Polyamine Oxidase Is Involved in Spermidine Reduction of Transglutaminase Type 2-Catalyzed ßH-Crystallins Polymerization in Calcium-Induced Experimental Cataract.

In an in vitro Ca2+-induced cataract model, the progression of opacification is paralleled by a rapid decrease of the endogenous levels of spermidine (SPD) and an increase of transglutaminase type 2 (TG2, EC 2.3.2.13)-catalyzed lens crystallins cross-linking by protein-bound N1-N8-bis(γ-glutamyl) SPD. This pattern was reversed adding exogenous SPD to the incubation resulting in a delayed loss of transparency of the rabbit lens. The present report shows evidence on the main incorporation of SPD by the catalytic activity of TG2, toward ßH-crystallins and in particular to the ßB2- and mostly in ßB3-crystallins. The increase of endogenous SPD in the cultured rabbit lens showed the activation of a flavin adenine dinucleotide (FAD)-dependent polyamine oxidases (PAO EC 1.5.3.11). As it is known that FAD-PAO degrades the N8-terminal reactive portion of N1-mono(γ-glutamyl) SPD, the protein-bound N8-mono(γ-glutamyl) SPD was found the mainly available derivative for the potential formation of ßB3-crystallins cross-links by protein-bound N1-N8-bis(γ-glutamyl)SPD. In conclusion, FAD-PAO degradation of the N8-terminal reactive residue of the crystallins bound N1-mono(γ-glutamyl)SPD together with the increased concentration of exogenous SPD, leading to saturation of glutamine residues on the substrate proteins, drastically reduces N1-N8-bis(γ-glutamyl)SPD crosslinks formation, preventing crystallins polymerization and avoiding rabbit lens opacification. The ability of SPD and MDL 72527 to modulate the activities of TG2 and FAD-PAO involved in the mechanism of lens opacification suggests a potential strategy for the prevention of senile cataract.

Estimation of Intra-vitreal Half-Lifes in the Rabbit Eye with Semi-mechanistic Equations.

PURPOSE: To develop an alternative method for estimating vitreal half-lifes in the rabbit eye based on simple equations for the physical processes of dissipation and the physiochemical properties of therapeutic substances applied by intravitreal drug administration. METHODS: Equations were derived to describe diffusion in the vitreous humor and permeation through the back-of-the-eye tissue, and the volume of distribution. The model was validated using reported half-life values from 83 compounds collected from literature. RESULTS: The rate limiting step for dissipation from the vitreous depends mainly on the molecular weight. Dissipation of very low molecular weight (MW) substances (<350 Da) is limited by diffusional transport to the back of the eye, for substances with a MW >350 Da uptake into the back of the eye tissue becomes limiting, and large molecules >500 Da predominantly take an alternative path being cleared through the front of the eye for which diffusion towards the posterior chamber turns out to be limiting. Taking the three rate determining processes into account, the derived model can estimate dissipation rates and respectively vitreal half-life values of small compounds and macromolecules from their molecular weight with very few exceptions. CONCLUSIONS: The equations derived in this analysis provide a simple method to predict vitreal half-lifes for a diverse group of molecules and can be easily implemented in early drug development.

Scleral cross-linking by riboflavin and blue light application in young rabbits: damage threshold and eye growth inhibition.

