Integrated high-throughput drug screening microfluidic system for comprehensive ocular toxicity assessment.

Traditional experimental methodologies suffer from a few limitations in the toxicological evaluation of the preservatives added to eye drops. In this study, we overcame these limitations by using a microfluidic device. We developed a microfluidic system featuring a gradient concentration generator for preservative dosage control with microvalves and micropumps, automatically regulated by a programmable Arduino board. This system facilitated the simultaneous toxicological evaluation of human corneal epithelial cells against eight different concentrations of preservatives, allowing for quadruplicate experiments in a single run. In our study, the IC50 values for healthy eyes and those affected with dry eyes syndrome showed an approximately twofold difference. This variation is likely attributable to the duration for which the preservative remained in contact with corneal cells before being washed off by the medium, suggesting the significance of exposure time in the cytotoxic effect of preservatives. Our microfluidic system, automated by Arduino, simulated healthy and dry eye environments to study benzalkonium chloride toxicity and revealed significant differences in cell viability, with IC50 values of 0.0033% for healthy eyes and 0.0017% for dry eyes. In summary, we implemented the pinch-to-zoom feature of an electronic tablet in our microfluidic system, offering innovative alternatives for eye research.

Pharmacokinetic and Ocular Toxicity Evaluation of Latanoprost Ophthalmic Solution, 0.005%, with Preservative Level Reduced to Below the Limit of Quantitation.

Purpose: The systemic and ocular pharmacokinetics (PK), and ocular toxicity of benzalkonium chloride (BAK)-free TearClear latanoprost ophthalmic solution, 0.005% formulation (TC-002) were evaluated. TC-002 is designed to selectively capture BAK at the time of drug administration; therefore, the dose delivered to the eye contains no quantifiable level of preservative. Methods: The systemic and ocular PK of TC-002 were compared to a BAK containing reference listed drug (RLD, Xalatan™) over a 24-h period, after a single topical ocular dose to 1 eye of male Dutch Belted (DB) rabbits (n = 3/timepoint). Latanoprost acid concentrations were measured in plasma and ocular tissues. The ocular toxicity was evaluated in a separate study and included toxicokinetic evaluation of TC-002 after once daily topical ocular dosing into each eye of DB rabbits (n = 8/group) for at least 28 days. Toxicity endpoints included ophthalmic and clinical evaluations, necropsy, and microscopic evaluation of ocular tissues. Results: Average ratios of Cmax values for TC-002/RLD ranged from 0.6 to 1.6, and Cmax and area under the concentration-time curve of last observed concentration (AUClast) exposures to latanoprost acid were similar (<2-fold) between the 2 treatments. In the 28-day study, the Tmax was achieved in both groups in <0.5 h. There were no abnormal ocular findings. Conclusions: TC-002 with no quantifiable preservative or BAK-containing RLD exhibited similar ocular and systemic PK profiles. TC-002 was well tolerated and comparable to RLD. TC-002 retains the safety and PK characteristics of RLD without the added concern of long-term exposure of the eye to preservatives.

Prediction of serious eye damage or eye irritation potential of compounds via consensus labelling models and active learning models based on uncertainty strategies.

Serious eye damage and eye irritation have been authenticated to be significant human health issues in various fields such as ophthalmic pharmaceuticals. Due to the shortcomings of traditional animal testing methods, in silico methods have advanced to study eye toxicity. The models for predicting serious eye damage and eye irritation potential of compounds were developed using 2299 and 5214 compounds, respectively. The 40 global single models and 40 local models were developed by combining 5 molecular description methods and 4 machine learning methods. The 40 active learning models were developed by adopting uncertainty-based active learning strategies and taking local models as initial models. The 110 global consensus models based on 40 global single models were developed using a consensus strategy. Active learning models and global consensus models performed high prediction accuracy. The test accuracy of the best serious eye damage model and eye irritation model reached 0.972 and 0.959, respectively. The applicability domains for all models were calculated to verify the rationality of prediction effect. In addition, 8 structural alerts probably causing serious eye damage or eye irritation were sought out. The prediction models and structural alerts contributed to providing hazard identification and assessing chemical safety.

A novel ophthalmic latanoprost 0.005% nanoemulsion: a cytotoxicity study.

