Corneal Barrier Research Articles

Solutol HS15 based binary mixed micelles with penetration enhancers for augmented corneal delivery of sertaconazole nitrate: optimization, in vitro, ex vivo and in vivo characterization

Keratomycosis is a serious corneal disease that can cause a permanent visual disability if not treated effectively. Sertaconazole nitrate (STZ), a novel broad spectrum antifungal drug, was suggested as a promising treatment. However, its utility in the ocular route is restricted by its poor solubility, along with other problems facing the ocular delivery like short residence time, and the existing corneal barrier. Therefore, the objective of this study was to formulate STZ loaded binary mixed micelles (STZ-MMs) enriched with different penetration enhancers using thin-film hydration method, based on a 31.22 mixed factorial design. Different formulation variables were examined, namely, type of auxiliary surfactant, type of penetration enhancer, and total surfactants: drug ratio, and their effects on the solubility of STZ in MMs (SM), particle size (PS), polydispersity index (PDI), and zeta potential (ZP) were evaluated. STZ-MMs enhanced STZ aqueous solubility up to 338.82-fold compared to free STZ. Two optimized formulations (MM-8 and MM-11) based on the desirability factor (0.891 and 0.866) were selected by Design expert® software for further investigations. The optimized formulations were imaged by TEM which revealed nanosized spherical micelles. Moreover, they were examined for corneal mucoadhesion, stability upon dilution, storage effect, and ex vivo corneal permeation studies. Finally, both in vivo corneal uptake and in vivo corneal tolerance were investigated. MM-8 showed superiority in the ex vivo and in vivo permeation studies when compared to the STZ-suspension. The obtained results suggest that the aforementioned STZ loaded mixed micellar system could be an effective candidate for Keratomycosis-targeted therapy.

Topical Application of Mizoribine Suppresses CD4+ T-cell-Mediated Pathogenesis in Murine Dry Eye.

We investigate the effect of topical application of mizoribine (MZR) eye drops on CD4+ T-cell-mediated immunity and epithelial damage in ocular surface of dry eye disease (DED). Topical application of MZR or vehicle eye drops was performed in mice subjected to desiccating stress (DS). The phenol red cotton test was used to measure tear production, and Oregon-green-dextran (OGD) staining was performed to assess corneal epithelial barrier function. PAS staining was used to quantify conjunctival goblet cells. Immunofluorescent staining and quantitative (q) RT-PCR were used to assess the expression of matrix metalloproteinase (MMP)-9 in corneal epithelium. qRT-PCR and ELISA were used to assess the production of TNF-α and IL-1β in conjunctiva. Apoptosis in ocular surface was assessed by TUNEL and activation of caspase-8. CD4+ T-cell-mediated immunity was evaluated by CD4 immunohistochemistry and production of T helper cytokines, including IFN-γ, IL-13, and IL-17A in conjunctiva. Compared to vehicle control mice, topical MZR-treated mice showed increased tear production, decreased goblet cell loss, and improved corneal barrier function. Topical application of MZR suppressed the expression of MMP-9 in corneal epithelium and apoptosis in ocular surface, while it had no obvious effect on production of TNF-α and IL-1β in conjunctiva. Topical application of MZR decreased CD4+ T cells infiltration, with decreased production of IFN-γ and IL-17A, and increased production of IL-13 in conjunctiva. Topical application of MZR could alleviate epithelial damage and CD4+ T-cell-mediated immunity in ocular surface of DED.

Therapeutic Effect of MK2 Inhibitor on Experimental Murine Dry Eye.