BACKGROUND: Scleral cross-linking (SXL) by riboflavin and light application has been introduced as a possible treatment to increase scleral tissue stiffness and to inhibit excessive axial elongation of highly myopic eyes. We evaluated an ocular tissue damage threshold for blue light irradiation, and used SXL treatment to induce eye growth inhibition. METHODS: The sclera of 3-week-old rabbits (39 pigmented and 15 albino rabbits) were treated with different blue light intensities (450 ± 50 nm) and riboflavin. Alterations and a damage threshold were detected in ocular tissues by means of light microscopy and immunohistochemistry. The influence of SXL on the eye growth was examined in 21 young rabbits and was measured by using A-scan ultrasonography, micrometer caliper, and for selected eyes additionally by MR imaging. RESULTS: Light microscopic examinations demonstrated degenerative changes in ocular tissue after irradiation with blue light intensities above 400 mW/cm(2) (with and without riboflavin application). Therefore, that light intensity was defined as the damage threshold. Tissue alteration in retina, choroid, and sclera and activation of retinal microglia cells and Müller cells could be earlier observed at blue light intensities of 150 and 200 mW/cm(2). Albino rabbits were less sensitive to this SXL treatment. A significant reduction of the eye growth could be detected by SXL treatment with the minimal efficient blue light intensity of 15 mW/cm(2) and maintained stable for 24 weeks. CONCLUSIONS: SXL with riboflavin and blue light intensities below a defined damage threshold can induce a long lasting growth inhibitory effect on young rabbit eyes. Therefore, SXL might be a realistic approach to inhibit eye elongation in highly myopic eyes.

Intravitreal controlled release of dexamethasone from engineered microparticles of porous silicon dioxide.

Dexamethasone is a glucocorticoid that is widely used in the ophthalmic arena. The recent FDA approved dexamethasone implant can provide a three month efficacy but with high rate of drug related cataract and high intraocular pressure (IOP). It seems that higher steroid in aqueous humor and around lens may be associated with these complications based on clinical fact that higher IOP was observed with intravitreal triamcinolone acetonide (TA) than with subtenon TA. We hypothesize that placing a sustained dexamethasone release system near back of the eye through a fine needle can maximize efficacy while mitigate higher rate of IOP rise and cataract. To develop a sustained intravitreal dexamethasone delivery system, porous silicon dioxide (pSiO2) microparticles were fabricated and functionalized with amines as well as carboxyl groups. Dexamethasone was conjugated to pSiO2 through the Steglich Esterification Reaction between hydroxyl of dexamethasone and carboxyl groups on the pSiO2. The drug loading was confirmed by Fourier transform infrared spectroscopy (FTIR) and loading efficiency was quantitated using thermogravimetric analysis (TGA). In vitro release was conducted for three months and dexamethasone was confirmed in the released samples using liquid chromatography-tandem mass spectrometry (LC/MS/MS). A pilot ocular safety and determination of vitreous drug level was performed in rabbit eyes. The drug loading study demonstrated that loading efficiency was from 5.96% to 10.77% depending on the loading reaction time, being higher with longer loading reaction time before reaching saturation around 7 days. In vitro drug release study revealed that dexamethasone release from pSiO2 particles was sustainable for over 90 days and was 80 days longer than free dexamethasone or infiltration-loaded pSiO2 particle formulation in the same setting. Pilot in vivo study demonstrated no sign of ocular adverse reaction in rabbit eyes following a single 3 mg intravitreal injection and free drug level at 2-week was 107.23 ± 10.54 ng/mL that is well above the therapeutic level but only around 20% level of dexamethasone released from OZURDEX(®) (dexamethasone intravitreal implant) in a rabbit eye model. In conclusion, dexamethasone is able to covalently load to the pSiO2 particles and provide sustained drug release for at least 3 months in vitro. Intravitreal injection of these particles were well tolerated in rabbit eyes and free drug level in vitreous at 2-week was well above the therapeutic level.

Ocular silicon distribution and clearance following intravitreal injection of porous silicon microparticles.