BACKGROUND: Benzalkonium chloride (BAK), the most commonly used preservative in anti-glaucoma eye drops, inflicts damage to the ocular surface. A novel anti-glaucoma formulation that avoids the use of BAK has been developed. The aim of this study was to evaluate the cytotoxicity of this formulation and to compare it with an ophthalmic solution containing BAK. METHODS: Two different latanoprost eye drops were used: one ophthalmic solution (LSc) containing BAK 0.02% and one ophthalmic nanoemulsion (LNe) with a soft preservative (potassium sorbate 0.18%). Human epithelial conjunctival cells were incubated for 15, 30, and 60 min with either LSc or LNe. The cytotoxicity was determined by MTT assay. Cell death was measured by flow cytometry using annexin V-FITC and propidium iodide. RESULTS: The values of cell viability and proliferation obtained from cells exposed to LNe were between 80 and 90% relative to the control group, whereas values obtained from cells exposed to LSc were around 30% at all study times (p < 0.05 at 15 and 30 min; p < 0.01 at 60 min). The percentage of viable cells decreased significantly when cells were incubated with LSc compared with cells incubated with LNe at all the study times, while the percentage of cells in late apoptosis/necrosis increased significantly in cells exposed to LSc compared to LNe. CONCLUSIONS: The new latanoprost nanoemulsion is significantly less cytotoxic on human conjunctival cells than LSc. These results suggest that the new formulation might be gentler on the eye surface than currently available BAK-preserved latanoprost solutions.

Phosphorylated xanthan gum-Ag(I) complex as antibacterial viscosity enhancer for eye drops formulation.

Topical instillation of eye drops represents the treatment of choice for many ocular diseases. Ophthalmic formulations must meet general requirements, i.e. pH, osmolality, transparency and viscosity to ensure adequate retention without inducing irritation and the development of eye infections. We developed a phosphorylated xanthan gum-Ag(I) complex (XGP-Ag) showing pH (pH = 7.1 ± 0.3) and osmolality values (311 ± 2 mOsm/kg) close to that of human tears (pH = 6.5-7.6 and 304 ± 23 mOsm/kg) thanks to the presence of phosphate moieties along the chain. The presence of phosphate groups covalently bound to the XG chains avoids their dispersion in fluid, thus reducing the risk of corneal calcification. 0.02% w/v XGP-Ag solution showed high transparency (higher than 95% along the entire visible range), adequate refractive index (1.334 ± 0.001) and viscosity in the range: γ 1 s-1-10,000 s- 1 (26.4 ± 0.8-2.1 ± 0.4 mPa·s). Its cytotoxicity and capability to hinder bacterial proliferation was also verified.

Enhancement in corneal permeability of riboflavin using cyclodextrin derivates complexes as a previous step to transepithelial cross-linking.

Corneal cross-linking has been described as an effective treatment to slow the progression of keratoconus. The standard protocol entails corneal epithelial removal to allow the diffusion of riboflavin into the stroma. Although, de-epithelization can generate risks or complications that transepithelial cross-linking tries to solve or avoid. Different formulations were developed after verifying that hydroxypropyl-ß-cyclodextrin (HPßCD) and sulfobuthylether-ß-cyclodextrin (SBEßCD) in a 20% concentration, increased the solubility of practically insoluble in water drugs such as riboflavin from 0.12 mg/mL to 0.35 mg/mL and 0.29 mg/mL respectively. These values were higher when chitosan and arginine were added to the formulation, showing solubility of 0.78 mg/mL when HPßCD concentration was not modified. Ex vivo corneal permeability was measured after having kept in contact bovine corneas with intact epithelium for 5 h with the 0.1 mg/mL riboflavin solution, the formulations developed and a reproduced nanoemulsion from another work. Riboflavin's permeability was increased when cyclodextrins, chitosan, and arginine were part of the formulations, compared to the control drug solution. The best permeability coefficient was reached when riboflavin was combined with 40% (w/v) HPßCD, 0.5% (w/w) arginine, and 0.5% (w/w) chitosan. After having carried out toxicity studies as bovine corneal opacity and permeability (BCOP) and Hens Egg Test - Chorioallantoic Membrane Test (HET-CAM) it was verified that both, the active ingredients and the excipients of the different formulations were not harmful without generating irritation, loss of transparency or corneal permeability alterations. The results show the great potential of the ocular developed solution for their use in transepithelial cross-linking for keratoconus treatment.

