects can occur. Intravitreal medication achieves a therapeutic level in the vitreous while minimizing systemic complications and is thus used as an exciting alternative. Corticosteroids, antivascular endothelial growth factors, immunomodulators such as methotrexate and sirolimus, and nonsteroidal anti-inflammatory drugs are currently available for intravitreal therapy. This article reviews the existing literature for efficacy and safety of these various options for intravitreal drug therapy for the management of noninfectious uveitis (mainly intermediate, posterior, and panuveitis).

The important causes of loss of vision are ocular infections, including keratitis and conjunctivitis. Attaining an adequate concentration of topically applied antibiotics to prevent or treat infections within the cornea is challenging. The study aimed to design and develop a drug-eluting polymeric contact lens for the effective delivery of moxifloxacin (MF) and dexamethasone (DM). The polymeric contact lens was prepared using chitosan, glycerol, and polyethylene glycol. MF and DM were loaded into the contact lens, both separately and in combination. The MF and DM loaded contact lenses were characterized for thickness, swelling index, surface topography, and mucoadhesion strength. Furthermore, studies were performed to understand the in vitro drug release behavior, ex vivo corneal permeation, and in vitro and in vivo antimicrobial activity. The drug-loaded contact lens was compared with the standard drug solutions. The physical characteristics of the polymeric contact lens were similar to the commercially available contact lens. Compared to the topically applied standard drug solutions, the drug-loaded contact lens showed significantly (p 0.05) greater corneal drug distribution after 24 h incubation. In vitro and in vivo antimicrobial activity of the MF loaded contact lens was superior to the standard drug solution. In vivo drug distribution studies showed greater tissue concentration of MF in cornea, sclera, and aqueous humor with contact lens application compared with drug solutions. Overall, the polymeric contact lens was efficient in delivering MF and DM at required therapeutic concentrations. The findings from the present study show that drug-eluting contact lenses could be used in post-operative conditions to prevent ocular infections.

To study the protection offered by empty liposomes (LPs) alone against acrolein-induced changes in urothelial cell viability and explored uptake of LPs by primary (rat) urothelial cells. Acrolein was used as a means to induce cellular damage and reduce urothelial cellular viability. The effect of acrolein or liposomal treatment on cellular proliferation was studied using 5-bromo-2′-deoxy-uridine assay. Cytokine release was measured after urothelial cells were exposed to acrolein. Temperature-dependent uptake study was carried out for fluorescent-labeled LPs using confocal microscopy. Liposome pretreatment protected against acrolein-induced decrease in urothelial cell proliferation. LPs also significantly affected the acrolein-induced cytokine (interferon-gamma) release offering protection to the urothelial cells against acrolein damage. We also observed a temperature-dependent urothelial uptake of fluorescent-labeled LPs occurred at 37 °C (but not at 4 °C). Empty LPs alone provide a therapeutic efficacy against acrolein-induced changes in urothelial cell viability and may be a promising local therapy for bladder diseases. Hence, our preliminary evidence provides support for liposome-therapy for urothelial protection and possible repair.

We hypothesize organic cation transporters (OCT) may have a potential role in determining the pharmacokinetics and toxicity of organic cation drugs applied topically. Hence, in the present in vivo study, we attempted to evaluate the role of OCT in modulating the transport of its substrates after topical application. New Zealand albino rabbits of either sex were used. Transcorneal penetration of OCT substrates tetraethylammonium and metformin after single instillation was evaluated in the absence and presence of OCT blockers (quinidine and atropine). Aqueous humor (AH) samples were collected through paracentesis amounting to 70–100 μl under topical anesthesia at various time intervals. The samples were subjected for estimation of both substrate as well as blocker concentrations using liquid chromatography mass spectrometry. Topical pre-treatment (30 min before substrate) of OCT blockers significantly decreased the transcorneal penetration of OCT substrates after single topical administration. The levels of blockers reaching AH in the presence of substrates were also modulated at 60 min after its administration as compared with its control. OCT are functionally active in the uptake of their substrates from tear to AH. Therefore, OCT in the corneal epithelium may be positioned from apical to basolateral. When administering their substrates/blockers topically, both may be competing for OCT for their uptake across the cornea, thereby decreasing the corneal penetration. Hence OCT can have a potential pharmacokinetic role in modulating the ocular bioavailability of their substrates administered topically, which are used as ocular therapeutics.

Background: This paper describes the synthesis of thiolated chitosan-based hydrogels with varying degrees of crosslinking that has been utilized to modulate release kinetics of two clinically relevant FDA-approved anti-VEGF protein drugs, ranibizumab and aflibercept. These hydrogels have been fabricated into disc shaped structures for potential use as patches on ocular surface. Methods: Protein conformational changes and aggregation after loading and release was evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, electrophoresis and size-exclusion chromatography (SEC). Finally, the capacity of both released proteins to bind to VEGF was tested by ELISA and surface plasmon resonance (SPR) technology. Results: The study demonstrates the versatility of thiolated chitosan-based hydrogels for delivering proteins. The effect of various parameters of the hydrogel on protein release kinetics and mechanism of protein release was studied using the Korsmeyer-Peppas release model. Furthermore, we have studied the stability of released proteins in detail while comparing it with non-entrapped proteins under physiological conditions to understand the effect of formulation conditions on protein stability. Conclusions: The disc-shaped thiolated chitosan-based hydrogels provide a potentially useful platform to deliver ranibizumab and aflibercept for the treatments of ocular diseases such as wet AMD, DME and corneal neovascularization

