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.

Influence of different additives and their concentrations on corneal toxicity and antimicrobial effect of benzalkonium chloride.

PURPOSE: The aim of this study was to examine the ophthalmic additives responsible for modulating acute corneal epithelial toxicity induced by benzalkonium chloride (BAC) and investigate the ability of polyoxyethylene hydrogenated castor oil 40 (HCO-40) and polysorbate 80 (PS-80) to reduce the corneal toxicity and antimicrobial effects of BAC. METHODS: Cytotoxicity of the additives, which included glycerin, polyvinyl alcohol, propylene glycol, polyethylene glycol, and PS-80, on rabbit corneal epithelial cells was examined using the cell proliferation assay in the presence and absence of 0.02% BAC. The corneal transepithelial electrical resistance change after a 60-second exposure to HCO-40 or PS-80 mixed with 0.02% BAC was measured in living rabbits. Corneal damage was examined using scanning electron microscopy. The antimicrobial activities of HCO-40 and PS-80 with 0.02% BAC against Staphylococcus aureus, Propionibacterium acnes, Pseudomonas aeruginosa, Escherichia coli, and Streptococcus pneumoniae were assessed. RESULTS: Of all the tested additives, only PS-80 could prevent the BAC-induced cytotoxicity. Corneal epithelial barrier function disorder caused by 0.02% BAC was significantly alleviated by either PS-80 or HCO-40 in a concentration-dependent manner. Scanning electron microscopy images showed an improvement of BAC-induced corneal epithelial toxicity after the addition of HCO-40 or PS-80. The antimicrobial effect of the BAC against P. aeruginosa, E. coli, and S. pneumoniae was reduced after adding HCO-40 or PS-80. CONCLUSIONS: HCO-40 and PS-80 reduce acute corneal toxicity and the antimicrobial effect of BAC. Possible interactions between BAC and other additives should be taken into consideration when evaluating the toxicity and antibacterial properties of BAC.

Castor oil and mineral oil nanoemulsion: development and compatibility with a soft contact lens.

CONTEXT: The non-invasive ophthalmic therapy has a drawback: low residence time in the eye socket. Nanoparticles and contact lenses have been studied as promising ocular drug delivery systems. OBJECTIVE: To develop a nanoemulsion and evaluate its compatibility with a soft contact lens as a potential strategy for ocular delivery. MATERIALS AND METHODS: The formulations were developed by spontaneous emulsification and fully characterized. Two drops of nanoemulsion were instilled on the surface of a commercial contact lens and its transparency was measured using a UV-Vis spectrophotometer. Before and after the instillation of the drops, the morphology (scanning electron microscopy - SEM) and ion permeability of the lenses were analyzed. RESULTS: The formulations had a mean particle size of 234 nm, polydispersity below 0.16, zeta potential of -8.56 ± 3.49 mV, slightly acid pH, viscosity ≈1.2 mPa s(-1) and spherical-shaped particles. Nanoemulsion was non-irritant (hen's egg test-chorioallantoic membrane), which was confirmed by the cytotoxicity studies in the SIRC cell cultures. After instillation, SEM analysis showed nanodroplets inside and on the surface of the lenses, although their transparency remained near 100%. No significant differences were found between lens ion permeability coefficients before and after instillation. CONCLUSIONS: Formulations presented appropriate physicochemical characteristics and suitability for ocular application. The contact lens remained transparent and ion-permeable after association with the formulation.

Occludin gene expression as an early in vitro sign for mild eye irritation assessment.

PURPOSE: To test a new multiple endpoint analysis (MEA) including occludin gene expression for screening the ocular irritation potential of tear substitutes on human corneal epithelium (HCE), an in vitro model proposed to limit the use of animal testing in pre-clinical studies. METHODS: Four chemically-preserved and two non chemically-preserved tear substitutes were tested after acute (24h, 24h+24h post incubation) and repeated applications (for 72h) and compared to the positive control, benzalkonium chloride (BAK) at 0.1% and 0.01%, by assessing complementary parameters. Cellular viability was evaluated using MTT, histomorphologic analysis was performed on H&E stained vertical sections, IL-8 release was measured by ELISA, and occludin gene expression was quantified using qRT-PCR. RESULTS: Cellular viability was moderately reduced by Perborate and Polyquad-preserved tear substitutes and dramatically reduced by BAK and by Thiomersal and Oxyd preserved tear substitutes. Thiomersal also increased IL-8 release. Occludin expression profiles were modified by the four chemically-preserved tear substitutes and by the mechanically-preserved Comod, but not by the mechanically-preserved Abak. The behavior of BAK and tear substitutes led us to propose a prediction model for the classification of different levels of irritants, mainly based on the occludin transcriptional study. CONCLUSION: The versatility and sensitivity of the HCE model allowed the modeling of cumulative effects that may approach conditions obtained after long term application of tear substitutes. Thus, the modified MEA proposed in this study represents a valuable tool for in vitro eye irritation assessment with the power to detect mild irritants and subclinical eye irritant potential.