Ocular toxicology: synergism of UV radiation and benzalkonium chloride.

PURPOSE: To investigate the combined toxic effect of ultraviolet (UV) radiation and benzalkonium chloride (BAK), a common preservative in ophthalmic eye drops, on human corneal epithelial cells (HCEC). METHODS: Cultured HCEC were exposed to different combined and separate UV (280-400 nm) and BAK solutions at relevant human exposure levels. Human exposure to UV can occur before, during, or after eye drop installation, therefore, three different orders of ocular exposures were investigated: UV and BAK at the same time, UV first followed by BAK, and BAK first followed by UV. Control treatments included testing HCEC exposed to BAK alone and also HCEC exposed to UV alone. In addition, phosphate-buffered saline (PBS) was used as a negative control. After exposure, cell metabolic activity of the cultures was measured with PrestoBlue, and cell viability was determined using confocal microscopy with viability dyes. RESULTS: BAK alone reduced the metabolic activity and cell viability of HCEC in a dose- and time-dependent manner. UV alone at a low dose (0.17 J/cm2) had little toxicity on HCEC and was not significantly different from PBS control. However, UV plus BAK showed combined effects that were either greater than (synergistic) or equal to (additive) the sum of their individual effects. The synergistic effects occurred between low dose UV radiation (0.17 J/cm2) and low concentrations of BAK (0.001%, 0.002%, 0.003%, and 0.004%). CONCLUSIONS: This investigation determined that at relevant human exposure levels, the combination of UV radiation (280-400 nm) and BAK can cause synergistic and additive toxic effects on human corneal epithelial cells. This finding highlights the importance of considering the combined ocular toxicity of BAK and solar radiation in the risk assessment of BAK-preserved ophthalmic solutions.

Corneal damage and its recovery after instillation of preservative-free versus preserved latanoprost eye drops.

Purpose: Latanoprost ophthalmic solution is highly effective as a therapeutic agent for glaucoma and is applied worldwide. However, harmful effects on the corneal surface have been reported regarding the commercially available latanoprost ophthalmic solution. Corneal surface toxicity may be caused by the added preservative of the ophthalmic solution. In order to ascertain whether latanoprost itself can damage the cornea or if this is solely due to the added preservatives, this study attempted to determine the corneal changes that occur at different time periods following usage of preservative-free versus preserved latanoprost eye drops.Materials and methods: Preservative-free latanoprost eye drops (Monoprost®) or preserved latanoprost eye drops (Xalatan®) containing 0.02% benzalkonium chloride (BAC) were instilled in the corneas of rabbits. For each of the two different eye drop solutions, the rabbits used in this experiment were divided into three exposure groups: 1 minute, 24 hour, and 1 week groups. Corneal transepithelial electrical resistance (TER) and scanning electron microscopy (SEM) were examined immediately (1 minute) after instillation, at 24 hours after instillation, and at 24 hours after 1 week of daily instillations of latanoprost. Hank's balanced salt solution was used in the control group.Results: The mean corneal TER of the control group was 933.8 ± 279.0 Ω cm2. In preservative-free latanoprost instilled corneas, there was no significant decrease in the TER or morphological changes at any of the time points, with the relative TER values of 117 ± 38%, 100 ± 34%, and 93 ± 21% for 1 minute, 1 day, and 1 week time points, respectively. In preserved latanoprost instilled corneas, SEM showed that only the immediate group exhibited superficial cell damage and a significant decrease in the corneal TER compared to the controls and other time points and to the immediate preservative-free latanoprost corneas. In the preserved latanoprost groups, the relative TER values were 18 ± 5%, 110 ± 28%, and 92 ± 10%, for the three respective observation time points.Conclusions: Preservative-free latanoprost can be safely instilled to the corneal epithelium. Latanoprost with 0.02% BAC has an immediate deleterious impact on the corneal epithelium; however, it disappears within 24 hours after instillation.

Investigation of comet assays under conditions mimicking ocular instillation administration in a three-dimensional reconstructed human corneal epithelial model.

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration. Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1'-dimethyl-4,4'-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison. Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model. Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.

A New Topical Eye Drop Containing LyeTxI-b, A Synthetic Peptide Designed from A Lycosa erithrognata Venom Toxin, Was Effective to Treat Resistant Bacterial Keratitis.