To investigate the role of mitogen-activated protein kinase-activated protein kinase-2 (MK2) in ocular surface damage of dry eye. MK2 inhibition was performed in mice subjected to desiccating stress (DS) by topical application of MK2 inhibitor (MK2i) or vehicle eye drops. The total and phosphorylated MK2 in conjunctiva were detected by Western blot. The phenol red cotton test was used to measure tear production, and Oregon green dextran staining was performed to assess corneal epithelial barrier function. PAS staining was used to quantify conjunctival goblet cells. Immunofluorescent staining and quantitative RT-PCR were used to assess the expression of matrix metalloproteinase (MMP)-3 and -9 in corneal epithelium. Apoptosis in ocular surface was assessed by TUNEL and immunofluorescent staining for activated caspase-3 and -8. Inflammation was evaluated by CD4+ T-cell infiltration and production of T helper (Th) cytokines, including IFN-γ, IL-13, and IL-17A in conjunctiva. DS promoted MK2 activation in conjunctiva. Compared with vehicle control mice, MK2i-treated mice showed increased tear production, decreased goblet cell loss, and improved corneal barrier function. Topical MK2 inhibition decreased the expression of MMP-3 and -9 in corneal epithelium, and suppressed cell apoptosis in ocular surface under DS. Topical MK2 inhibition decreased CD4+ T-cell infiltration, with decreased production of IFN-γ and IL-17A and increased production of IL-13 in conjunctiva. Topical MK2 inhibition effectively alleviated ocular surface damage via suppressing cell apoptosis and CD4+ T-cell-mediated inflammation in ocular surface of dry eye.

Ectodysplasin A regulates epithelial barrier function through sonic hedgehog signalling pathway

Ectodysplasin A (Eda), a member of the tumour necrosis factor superfamily, plays an important role in ectodermal organ development. An EDA mutation underlies the most common of ectodermal dysplasias, that is X‐linked hypohidrotic ectodermal dysplasia (XLHED) in humans. Even though it lacks a developmental function, the role of Eda during the postnatal stage remains elusive. In this study, we found tight junctional proteins ZO‐1 and claudin‐1 expression is largely reduced in epidermal, corneal and lung epithelia in Eda mutant Tabby mice at different postnatal ages. These declines are associated with tail ulceration, corneal pannus formation and lung infection. Furthermore, topical application of recombinant Eda protein markedly mitigated corneal barrier dysfunction. Using cultures of a human corneal epithelial cell line and Tabby mouse skin tissue explants, Eda up‐regulated expression of ZO‐1 and claudin‐1 through activation of the sonic hedgehog signalling pathway. We conclude that EDA gene expression contributes to the maintenance of epithelial barrier function. Such insight may help efforts to identify novel strategies for improving management of XLHED disease manifestations in a clinical setting.

Wound-Healing Studies in Cornea and Skin: Parallels, Differences and Opportunities.

The cornea and the skin are both organs that provide the outer barrier of the body. Both tissues have developed intrinsic mechanisms that protect the organism from a wide range of external threats, but at the same time also enable rapid restoration of tissue integrity and organ-specific function. The easy accessibility makes the skin an attractive model system to study tissue damage and repair. Findings from skin research have contributed to unravelling novel fundamental principles in regenerative biology and the repair of other epithelial-mesenchymal tissues, such as the cornea. Following barrier disruption, the influx of inflammatory cells, myofibroblast differentiation, extracellular matrix synthesis and scar formation present parallel repair mechanisms in cornea and skin wound healing. Yet, capillary sprouting, while pivotal in proper skin wound healing, is a process that is rather associated with pathological repair of the cornea. Understanding the parallels and differences of the cellular and molecular networks that coordinate the wound healing response in skin and cornea are likely of mutual importance for both organs with regard to the development of regenerative therapies and understanding of the disease pathologies that affect epithelial-mesenchymal interactions. Here, we review the principal events in corneal wound healing and the mechanisms to restore corneal transparency and barrier function. We also refer to skin repair mechanisms and their potential implications for regenerative processes in the cornea.

Ocular Surface and External Filtration Surgery: Mutual Relationships.