Porous silicon (pSi) microparticles have been investigated for intravitreal drug delivery and demonstrated good biocompatibility. With the appropriate surface chemistry, pSi can reside in vitreous for months or longer. However, ocular distribution and clearance pathway of its degradation product, silicic acid, are not well understood. In the current study, rabbit ocular tissue was collected at different time point following fresh pSi (day 1, 5, 9, 16, and 21) or oxidized pSi (day 3, 7, 14, 21, and 35) intravitreal injection. In addition, dual-probe simultaneous microdialysis of aqueous and vitreous humor was performed following a bolus intravitreal injection of 0.25 mL silicic acid (150 µg/mL) and six consecutive microdialysates were collected every 20 min. Silicon was quantified from the samples using inductively coupled plasma-optical emission spectroscopy. The study showed that following the intravitreal injection of oxidized pSi, free silicon was consistently higher in the aqueous than in the retina (8.1 ± 6.5 vs. 3.4 ± 3.9 µg/mL, p = 0.0031). The area under the concentration-time curve (AUC) of the retina was only about 24% that of the aqueous. The mean residence time was 16 days for aqueous, 13 days for vitreous, 6 days for retina, and 18 days for plasma. Similarly, following intravitreal fresh pSi, free silicon was also found higher in aqueous than in retina (7 ± 4.7 vs. 3.4 ± 4.1 µg/mL, p = 0.014). The AUC for the retina was about 50% of the AUC for the aqueous. The microdialysis revealed the terminal half-life of free silicon in the aqueous was 30 min and 92 min in the vitreous; the AUC for aqueous accounted for 38% of the AUC for vitreous. Our studies indicate that aqueous humor is a significant pathway for silicon egress from the eye following intravitreal injection of pSi crystals.

Device Development for Ocular Surface Temperature and Heat Flux Density Measurement.

PURPOSE: Currently, much attention is paid to measuring the temperature of the ocular surface in various ophthalmic diseases. However, for a comprehensive assessment of heat transfer of the eye, it is advisable to measure both the ocular surface temperature and the heat flux (HF) density. This will expand our knowledge of the physiology of the eye and create new possibilities for diagnosing ocular pathology. The present study aimed to develop a thermoelectric device to study rabbits' ocular surface temperature and HF density. METHODS: The multichannel thermoelectric device was developed to measure the ocular surface temperature and HF density. This study included ten rabbits (20 eyes). In all animals, the temperature and the HF density were measured on the surface of the central cornea of both eyes. The measurement was repeated after pupils' dilation. RESULTS: The corneal surface temperature of rabbits was 33.1 ± 0.8 °C, and the HF density of the surface of the cornea was 8.3 ± 0.6 mW/cm2. Our results revealed a high degree of interocular symmetry in the surface HF density in healthy rabbits. After pupil dilation, an increase in the HF density on the surface of the rabbit cornea compared with the initial data and control eye was noted. CONCLUSIONS: The application of the developed device showed that it is safe and allows for the measurement of the ocular surface temperature and the HF density. We believe that further studies on the ocular surface HF density measurement in various eye diseases will allow us to evaluate the possibilities of this device and technique for diagnostic purposes.

Ocular pharmacokinetics and toxicity of nanoparticular acetazolamide: In vivo distribution and safety of PHBV-ACZ nanoparticle.

Diabetic macular edema (DME) is defined as fluid accumulation in the macular region, between the retinal layers, due to many diseases, especially diabetes. DME is one of the major complications of diabetic retinopathy (DRP). Carbonic anhydrase inhibitors (CAI) are a pharmaceutical agent used in different fields, especially glaucoma treatment. Acetazolamide (ACZ), which is a CAI, is an active substance that has been used off-label for many years in the treatment of macular edema due to diabetes and many other diseases. The low solubility and bioavailability of ACZ limit its use in the treatment of DME. In this study, a nanoparticulate formulation was developed that would increase the solubility and bioavailability of ACZ and allow it to be administered intravitreally. ACZ was loaded on poly(3-hydroxybutyrate-co-3-Hydroxyvalerate) (PHBV) nanoparticles and the loading efficiency was 71.58 ± 1.22%. Toxicity of nanoparticles after intravitreal application was evaluated with anterior segment and posterior segment examination findings, intraocular pressure (IOP) measurements and electrophysiological tests. At the end of the 3-month follow-up, electroretinography (ERG) measurements demonstrated that ACZ loaded PHBV (PHBV-ACZ) nanoparticles did not cause loss of function in retinal cells. On histological examination, rare degenerative changes were observed in several cell groups. In addition, pharmacokinetic studies were performed to determine the tissue distribution of ACZ at various periods. ACZ was identified in vitreous humor and retina at the highest concentration. Based on our results, the prepared nanoparticle formulation can release long-term CAI for DRP therapy and accordingly can reduce the need for monthly intravitreal injections.