Bacterial keratitis is an ocular infection that can lead to severe visual disability. Staphylococcus aureus is a major pathogen of the eye. We recently demonstrated the strong antimicrobial activity of LyeTxI-b, a synthetic peptide derived from a Lycosa erithrognatha toxin. Herein, we evaluated a topical formulation (eye drops) containing LyeTxI-b to treat resistant bacterial keratitis. Keratitis was induced with intrastromal injection of 4 × 105 cells (4 µL) in New Zealand female white rabbits. Minimum inhibitory concentration (MIC) and biofilm viability were determined. LyeTxI-b ocular toxicity was evaluated through chorioallantoic membrane and Draize tests. One drop of the formulation (LyeTxI-b 28.9 µmol/L +0.5% CMC in 0.9% NaCl) was instilled into each eye four times a day, for a week. Slit-lamp biomicroscopy analysis, corneal histopathological studies and cellular infiltrate quantification through myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) detection were performed. LyeTxI-b was very effective in the treatment of keratitis, with no signs of ocular toxicity. Planktonic bacteria MIC was 3.6 µmol/L and LyeTxI-b treatment reduced biofilm viability in 90%. LyeTxI-b eliminated bacteria and reduced inflammatory cellular activity in the eyes. Healthy and treated animals showed similar NAG and MPO levels. LyeTxI-b is a potent new drug to treat resistant bacterial keratitis, showing effective antimicrobial and anti-inflammatory activity.

Toxicology study of long-term administration of rhKGF-2 eye drops on rabbit corneas.

Keratinocyte growth factor -2 promotes corneal repair. Its mechanism of action involves regulating regeneration and migration of corneal cells, as well as activating corneal limbal stem cells. However, KGF-2 being a carcinogenic growth factor and its potential adverse effect in over dosage long-term treatment had not yet been reported. In this study, we used New Zealand white rabbits to study possible toxic effects of ocular administration of recombinant human keratinocyte growth factor-2 eye drops. Animals in the medium- and high-dose groups had some ocular irritant reactions during the course of drug administration; however this reaction was harmless to the cornea and it ended up when administration was stopped. Serum biochemistries were largely unaffected by treatment. Pathological examinations were unremarkable. We found that over-dosed administration of these eye drops caused some ocular irritation, but this irritant reaction was harmless to the eye, and it reversed after the drug was stopped. There were no apparent systemic effects of the drug.

Impact of Nanostructured Lipid Carriers as an Artificial Tear Film in a Rabbit Evaporative Dry Eye Model.

PURPOSE: To characterize formulations of nanostructured lipid carriers (NLCs) as an artificial tear film and evaluate their efficacy in protecting the ocular surface epithelial cells from desiccating stress in vivo. METHODS: The physicochemical properties of NLCs, produced with components similar to the tear film such as phosphatidylcholine and squalene, were determined. In vitro cytotoxicity of NLCs was evaluated by a short-time exposure test in porcine corneal epithelial cells using a methyl thiazol diphenyl-tetrazolium bromide assay. The residence time of NLCs in rabbit eyes and the efficacy of NLCs eye drops in protecting the rabbit corneal epithelium from desiccating stress were assessed. RESULTS: Nanosized NLCs with a mean size of ∼39 ± 5 nm and a zeta potential of -30 mV could be produced and formulated into eye drop with a pH of 6.90 ± 0.01, osmolarity of 273 ± 1 mOsm/L, and surface tension of 39 ± 1 mN/m (for air interface). Eye drop formulations of NLCs were nontoxic to porcine corneal epithelial cells. NLCs drops showed higher ocular surface retention and formed a stable corneal film compared with a saline solution. Moreover, NLCs eye drops showed greater efficacy in protecting the corneal surface against desiccating stress compared with a polymer-based commercial artificial tear. CONCLUSIONS: NLCs eye drops are biocompatible in rabbit eyes and show potential as a tear replacement vehicle for the treatment of dry eye disease.

Safety of sodium hyaluronate eye drop with C12-benzalkonium chloride.