There is a large body of evidence from clinical and experimental studies indicating that the long-term use of topical drugs may induce ocular surface changes, causing ocular discomfort, dry eye, conjunctival inflammation, subconjunctival fibrosis, corneal surface impairment, and, as a consequence of chronic ocular surface changes, the potential risk of failure for further glaucoma surgery. Subclinical inflammation has also been widely described in patients receiving antiglaucoma treatments for long periods of time, with inflammatory cell infiltration and fibroblast activation in the conjunctiva and subconjunctival space. Preservatives, especially benzalkonium chloride, which has consistently demonstrated its toxic effects in laboratory, experimental, and clinical studies, could induce or enhance such inflammatory changes, and has been recently confirmed as a direct risk factor of surgical failure. As a quaternary ammonium, this compound causes tear film instability, loss of goblet cells, conjunctival squamous metaplasia and apoptosis, disruption of the corneal epithelium barrier, corneal nerve impairment, chronic inflammation and potential damage to deeper ocular tissues. Drug-induced adverse effects are therefore far from restricted to only allergic reactions, but they are often very difficult to identify because they mostly occur in a delayed or poorly specific manner, and result from complex and multifactorial interactions between the drugs and the ocular surface. Postoperatively, the ocular surface also plays an important role, as the conjunctiva interacts with aqueous humor and subconjunctival fibrosis may block aqueous outflow and cause surgical failure. As preoperative inflammation underlies postoperative fibrosis and therefore surgical outcome, a better knowledge of ocular surface changes with appropriate evaluation and management should thus become a new paradigm in glaucoma care over the long term.

Silver Sulfadiazine-Impregnated Hydrocolloid Dressing Is Beneficial in Split-Thickness Skin-Graft Donor Wound Healing in a Small Randomized Controlled Study.

Donor-site wound healing was tested with a silver sulfadiazine (SSD)-impregnated hydrocolloid dressing and hydrocolloid dressing applied manually by a physician on site. A total of 14 patients, 5 woman and 9 men (23-89 years old, average = 61.6 ± 18.70 years), were enrolled in this prospective controlled study. The degree of bleeding was significantly less with the SSD-impregnated than with the hydrocolloid dressing (P < .01). In the moisture meter analysis, the values of the effective contact coefficient and corneal thickness were significantly smaller with the SSD-impregnated dressing (P < .05). In the color analysis, the clarity was significantly lower with the hydrocolloid dressing after 3 months than that of intact neighboring skin (P < .01). Regarding red-green color, SSD-impregnated and hydrocolloid values were significantly greater than the intact skin value immediately after and at 3 months, and the hydrocolloid value was significantly greater than intact at 6 months (P < .01 immediately; P < .01 at 3 months and intact at 6 months) in redness. Regarding yellow-blue color, the hydrocolloid value was significantly lower than the intact skin value at 3 months (P < .05 and intact) in yellow. The extensibility was significantly lower with the hydrocolloid dressing than in intact skin immediately (P < .01), and viscoelasticity was significantly lower with the hydrocolloid dressing than in intact skin immediately and after 3 and 6 months (P < .01 immediately and at 6 months and P < .05 at 3 months). The SSD-impregnated hydrocolloid dressing led to superior wound healing, decreased the degree of bleeding, and demonstrated better corneal barrier function, clarity, color matching, and viscoelasticity in split-thickness donors.

Activation of Rac1 and RhoA Preserve Corneal Endothelial Barrier Function.