Limitations of Reconstructing Pentacam Rabbit Corneal Tomography by Zernike Polynomials.

The study aims to investigate the likelihood of Zernike polynomial being used for reconstructing rabbit corneal surfaces as scanned by the Pentacam segment tomographer, and hence evaluate the accuracy of corneal power maps calculated from such Zernike fitted surfaces. The study utilised a data set of both eyes of 21 rabbits using a reverse engineering approach for deductive reasoning. Pentacam raw elevation data were fitted to Zernike polynomials of orders 2 to 20. The surface fitting process to Zernike polynomials was carried out using randomly selected 80% of the corneal surface data points, and the root means squared fitting error (RMS) was determined for the other 20% of the surface data following the Pareto principle. The process was carried out for both the anterior and posterior surfaces of the corneal surfaces that were measured via Pentacam scans. Raw elevation data and the fitted corneal surfaces were then used to determine corneal axial and tangential curvature maps. For reconstructed surfaces calculated using the Zernike fitted surfaces, the mean and standard deviation of the error incurred by the fitting were calculated. For power maps computed using the raw elevation data, different levels of discrete cosine transform (DCT) smoothing were employed to infer the smoothing level utilised by the Pentacam device. The RMS error was not significantly improved for Zernike polynomial orders above 12 and 10 when fitting the anterior and posterior surfaces of the cornea, respectively. This was noted by the statistically non-significant increase in accuracy when the order was increased beyond these values. The corneal curvature calculations suggest that a smoothing process is employed in the corneal curvature maps outputted by the Pentacam device; however, the exact smoothing method is unknown. Additionally, the results suggest that fitting corneal surfaces to high-order Zernike polynomials will incur a clinical error in the calculation of axial and tangential corneal curvature of at least 0.16 ± 01 D and 0.36 ± 0.02 D, respectively. Rabbit corneal anterior and posterior surfaces scanned via the Pentacam were optimally fitted to orders 12 and 10 Zernike polynomials. This is essential to get stable values of high-order aberrations that are not affected by Zernike polynomial fittings, such as comas for Intracorneal Ring Segments (ICRS) adjustments or spherical aberration for pre-cataract operations. Smoothing was necessary to replicate the corneal curvature maps outputted by the Pentacam tomographer, and fitting corneal surfaces to Zernike polynomials introduces errors in the calculation of both the axial and tangential corneal curvatures.

Force-based Control for Safe Robot-assisted Retinal Interventions: In Vivo Evaluation in Animal Studies.

In recent years, robotic assistance in vitreoretinal surgery has moved from a benchtop environment to the operating rooms. Emerging robotic systems improve tool manoeuvrability and provide precise tool motions in a constrained intraocular environment and reduce/remove hand tremor. However, often due to their stiff and bulky mechanical structure, they diminish the perception of tool-to-sclera (scleral) forces, on which the surgeon relies, for eyeball manipulation. In this paper we measure these scleral forces and actively control the robot to keep them under a predefined threshold. Scleral forces are measured using a Fiber Bragg Grating (FBG) based force sensing instrument in an in vivo rabbit eye model in manual, cooperative robotic assistance with no scleral force control (NC), adaptive scleral force norm control (ANC) and adaptive scleral force component control (ACC) methods. To the best of our knowledge, this is the first time that the scleral forces are measured in an in vivo eye model during robot assisted vitreoretinal procedures. An experienced retinal surgeon repeated an intraocular tool manipulation (ITM) task 10 times in four in vivo rabbit eyes and a phantom eyeball, for a total of 50 repetitions in each control mode. Statistical analysis shows that the ANC and ACC control schemes restrict the duration of the undesired scleral forces to 4.41% and 14.53% as compared to 43.30% and 35.28% in manual and NC cases, respectively during the in vivo studies. These results show that the active robot control schemes can maintain applied scleral forces below a desired threshold during robot-assisted vitreoretinal surgery. The scleral forces measurements in this study may enable a better understanding of tool-to-sclera interactions during vitreoretinal surgery and the proposed control strategies could be extended to other microsurgery and robot-assisted interventions.