The anti-vascular endothelial growth factor (VEGF) agents such as ranibizumab (Lucentis®) and aflibercept (EyLea®) are currently used as monthly or bimonthly intravitreal injections to treat potentially retinal diseases such as wet age-related macular degeneration (AMD) or diabetic macular edema (DME). Because of the complications associated with repeated intra-vitreal injections, there is considerable interest in developing a sustained delivery system. The purpose of this study was to examine the stability of both therapeutic proteins under physiological conditions as well as when incorporated into drug delivery systems (DDS). First, thermotropic properties in physiological conditions and at different pH values were evaluated by differential scanning calorimetry (DSC) to determine the protein denaturation temperature. Second, the effects of pH and incubation time on conformational changes and aggregation were evaluated by circular dichroism (CD), steady-state tryptophan fluorescence spectroscopy, and size-exclusion chromatography (SEC). Also, the ability of both proteins to bind to VEGF was tested in the aforementioned experimental conditions for up to 30 days. Finally, we investigated the stability of both proteins after a rapid screening method that simulates the first homogenizing step during the protein microencapsulation process. This method allowed the development of stable ranibizumab and aflibercept formulations that may be useful to entrap these proteins into microparticles selecting the most convenient organic solvent and protein stabilizers.

Background: The functional and molecular alterations of nerve growth factor (NGF) and Prostaglandin E2 (PGE2) and its receptors were studied in bladder and urine in streptozotocin (STZ)-induced diabetic rats. Methodology/Principal Findings: Diabetes mellitus was induced with a single dose of 45 mg/kg STZ Intraperitoneally (i.p) in female Sprague-Dawley rats. Continuous cystometrogram were performed on control rats and STZ treated rats at week 4 or 12 under urethane anesthesia. Bladder was then harvested for histology, expression of EP receptors and NGF by western blotting, PGE2 levels by ELISA, and detection of apoptosis by TUNEL staining. In addition, 4-hr urine was collected from all groups for urine levels of PGE2, and NGF assay. DM induced progressive increase of bladder weight, urine production, intercontraction interval (ICI) and residual urine in a time dependent fashion. Upregulation of Prostaglandin E receptor (EP)1 and EP3 receptors and downregulation of NGF expression, increase in urine NGF and decrease levels of urine PGE2 at week 12 was observed. The decrease in ICI by intravesical instillation of PGE2 was by 51% in control rats and 31.4% in DM group at week 12. Conclusions/Significance: DM induced hyposensitive underactive bladder which is characterized by increased inflammatory reaction, apoptosis, urine NGF levels, upregulation of EP1 and EP3 receptors and decreased bladder NGF and urine PGE2. The data suggest that EP3 receptor are potential targets in the treatment of diabetes induced underactive bladder. © 2014 Nirmal et al.

The present study demonstrates the development and validation of a sensitive method for the quantification of homocysteine thiolactone (HCTL) in human plasma using the technique of LC–MS/MS. The gradient elution of HCTL was achieved within 5 min using ZIC HILIC column having acetonitrile with 0.1% formic acid and water with 0.1% formic acid. The method was validated for the linearity, sensitivity, accuracy, precision, recovery, matrix effect and stability. A good linearity was found within a range of 0.5–32.5 nmol/ml. Quantification was performed using multiple reaction monitoring (MRM) mode based on the molecular/fragment ion transitions for HCTL (118/56) and homatropine (276.1/142.2) as internal standard. Generally, HCTL levels in plasma were found to be highly unstable. In order to verify the stability of the HCTL levels in plasma for a longer period, the samples were extracted immediately and stored at −86 °C. Using the above method it was found to be stable for a period of 1 month. The method was well applied for quantification of HCTL in plasma of healthy human volunteers.

The present study was conducted to test the hypothesis; OCT may be active from blood-to-vitreous for the uptake of its substrates. Ocular uptake of Tetraethylammonium (TEA) across blood ocular barriers and the tissue distribution was evaluated in vivo in New Zealand albino rabbits after intravenous administration. Quinidine (blocker) pretreatment resulted in a significant (p

To evaluate the functional role of organic cation transporters (OCT) and ocular tissue distribution of intravitreally injected OCT substrate tetraethylammonium (TEA) in presence of OCT blocker (quinidine).New Zealand albino rabbits of either sex were used. Intravitreal quinidine pretreatment was made 30 min before the administration of TEA. Modulation of vitreous and ocular tissue kinetics of OCT substrate was evaluated with or without blocker pretreatment. Gamma scintigraphy was also performed to visualize the vitreous residence of (99m)Tc-labelled TEA in the presence and absence of blocker.Intravitreally injected quinidine did not significantly alter the ocular disposition of TEA. TEA showed less significant posterior elimination kinetics and slow anterior elimination which resulted in longer residence time of TEA in eye after intravitreal administration.Intravitreally injected OCT substrates may follow an anterior elimination pathway and prolonged residence time in vitreous humor. The present study shows that OCT may not be active from vitreous-to-blood route in the blood-retinal barrier.