The corneal endothelium is responsible for the correct hydration of the corneal stroma. Corneal endothelial cells have a low proliferative capacity, so preserving their barrier function under suboptimal conditions that cause osmotic imbalance, such as those arising from corneal pathologies, age, cryopreservation, and transplantation, is essential for maintaining corneal transparency. We have investigated the signaling induced by hyperosmotic shock that reversibly disrupts corneal endothelial barriers in human endothelial cells and in murine corneas. Endothelial barrier properties were analyzed in vitro by electric cell substrate impedance sensing (ECIS) and confocal microscopy of the human endothelial cell line B4G12-HCEC, and, ex vivo, by confocal microscopy and stimulated emission-depletion (STED) super-resolution microscopy of murine corneas. Cell signaling in response to hyperosmotic stress, induced with an excess of sodium chloride, was investigated in B4G12-HCECs. Rho GTPase activity was detected by pulldown assays with recombinant GST proteins fused to the Rho binding domains of Rho effectors. Hyperosmotic stress increased actin polymerization and activated the Rho GTPases Rac1 and RhoA, but not Cdc42. Rac1- and RhoA-mediated pathway inhibition had a minor effect on barrier disruption but partially delayed barrier reformation after stress withdrawal. In contrast, Rac1 and RhoA activation enhanced constitutive endothelial barrier function and accelerated barrier repair. Our results indicate that Rac1 and RhoA activation do not mediate stress-induced cell contraction but are endothelial responses that act to restore and maintain barrier homeostasis. Therefore, pharmacological activation of these two GTPases could be a therapeutic strategy for preserving corneal endothelial barrier function.

FUNDAMENTAL ASPECTS IN THE DEVELOPMENT OF MICROEMULSION SYSTEMS CONTAINING AMPHOTERICIN B FOR OPHTHALMIC USE

This is an overview of the development of microemulsion systems containing amphotericin B for ophthalmic use. The amphotericin B (AmB) is used to treat a fungal infection of eye and bioavailability of the AmB is the challenge for the research scientist. The poor bioavailability of AmB is due mainly to the corneal barrier, which eventually leads to a precorneal loss and subsequent decrease in the absorption of this drug into the intraocular tissues. Also, AmB has limited permeation across the intact corneal epithelium, which along with the toxicity associated with this molecule, epitomizes a major drawback to its clinical use. Therefore, new effective and safe drug vehicles for ocular delivery of AmB are urgently required. Recently, microemulsions (MEs) have been used in ophthalmic drug delivery system owing to their thermodynamic stability, transparent appearance, and favorable viscosity. Information and proper understanding of the physical chemistry and process of formation of AmB-containing MEs would provide reliable information on the best conditions for the use of these systems as eye drops. The aim of this research was thus to review the main studies on the use of MEs as delivery systems for AmB in topical eye treatment.

Interferon-gamma deficiency protects against aging-related goblet cell loss.

Aging is a well-recognized risk factor for dry eye. Interferon-gamma (IFN-γ) has been implicated in conjunctival keratinization and goblet cell loss in dry eye. We investigated the role of IFN-γ in age-related dry eye by evaluating young (8 weeks) and aged (15 months; 15M) C57BL/6 (B6) and IFN-γKO mice. Age effects on the conjunctiva and cornea epithelium were assessed with PAS staining and corneal staining, respectively. Expression of T cell-related cytokines (IL-17A, IFN-γ), chemokines (CXCL10 and CCL20), in the ocular surface epithelium was evaluated by real time PCR. A significant decrease in filled goblet cells was noted in 15M B6 mice and this was significantly lower than age and sex-matched IFN-γKO mice. Aged male B6 had significantly higher IFN-γ, and CXCL10 mRNA in their conjunctiva than female B6 mice. Aged IFN-γKO females had significantly higher IL-17A mRNA in conjunctiva than IFN-γKO males and B6 mice. Corneal barrier dysfunction was observed in 15M female B6 and aged IFN-γKO mice of both sexes; however it was significantly higher in IFN-γKO compared to B6 mice. While there was a significant increase in IL 17A, and CCL20 in corneas of aged female B6 and IFN-γKO mice compared to males, these changes were more evident in aged female IFN-γKO group.Partial resistance of IFN-γKO mice to aging-induced goblet cell loss indicates IFN-γ is involved in the age-related decline in conjunctival goblet cells. Increased corneal IL-17A expression paralleled corneal barrier disruption in aging female of both strains. IFN-γ appears to suppress IL-17A on the ocular surface.