Management and control of intraocular pressure applying macitentan hydrogel film formulation: improved effect of surfactant and cosurfactant system.

BACKGROUND: Macitentan blocks endothelin receptors in order to control the pulmonary arterial hypertension (PAH). Oral administration of macitentan is associated with painful urination and troubled breathing. OBJECTIVES: Formulated macitentan hydrogel film was used for examining the control of intraocular pressure, and the effect of surfactant and cosurfactant was studied. METHODS: Macitentan ocular film formulation has been prepared in hydroxypropyl methylcellulose (HPMC) matrix system using different surfactant/co-surfactant system, and intraocular pressure was monitored on normotensive rabbit eyes after application in the cul-de-sac. RESULTS: The solid state characterization of the film indicated amorphisation of macitentan and no issues regarding major incompatibility was observed. Combination of surfactant, co-surfactant and hydrophilic co-solvent systems in the said films markedly improved the drug release and mucosal tissue permeation. Presence of PEG and Transcutol significantly improved ex vivo corneal permeation of MP and MT respectively compared to other films. Transcutol (MT) exhibited greatest difference among the formulations by improving the vesicular bilayer fluidity and reducing the mucosal tissue barrier facilitating the transcorneal diffusion. A combination of diffusion and erosion control behavior was observed in drug release and corneal permeation of the films due to the balanced liquid penetration and polymeric chain relaxation rate. MP and MT films were used for further in vivo studies to achieve possible effective and prolonged control of intraocular pressure. In vivo study has revealed the reduction in intraocular pressure upto about 23 % when tested on normotensive rabbit model. The films has managed to lower the IOP upto 3 h. CONCLUSION: Developed macitentan hydrogel film containing Transcutol (MT) could have a high potential for the control and management of ocular hypertension after topical application.

In-vivo estimation of tissue electrical conductivities of a rabbit eye for precise simulation of electric field distributions during ocular iontophoresis.

Precise estimation of electrical conductivity of the eyes is important for the accurate analysis of electric field distributions in the eyes during ocular iontophoresis. In this study, we estimated the tissue electrical conductivities of a rabbit eye, which has been widely employed for neuro-ophthalmological experiments, through an in vivo experiment for the first time. Electrical potentials were measured at multiple locations on the skin, while weak currents were transmitted into the skin via two surface electrodes attached to the skin around the eye. A finite element model was constructed to calculate the electric potentials at the measurement locations. The conductivity values of different tissues were then estimated using an optimization procedure to minimize the difference between the measured and calculated electric potentials. The accuracy of the estimated tissue conductivity values of the rabbit eye was validated by comparing the measured and calculated electric potential values for different electrode montages. Further multi-physical analyses of iontophoretic drug delivery to the rabbit eye showed a significant influence of the conductivity profile on the resultant particle distribution. Overall, our results provide an important reference for the tissue electrical conductivity values of the rabbit eye, which could be further utilized for designing new medical devices for delivering electric fields to the eyes, such as transorbital and transscleral electrical stimulations.

Comparative Assessment of Distribution Characteristics and Ocular Pharmacokinetics of Norvancomycin Between Continuous Topical Ocular Instillation and Hourly Administration of Eye Drop.