PURPOSE: In this study, we investigated the effects of commercially available multi-dose sodium hyaluronate 0.1% (Hyalein®; Santen, Osaka, Japan) containing 0.003% C12-benzalkonium chloride (BAC) on the Corneal epithelium and its degree of safety. METHODS: Japanese white male rabbits were divided into four groups. The corneas of each group exposed to one of the following solutions: sodium hyaluronate 0.1%, C12-BAC, C12, 14, 16-BAC Mixture, and Hank's Balanced Salt Solution (HBSS) (as control), respectively. Corneal transepithelial electrical resistance (TER) changes after 60 s of exposure to the above solutions were measured in living rabbits. TER reflects the barrier function of the epithelium. In addition, scanning electron microscopy was used to examine the acute effects of the above solutions on the integrity of the corneal epithelium of four groups. RESULTS: There was no significant decrease in the corneal TER after exposure of the cornea to Hyalein® eye drops as compared to HBSS control eyes. Also, BAC mixture solution and C12-BAC did not produce any significant decrease in the corneal TER as compared to HBSS control eyes. All the corneal epithelium exposed to Hyalein®, 0.003% C12-BAC and 0.003% BAC mixture exhibited a regular appearance of the superficial cells with a high density of microvilli. CONCLUSION: This study confirms that Hyalein® has no acute hazardous effect on corneal epithelium.

Chemical and toxicological studies on different brands of Asmad (Antimony sulphide) available in Pakistan and Saudi Arabia.

Eye is the most beautiful, important and sensitive organ of human body. It is not only linked with visionary complex optical system also has the ability to differentiate among the millions of colors. The apparent human personality is also associated with it. Asmad/Antimony Sulfide/Kohl/Surma powder is one of the eye preparation has been used since ancient time. There are several aesthetic and ophthalmic preparations available for human eye and they have closed association between the aesthetic and medicinal significance such as cleansing, soothing, strengthening and anti-infectious actions along with beautifying purpose of eye. The main objective of present research is to provide scientific findings regarding beneficial and toxic effects of Asmad products available in market for the frequent users. The chemical and toxicological investigations on ten selected famous brands of Pakistan samples (PHS1, PHS2, PLS, PMS and PSS) and Saudi Arabia samples (SBS, SAS, SHS, SMS and STS) were carried out through advanced and sophisticated technique Scanning Electron Microscope (SEM) linked with Energy Dispersive X-ray Spectroscopy (EDS) which is used to determine the presence different percentages of organic and inorganic elements in all the brands of Pakistani and Saudi Arabian samples. The safety and toxicity depends on the Na, Mg, Ca, K, Al, Cu, Zn, Fe, Bi, Si, O, C, S and Pb percentages respectively of the Asmad products.

High Permeability and Intercellular Space Widening With Brimonidine Tartrate Eye Drops in Cultured Stratified Human Corneal Epithelial Sheets.

PURPOSE: To investigate the toxicity of topical glaucoma medications using cultured stratified human corneal epithelial sheets (HCES). METHODS: HCES were exposed for 30 minutes to the following glaucoma medications: 0.1% brimonidine with sodium chlorite as the preservative, 0.005% latanoprost with 0.02% benzalkonium chloride (BAC) as the preservative, and 0.5% timolol with 0.005% BAC as the preservative. Then, cell viability and barrier function were tested by the WST-1 assay and carboxyfluorescein permeability assay, respectively. After exposure to glaucoma medications, HCES were evaluated by hematoxylin and eosin staining, periodic acid-Schiff staining, scanning electron microscopy, and transmission electron microscopy. RESULTS: HCES exposed to brimonidine showed higher viability and better preservation of cell morphology and microvilli compared with cell sheets exposed to latanoprost or timolol. The carboxyfluorescein permeability assay demonstrated that the barrier function was preserved after HCES were exposed to timolol, but not after exposure to brimonidine or latanoprost. Transmission electron microscopy revealed widening of intercellular junctions with prominent deposits of glycogen or mucopolysaccharide (periodic acid-Schiff positive) after exposure of HCES to brimonidine. CONCLUSIONS: The toxicity of 0.1% brimonidine containing sodium chlorite for HCES was lower than that of ophthalmic preparations containing BAC. Reduction of the barrier function occurred after HCES were exposed to brimonidine because of widening of intercellular junctions.

El problema de la toxicidad aguda de algunos perfluoro-octanos / The acute toxicity problem with some perfluorooctanes

No disponible

Formulations and toxicologic in vivo studies of aqueous cyclosporin A eye drops with cyclodextrin nanoparticles.