Tat PTD-Endostatin-RGD: A novel protein with anti-angiogenesis effect in retina via eye drops

BackgroundDiabetic retinopathy is a leading cause of blindness. The objective was to design a novel fusion protein, Tat PTD-Endostatin-RGD, to treat retinal neovascularization via eye drops instead of traditional intravitreal injection trepapeutical methods. MethodThe anti-angiogenesis ability was evaluated in vitro by chick embryo chorioallantoic membrane assay, wound healing assay and tube formation assay. Corneal barrier and blood-retina barrier were constructed in vitro to investigate the penetration ability of Tat PTD-Endostatin-RGD. Western blot was used to detect the integrin αvβ3 expression level in rat retina microvascular endothelial cells which was stimulated by S-nitroso-N-acetylpenicillamine. The binding affinity of Tat PTD-Endostatin-RGD to integrin αvβ3 was investigated by evaluating the penetration ability on blood-retina barriers treated with S-nitroso-N-acetylpenicillamine. The pharmacodynamics and efficacy analysis were further carried out in the oxygen-induced retinopathy model in vivo. In addition, the pharmacokinetic profile via eye drops was studied on a C57BL/6 mice model. ResultTat PTD-Endostatin-RGD showed high anti-angiogenesis activity and high ability to penetrate these two barriers in vitro. The Western blot results indicated S-nitroso-N-acetylpenicillamine upregulated the expression level of integrin αvβ3 in a dose-dependent manner. Tat PTD-Endostatin-RGD showed a high affinity to rat retina microvascular endothelial cells treated with S-nitroso-N-acetylpenicillamine. The results showed that Tat PTD-Endostatin-RGD could inhibit abnormal angiogenesis in retina via eye drops. ConclusionTat PTD-Endostatin-RGD showed high penetration ability through ocular barriers, bound specifically to integrin αvβ3 and effectively inhibited the abnormal angiogenesis. General significanceTat PTD-Endostatin-RGD represents a potent novel drug applied via eye drops for fundus oculi neovascularization diseases.

In vitro and in vivo corneal effects of latanoprost combined with brimonidine, timolol, dorzolamide, or brinzolamide

To examine the relevance of concentration of benzalkonium chloride (BAK) on the cornea, we investigated the effects of latanoprost containing BAK alone and in combination with other antiglaucoma drug classes on corneal epithelium in vitro in a cultured rabbit corneal cell line (SIRC) and in vivo, using a corneal resistance device (CRD). [In vitro] staten's seruminstitut rabbit corneal cells were exposed to 0.005% latanoprost for 30s, followed by either phosphate buffered saline (control), 0.1% brimonidine, 0.5% timolol, 1% dorzolamide, or 1% brinzolamide. The number of viable cells was counted at 8, 15, and 30min. [In vivo] Albino rabbits were administered one drop of 0.005% latanoprost, followed 5min later by one drop of an agent from the in vitro trial. This was repeated every 15min for a total of three times. The change in corneal barrier function was assessed by measuring the corneal resistance at 2 and 30min after the final administration. [In vitro] At 8min, the viable cell count in the latanoprost+dorzolamide group was significantly lower than in the control group. At 15 and 30min, all treatment groups, except the latanoprost+brimonidine group, demonstrated significantly lower viable cell counts than the control group. [In vivo] At 2min after the final eye drop, the latanoprost+timolol group and the latanoprost+brinzolamide group demonstrated significantly lower corneal resistance than did the latanoprost+brimonidine group. No significant difference was observed between the agents at 30min. In conclusion, when combining latanoprost containing benzalkonium chloride with other classes of antiglaucoma drugs, brimonidine may cause the least corneal damage, and the number of drug administrations may be an important factor.