BACKGROUND: The aim of this study was to compare the distribution characteristics and ocular pharmacokinetics of norvancomycin (NVCM) in ocular tissues of the anterior segment between continuous topical ocular instillation and hourly administration of eye drop in rabbits. METHODS: Sixty rabbits were randomly divided into two groups: continuous topical ocular instillation drug delivery (CTOIDD) group and eye drop (control) group. In the CTOIDD group, NVCM solution (50 mg/mL) was perfused to the ocular surface using the CTOIDD system at 2 mL/h up to 10 h and the same solution was administered at one drop (50 µL) per hour for 10 h in the control group. Animals (N=6 per time-point per group) were humanely killed at 2, 4, 6, 10, and 24 h to analyze their ocular tissues and plasma. The concentrations of NVCM in the conjunctiva, cornea, aqueous humour, iris, ciliary body and plasma were measured by HPLC with photodiode array detector. The pharmacokinetic parameters were calculated by Kinetica 5.1. RESULTS: The highest concentrations of NVCM for the CTOIDD group and control group were 2105.45±919.89 µg/g and 97.18±43.14 µg/g in cornea, 3033.92±1061.95 µg/g and 806.99±563.02 µg/g in conjunctiva, 1570.19±402.87 µg/g and 46.93±23.46 µg/g in iris, 181.94±47.11 µg/g and 15.38±4.00 µg/g in ciliary body, 29.78±4.90 µg/mL and 3.20±1.48 µg/mL in aqueous humour, and 26.89±5.57 µg/mL and 1.90±1.87 µg/mL in plasma, respectively. The mean NVCM levels significantly increased at all time-points in cornea, iris, and ciliary body (p<0.05) in the CTOIDD group. The AUC0-24 values in the CTOIDD group were 27,543.70 µg·h/g in cornea, 32,514.48 µg·h/g in conjunctiva, 8631.05 µg·h/g in iris, 2194.36 µg·h/g in ciliary body and 343.9 µg·h/mL in aqueous humour, which were higher than for the eye drop group in all tissues. CONCLUSION: Since continuous instillation of NVCM with CTOIDD could reach significantly higher concentrations and was sustained for a longer period compared with hourly administration of eye drop, CTOIDD administered NVCM could be a possible method to treat bacterial keratitis.

Imaging depth extension of optical coherence tomography in rabbit eyes using optical clearing agents.

Optical coherence tomography has become an indispensable diagnostic tool in ophthalmology for imaging the retina and the anterior segment of the eye. However, the imaging depth of optical coherence tomography is limited by light attenuation in tissues due to optical scattering and absorption. In this study of rabbit eye both ex vivo and in vivo, optical coherence tomography imaging depth of the anterior and posterior segments of the eye was extended by using optical clearing agents to reduce multiple scattering. The sclera, the iris, and the ciliary body were clearly visualized by direct application of glycerol at an incision on the conjunctiva, and the posterior boundary of sclera and even the deeper tissues were detected by submerging the posterior segment of eye in glycerol solution ex vivo or by retro-bulbar injection of glycerol in vivo. The ex vivo rabbit eyes recovered to their original state in 60 s after saline-wash treatment, and normal optical coherence tomography images of the posterior segment of the sample eyes proved the self-recovery of in vivo performance. Signal intensities of optical coherence tomography images obtained before and after glycerol treatment were compared to analysis of the effect of optical clearing. To the best of our knowledge, this is the first study for imaging depth extension of optical coherence tomography in both the anterior and posterior segments of eye by using optical clearing agents.

Protocol for evaluation of topical ophthalmic drug products in different compartments of fresh eye tissues in a rabbit model.