Diphenhydramine as a topical ocular anesthetic.

OBJECTIVE: To determine whether 5% diphenhydramine solution has an anesthetic effect when administered topically to rabbit corneas. DESIGN: Experimental study. PARTICIPANTS: Twenty white New Zealand rabbits. METHODS: Twenty rabbits at the University of Arkansas for Medical Sciences received 1 drop of 5% diphenhydramine solution in the left eye and 1 drop of balanced salt solution in the right eye. Corneal sensation was then measured with a Cochet-Bonnet esthesiometer at 30-, 60-, and 90-minute intervals. Rabbits were observed for conjunctival reaction. Follow-up fluorescein and Rose Bengal slit-lamp examinations were then performed to assess toxicity. RESULTS: Diphenhydramine solution at a 5% concentration demonstrated a significant anesthetic effect 30, 60, and 90 minutes after instillation (p < 0.0001, p = 0.0001, p = 0.0164, respectively). Mild conjunctival injection occurred in all diphenhydramine-treated eyes. No toxic effects on the corneal epithelium were observed. CONCLUSIONS: When applied topically to rabbit corneas, 5% diphenhydramine solution has a significant anesthetizing effect compared with salt solution (control eyes). Topical diphenhydramine may be a safe alternative in patients requiring topical anesthesia who have multiple allergies to topical anesthetics. Additional studies are needed to determine a dose-response curve and to further evaluate corneal toxicity prior to use in humans.

Evaluation of toxicity of commercial ophthalmic fluoroquinolone antibiotics as assessed on immortalized corneal and conjunctival epithelial cells.

PURPOSE: To evaluate the toxicity of a variety of the fluoroquinolone antibiotics on the ocular surface by using tissue culture models of corneal epithelial cells and conjunctival epithelial cells. METHODS: Immortalized conjunctival (CCC) and human corneal (HCE) epithelial cells were grown and when confluent the cells allowed to air dry for 1 hour. Medium was then replaced with 100 microL of one of the following: 1) Vigamox [moxifloxacin (0.5%: MX)]; (2) Zymar [gatifloxacin (0.3%: GA)]; 3) Quixin [levofloxacin (0.5%: LE)]; 4) Ocuflox [ofloxacin (0.3%: OF)]; 5) Ciloxan [ciprofloxacin (0.3%: CP)]; 6) medium (viable control); 7) "normal"/physiologic saline; 8) formalin (dead control). After one hour, 150 microL of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazonium bromide was added and incubated for 4 hours. After decanting, precipitate was dissolved in 150 microL of isopropanol. Absorbance was determined at 572 nm. RESULTS: The lowest amount of cell death was associated with the viable control. All ophthalmic preparations showed both corneal and conjunctival cell toxicity. Aside from the viable control, normal saline showed the next lowest amount of toxicity. Of the topical ocular antibiotics tested, MX showed the least amount of toxicity. All of the other antibiotics tested were statistically indistinguishable from each other. CONCLUSIONS: All of the topical ocular antibiotics tested showed evidence of both corneal and conjunctival toxicity (MX < OF < or = LE < or = CP < or = GA), although only MX was statistically significant. Whether this finding reflects on in vivo wound healing remains to be determined. This model provides a rapid and cost-effective method to screen for surface toxicity of topical agents.

Intravitreal toxicity of garenoxacin.

PURPOSE: To assess the retinal toxicity of varying concentrations of intravitreally injected garenoxacin. METHODS: Twenty eyes of 20 New Zealand albino rabbits were used for this study. The animals were anesthetized with ketamine (35-50 mg/kg) and xylazine (3-5 mg/kg). Garenoxacin was titrated using distilled water to the following concentrations: 4,000, 2,000, 1,000, 400, 200, and 100 microg/0.1 mL. Each concentration was injected intravitreally (0.1 mL) into three rabbit eyes. Three control eyes were injected with 0.1 mL of balanced saline solution. All animals were examined before and after injection by indirect ophthalmoscopy and slit-lamp biomicroscopy. Electroretinography was performed on all animals before intravitreal injection and 14 days after injection. The animals were examined by indirect ophthalmoscopy and slit-lamp biomicroscopy before they were killed; the eyes were enucleated and examined with light microscopy. RESULTS: No electroretinographic changes or signs of retinal toxicity by slit-lamp examination, indirect ophthalmoscopy, or light microscopy were seen in any eyes 14 days after intravitreal injection of garenoxacin (< or =4,000 microg/0.1 mL). CONCLUSIONS: Garenoxacin injected intravitreally appeared safe at concentrations of < or =4,000 microg/0.1 mL.