Less Invasive Corneal Transepithelial Electrical Resistance Measurement Method

PurposeTo evaluate acute corneal permeability changes after instillation of benzalkonium chloride (BAC) using a newly developed in vivo less invasive corneal transepithelial electrical resistance (TER) measurement method in animals and humans. MethodsWe previously developed an in vivo method for measuring corneal TER using intraocular electrodes in animals. This method can be used to precisely measure the decline of the corneal barrier function after instillation of BAC. To lessen the invasiveness of that procedure, we further refined the method for measuring the corneal TER by developing electrodes that could be placed on the surface of the cornea and in the conjunctival sac instead of inserting them into the anterior chamber. Corneal TER changes before and after exposure to 0.02% BAC were determined in this study using the new device in both rabbits and humans. ResultsThere was a significant decrease in the corneal TER after exposure of the cornea to 0.02% BAC solution in both rabbits and humans (P<.01). The results of this new less invasive method agreed with those of formerly established anterior chamber methods in rabbit experiments. ConclusionThis new less invasive corneal TER measurement method enables us for the first time to measure TER of the human cornea, allowing safe and reliable investigation of the direct effect of different eye drop treatments on the corneal epithelium.

Sjögren's syndrome associated dry eye in a mouse model is ameliorated by topical application of integrin α4 antagonist GW559090

Sjögren's syndrome is an autoimmune disease associated with inflammation of exocrine glands with clinical manifestations of dry eye and dry mouth. Dry eye in this disease involves inflammation of the ocular surface tissues – cornea and conjunctiva. While systemic blockade of adhesion molecules has been used to treat autoimmune diseases, the purpose of this study was to determine the therapeutic efficacy of topical application of an integrin α4 adhesion molecule antagonist in a mouse model of dry eye associated with Sjögren's syndrome. To assess this spontaneously developed ocular surface inflammation related to Sjögren's syndrome in TSP-1null mice (12 wks) was evaluated. Mice were treated with topical formulations containing 0.1% dexamethasone or 30 mg/ml GW559090 or vehicle control. Corneal fluorescein staining and conjunctival goblet cell density were assessed. Real-time PCR analysis was performed to assess expression of the inflammatory marker IL-1β in the cornea and Tbet and RORγt in the draining lymph nodes. Ocular surface inflammation was detectable in TSP-1null mice (≥12 wk old), which resulted in increased corneal fluorescein staining indicative of corneal barrier disruption and reduced conjunctival goblet cell density. These changes were accompanied by increased corneal expression of IL-1β as compared to WT controls and an altered balance of Th1 (Tbet) and Th17 (RORγt) markers in the draining lymph nodes. Topically applied dexamethasone and GW559090 significantly reduced corneal fluorescein staining compared to vehicle treatment (p = 0.023 and p < 0.001, respectively). This improved corneal barrier integrity upon adhesion molecule blockade was consistent with significantly reduced corneal expression of pro-inflammatory IL-1β compared to vehicle treated groups (p < 0.05 for both treatments). Significant improvement in goblet cell density was also noted in mice treated with 0.1% dexamethasone and GW559090 (p < 0.05 for both). We conclude that similar to topical dexamethasone, topically administered GW559090 successfully improved corneal barrier integrity and inflammation in an established ocular surface disease associated with Sjögren's syndrome.

Epithelial cell adhesion molecule-1 (ECAM1) is required in the maintenance of corneal epithelial barrier integrity.

Corneal epithelial barrier integrity is critical in the maintenance of the corneal homeostasis. The corneal barrier dysfunction may be associated with the pathogenesis of a number of eye diseases. In this study, we assessed the expression of epithelial cell adhesion molecule-1 (ECAM1) in human corneal epithelial cells (HCE). The epithelial barrier function of the corneal epithelial monolayer was determined in Transwells. We found that the HCE cells expressed ECAM1. Knockdown of ECAM1 markedly compromised the HCE monolayer barrier function. A complex of ECAM1, claudin1, and occludin was detected in the HCE monolayers, which was not detected in the ECAM1-null HCE monolayers. Exposure to the proinflammatory cytokine, interleukin-13, inhibited the expression of ECAM1 in HCE cells and compromised the barrier function, which was prevented in the HCE monolayer with the ECAM1 overexpression. In conclusion, ECAM1 is required in the formation of the tight junction complex and maintaining the corneal epithelial barrier function.