Cyclosporin A (CyA) is an immunosuppressive drug used topically to treat ocular inflammatory disorder such as dry eye disease (DES). It is a lipophilic cyclic peptide with molecular weight of 1202.6Da. The aim of this study was to develop surfactant free aqueous 0.2% (w/v) CyA eye drops where the drug is present in an aqueous vehicle containing CyA/cyclodextrin (CyA/CD) nanoparticles and then do three-month toxicological testing in rabbits. Five formulations of different CD concentrations were studied, all of them contained 12.5% (w/v) of α-cyclodextrin (αCD) and various amounts of γ-cyclodextrin (γCD) (ranging from 0 to 12.5% w/v). αCD was used to solubilize the drug and γCD to promote formation of complex aggregates. CyA/CD complex aggregates were formed in all the formulations tested. However, the formulation containing 12.5% (w/v) αCD and 12.5% (w/v) γCD created more CyA/CD nanoparticles of suitable size and was therefore tested in vivo. The eye drops did not cause ocular irritation or toxic side effects upon topical administration to rabbits once or twice a day for three months.

Ocular Cubosome Drug Delivery System for Timolol Maleate: Preparation, Characterization, Cytotoxicity, Ex Vivo, and In Vivo Evaluation.

Glaucoma is an ocular disease featuring increased intraocular pressure (IOP) and its primary treatment strategy is to lower IOP by medication. Current ocular drug delivery in treating glaucoma is confronting a variety of challenges, such as low corneal permeability and bioavailability due to the unique anatomical structure of the human eye. To tackle these challenges, a cubosome drug delivery system for glaucoma treatment was constructed for timolol maleate (TM) in this study. The TM cubosomes (liquid crystalline nanoparticles) were prepared using glycerol monooleate and poloxamer 407 via high-pressure homogenization. These constructed nanoparticles appeared spherical using transmission electron microscopy and had an average particle size of 142 nm, zeta potential of -6.27 mV, and over 85% encapsulation efficiency. Moreover, using polarized light microscopy and small-angle X-ray scattering (SAXS), it was shown that the TM cubosomes have cubic liquid crystalline D-type (Pn3m) structure, which provides good physicochemical stability and high encapsulation efficiency. Ex vivo corneal permeability experiments showed that the total amount of TM cubosomes penetrated was higher than the commercially available eye drops. In addition, in vivo studies revealed that TM cubosomes reduced the IOP in rabbits from 27.8∼39.7 to 21.4∼32.6 mmHg after 1-week administration and had a longer retention time and better lower-IOP effect than the commercial TM eye drops. Furthermore, neither cytotoxicity nor histological impairment in the rabbit corneas was observed. This study suggests that cubosomes are capable of increasing the corneal permeability and bioavailability of TM and have great potential for ocular disease treatment.

The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant Cells.

Purpose: Benzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as "dry eye" and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects. Method: Mitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3). Results: Benzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 µM) and O2 consumption (IC50, 10.9 µM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107-667 µM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 µM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 µM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect. Conclusions: Benzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients.

Nanoemulsion-based electrolyte triggered in situ gel for ocular delivery of acetazolamide.

In the present work the antiglaucoma drug, acetazolamide, was formulated as an ion induced nanoemulsion-based in situ gel for ocular delivery aiming a sustained drug release and an improved therapeutic efficacy. Different acetazolamide loaded nanoemulsion formulations were prepared using peanut oil, tween 80 and/or cremophor EL as surfactant in addition to transcutol P or propylene glycol as cosurfactant. Based on physicochemical characterization, the nanoemulsion formulation containing mixed surfactants and transcutol P was selected to be incorporated into ion induced in situ gelling systems composed of gellan gum alone and in combination with xanthan gum, HPMC or carbopol. The nanoemulsion based in situ gels showed a significantly sustained drug release in comparison to the nanoemulsion. Gellan/xanthan and gellan/HPMC possessed good stability at all studied temperatures, but gellan/carbopol showed partial drug precipitation upon storage and was therefore excluded from the study. Gellan/xanthan and gellan/HPMC showed higher therapeutic efficacy and more prolonged intraocular pressure lowering effect relative to that of commercial eye drops and oral tablet. Gellan/xanthan showed superiority over gellan/HPMC in all studied parameters and is thus considered as a promising mucoadhesive nanoemulsion-based ion induced in situ gelling formula for topical administration of acetazolamide.