Intravitreal toxicity of moxifloxacin.

PURPOSE: To assess the retinal toxicity of various concentrations of intravitreally administered moxifloxacin, a fourth-generation fluoroquinolone. METHODS: Ten New Zealand albino rabbits were divided into five groups. The initial concentration of moxifloxacin (400 mg/250 mL) was titrated using 5% dextrose solution to concentrations (320 microg/0.1 mL, 160 microg/0.1 mL, 100 microg/0.1 mL, and 50 microg/0.1 mL) that were injected intravitreally into 1 eye of each rabbit. Two control eyes were injected intravitreally with 0.1 mL of 5% dextrose solution. All animals were examined before and after injection by indirect ophthalmoscopy and slit-lamp biomicroscopy; electroretinography (ERG) was performed on all animals. The animals were killed, and their eyes were enucleated and examined with light microscopy. RESULTS: Remarkable decreases in ERG findings were noted in the group injected with moxifloxacin at a concentration of 320 microg/0.1 mL. No meaningful ERG changes were observed in eyes injected with moxifloxacin at other concentrations. There were no signs of retinal toxicity during slit-lamp examination, indirect ophthalmoscopy, or light microscopy in any eyes injected with moxifloxacin concentrations of < or =160 microg/0.1 mL. CONCLUSIONS: Intravitreal injection of moxifloxacin at a concentration of < or =160 microg/0.1 mL appeared nontoxic in the rabbit eye.

Alternatives to ocular irritation testing in animals.

The preliminary conclusions of a survey of possible non-animal alternatives to the Draize rabbit eye irritancy test, recently conducted for the Commission of the European Communities, are presented. The various types of alternatives to animal tests are reviewed in terms of their current state of development and validation, and also their potential in relation to the type of exposure, level of testing, type of testing, type of effect, location of effect, and type of test material. Various problems concerning the availability and quality of in vivo eye irritation data, and the use of this data in in vitro/in vivo comparisons, are highlighted. Finally, the use of step-wise and integrated animal/non-animal and non-animal/non-animal test systems and strategies are discussed.

Nonclinical study requirements for ophthalmic drugs and devices in the United States.

The eye is a unique organ embryologically, immunologically, physiologically and consequently exhibits unique toxicological responses. Toxic responses in the eye may result from topical ocular administration of drugs, intraocular administration or implantation of drugs or devices or may be the result of target organ toxicity following systemic administration of a drug. The primary responsibility of the toxicologist is to establish the safety/toxicity profile for a drug or device under development and thus provide an appropriate risk analysis of the drug/device for human use. For the ophthalmic toxicologist this safety profile must include the appropriate toxicological evaluations to place in perspective the intended use of the drug or device, its effect on the relevant ocular tissues, its potential for adverse systemic effects, if warranted, and the potential risk to the patient in the clincal setting.

Requirements of preclinical examinations.

The eye is an important and sensitive target organ not only for ophthalmic drugs but also generally for adverse drug effects and has to be taken into consideration accordingly when performing toxicological studies. Depending on the drug in question protocols for oculotoxicity studies have to be determined on a case by case basis. The test battery used to detect possible eye damage should be an intelligent combination of pharmacokinetic/metabolic studies, clinical examinations, pathomorphologic examinations, and biochemical tests. For special studies on oculotoxicity only pigmented animals should be used. The legal basis for the necessity to conduct oculotoxicity studies and ophthalmologic examinations in toxicity studies is given by national law (AMG) and guidelines and by EEC-Guidelines. Studies have to be performed according to the principles of Good Laboratory Praxis.

Albino versus pigmented animals for ocular toxicity testing.

The capability of uveal and retinal tissue to accumulate exogenously administered materials, coupled with the existence of anatomic, physiologic and biochemical ocular abnormalities in albino animals, evokes the question of whether albino animals are suitable for testing novel compounds for untoward ocular effects. Examples of special susceptibilities of either pigmented or non-pigmented animals to novel compounds exist. The best way to avoid unanticipated oculotoxic effects from the administration of novel compounds is to use both pigmented and unpigmented strains.

Screening for potential in vivo phototoxicity in the lens/retina.

The eye is particularly vulnerable to damage from the phototoxic side effects of drugs since it is constantly subjected to ambient light. Presented here are several screens that can be used to determine the potential of drugs to cause light damage to the eye. These include a simple screen that takes into account the optical properties of the eye and the absorption spectra of the various drugs. This can be used to omit various drugs as potential photooxidants in the eye. In addition a second, more detailed screen is presented. This can be used to determine a) the quantitative potential for a drug to cause photooxidative damage in the eye, b) the mechanism by which it occurs and c) the in vivo verification of phototoxicity.