A novel in vivo corneal trans-epithelial electrical resistance measurement device

PurposeTo develop a device that is capable of easily measuring corneal transepithelial electrical resistance (TER) and changes in the corneal barrier function. MethodsWe had previously developed an in vivo method for measuring corneal TER using intraocular electrode. This method can be used to precisely measure the decline of the corneal barrier function after instillation of benzalkonium chloride (BAC). In order to lessen the invasiveness of that procedure, we further refined the method for measuring the corneal TER by developing electrodes that could be placed on the cornea and in the conjunctival sac instead of inserting them into the anterior chamber. TER was then calculated by subtracting the electrical resistance, which lacked the corneal epithelial input, from the whole electrical resistance that was measured between the electrodes. Slit lamp examination and scanning electron microscopy (SEM) were used to determine safety of the new device. Corneal TER changes after exposure to 0.02% BAC were determined using the new device as well as SEM and transmission electron microscopy (TEM). ResultsSlit lamp examination before and after exposure of rabbits' corneas to the sensor confirmed safety of the device. SEM examination revealed no difference of the corneal epithelium which exposed to the new device with normal corneas. SEM and TEM pictures revealed damaged microvilli and tight junctions after instillation of 0.02% BAC. TER change after treatment with 0.02%BAC was similar to those determined by the established anterior chamber method. ConclusionWe succeeded to develop a less invasive device for corneal TER measurement in vivo in animals. This new device may be applicable in the future for clinical use in humans.

Dexamethasone nanowafer as an effective therapy for dry eye disease

Dry eye disease is a major public health problem that affects millions of people worldwide. It is presently treated with artificial tear and anti-inflammatory eye drops that are generally administered several times a day and may have limited therapeutic efficacy. To improve convenience and efficacy, a dexamethasone (Dex) loaded nanowafer (Dex-NW) has been developed that can release the drug on the ocular surface for a longer duration of time than drops, during which it slowly dissolves. The Dex-NW was fabricated using carboxymethyl cellulose polymer and contains arrays of 500nm square drug reservoirs filled with Dex. The in vivo efficacy of the Dex-NW was evaluated using an experimental mouse dry eye model. These studies demonstrated that once a day Dex-NW treatment on alternate days during a five-day treatment period was able to restore a healthy ocular surface and corneal barrier function with comparable efficacy to twice a day topically applied dexamethasone eye drop treatment. The Dex-NW was also very effective in down regulating expression of inflammatory cytokines (TNF-α, and IFN-γ), chemokines (CXCL-10 and CCL-5), and MMP-3, that are stimulated by dry eye. Despite less frequent dosing, the Dex-NW has comparable therapeutic efficacy to topically applied Dex eye drops in experimental mouse dry eye model, and these results provide a strong rationale for translation to human clinical trials for dry eye.

Osmolarity of prevalent eye drops, side effects, and therapeutic approaches.

Little is known about how the osmolarity of ophthalmic formulations affects the ocular surface. Because hyperosmolar eye drops could be therapeutic for treating corneal edema, this article presents an ex vivo model of corneal edema for testing ophthalmic drugs based on their osmolarity. The respective osmolarity of common eye drops found in the German market is also analyzed here. For modeling corneal edema, an Ex Vivo Eye Irritation Test was used to simulate an ocular anterior chamber with a physiological corneal barrier. To induce corneal edema, the anterior chamber was supplied with a hypoosmolar medium (148 mOsm/L) for 24 hours. Preserved and preservative-free 5% sodium chloride (hyperosmolar Omnisorb and Ocusalin 5% UD) were used for 1 hour, on 5 corneas each, to test their efficiency to reduce corneal edema in this model. Corneal thickness was determined by optical coherence tomography. Osmolarity of 87 common eye drops was measured by freezing point osmometry. Ex vivo, the tested hypoosmolar condition induced corneal edema from 450 μm (±50 μm) at baseline to 851 μm (±94 μm, P < 0.0001). Omnisorb and Ocusalin 5% UD significantly reduced the corneal thickness by 279 μm (±28 μm, P < 0.001) for Omnisorb and 258 μm (±29 μm, P < 0.001) for Ocusalin 5% UD. Forty-three (49%) of the tested products had an osmolarity below and 44 (51%) above the physiological tear osmolarity of 289 mOsm/L. Osmolarity values of less than 200 mOsm/L were found in lubricant drops. The highest osmolarity was detected in Omnisorb (1955 mOsm/L). The Ex Vivo Eye Irritation Test has proven to be a reliable novel model of corneal edema for evaluating osmotic eye drops. Osmolarity measurements revealed a wide range from hypotonic to hypertonic formulations for commonly marketed ophthalmic drugs.

Design and Evaluation of Ocular Controlled Delivery System for Diclofenac Sodium.

Diclofenac sodium as ophthalmic dosage form is used for the treatment of the pain, swelling and redness of patients' eyes recovering from cataract surgery; however, it faces the bioavailability limitation of eye drops due to effective protective mechanisms and corneal barrier functions in the eyes. Therefore, this investigation was aimed to develop ocular film formulations to achieve controlled drug release. Drug films were prepared using polymers, namely hydroxypropyl methylcellulose (HPMC) and polyvinyl pyrrolidone (PVP), Eudragit RL PO, and Eudragit RS PO by solvent casting method considering parameters such as drug: polymer ratio, different polymer combinations as well as plasticizer effect. Ocular films were evaluated for various physicochemical parameters such as physical characters, film thickness, uniformity of weight, drug content, swelling index, mucoadhesion time and in-vitro release study. Ocular films complied with all physicochemical parameters underwent in-vitro release study. Finally, the film formulation with HPMC: Eudragit RS PO 1:1 ratio, Drug: Polymer ratio 1:45 and glycerin as plasticizer showed controlled and prolonged release following the zero order and non-Fickian transport.

Diadenosine tetraphosphate improves adrenergic anti-glaucomatous drug delivery and efficiency

The effect of the dinucleotide P1, P4-Di (adenosine-5') tetraphosphate (Ap4A) in improving adrenergic anti-glaucomatous delivery by modifying the tight junction proteins of the corneal epithelium was evaluated. Stratified human corneal epithelial cells (HCLE) were treated with Ap4A (100 μM) for 5 min and TJ protein levels and barrier function were analysed by western blotting and transepithelial electrical resistance (TEER), respectively. Western blot experiments showed a significant reduction at 2 h (45% reduction of ZO-1 and 65% reduction of occludin protein levels) as compared to non-treated (control) cells. Two hours after Ap4A treatment, TEER values were significantly reduced (65% as compared to control levels (p < 0.001)), indicating an increase in corneal barrier permeability. Topical application of Ap4A in New Zealand white rabbits two hours before the instillation of the hypotensor compounds (the α2-adrenergic receptor agonist, brimonidine and the β-adrenergic receptor antagonist, timolol), improved the delivery of these compounds to the anterior chamber as well as their hypotensive action on the intraocular pressure. The results obtained showed that, when Ap4A was topically applied two hours before the adrenergic compounds, the concentration of brimonidine in the aqueous humour increased from 64.3 ± 5.3 nM to 240.6 ± 8.6 nM and from 58.9 ± 9.2 nM to 183.7 ± 6.8 nM in the case of timolol, which also produces a more profound effect on IOP. Therefore, Ap4A treatment results in a better entrance of adrenergic anti-glaucomatous compounds within the eye and consequently improved therapeutic efficiency by increasing corneal epithelial barrier permeability.