Topical ophthalmic drugs are the most commonly used dosage form to treat diseases of the anterior segment of the eye. Although this dosage form has the advantages of ease of application, small volume dose, and rapid action and is largely devoid of systemic adverse effects, the bioavailability is low due to pre-corneal anatomical barriers and the nature of the drug formulation itself. Some complex generic formulations (suspensions, ointments, gels) for topical ophthalmic products face impediments to rapid regulatory approval because of the complex nature of the formulations and difficulties in determining bioequivalence with the innovator product. Clinical endpoint bioequivalence studies of ophthalmic products in humans are challenging due to inaccessibility of internal compartments of eye, large inter-subject variability that reduces study sensitivity, patient safety issues, and the prohibitively high costs of these types of clinical studies. Because of its ocular anatomical similarity to human eye, rabbits are frequently used as a model in early product development. Generating appropriate animal model data can inform physiological-based pharmacokinetic (PBPK) model building that might eventually replace the need for extensive, expensive preclinical and clinical testing. Little detail was found in the existing literature on sampling and bioanalytical protocols for determining drug concentration in different compartments of fresh eye tissues. This study describes in detail a sampling protocol for evaluating dexamethasone concentration in different tissues of freshly harvested eyes using TobraDex ST topical ophthalmic drug product in a rabbit model.

Acute Rabbit Eye Model for Testing Subretinal Prostheses.

PURPOSE: Subretinal prostheses are a novel technology for restoring useful vision in patients with retinitis pigmentosa or age-related macular degeneration. We characterize the surgical implantation technique and functional time window of an acute rabbit eye model for testing of human subretinal prostheses. METHODS: Retinal prostheses were implanted subretinally in 26 rabbits using a two-step technique. Fundus imaging, fluorescein fundus angiography, and optical coherence topography (OCT) were conducted postoperatively from days 1 to 21 to monitor prosthesis positioning and retinal anatomic changes. RESULTS: Successful implantation and excellent retina apposition were achieved in 84.6% of the rabbits. OCTs showed the overlying retina at full thickness for the first 2 days after implantation. Histology confirmed intact inner layers of the overlying retina until day 3. Progressive atrophy of the overlying retina was revealed by repeated OCTs; approximately 40% of the retina thickness remained on postoperative days 5 and 6. CONCLUSIONS: The two-step implantation technique works well for the rabbit eye model with human prostheses. Rabbit retina may be used for acute electrophysiologic testing of a retinal prosthesis, but is unsuitable for chronic studies due to the merangiotic retina and its limited time window of validity. TRANSLATIONAL RELEVANCE: The improved efficacy in prosthesis surgery using this technique will circumvent the challenges in animal models that provide human-like features critical for the transition into human clinical trials.

An in vitro depth of injury prediction model for a histopathologic classification of EPA and GHS eye irritants.

The purpose of this study was to develop Globally Harmonized System (GHS) and U.S. Environmental Protection Agency (EPA) prediction models for classifying irritant materials based on histopathologic in vitro depth of injury (DoI) measurements. Sixteen different materials were selected, representing all classes of toxicity, according to the GHS and EPA classification systems. Food-source rabbit eyes, similar to eyes used for the widely accepted Bovine Corneal Opacity and Permeability and Isolated Chicken Eye ocular irritation tests, were used. Tissues were exposed to test material for 1 min, and corneas were collected at 3- and 24-hours post-exposure. Tissues were then fixed and processed for live/dead biomarker fluorescent staining using phalloidin. DoI was then measured, and the percent DoI values for the epithelium and stroma were compared to the EPA and GHS classifications. Excluding surfactants, EPA nonclassified (category IV) materials showed no stromal and very slight epithelial damage (≤10%) to the cornea, whereas EPA corrosive (category I) materials showed significantly greater damage (P < 0.001), ranging from 39% to 100% of the stromal depth. Importantly, EPA reversible (categories II and III) materials showed significant damage to the epithelium (>10%, P < 0.005) but significantly less severe damage to the corneal stroma (P < 0.001), ranging from 1% to 38% of the stromal depth. GHS nonclassified (category NC) irritants caused damage to the epithelium but not to the stroma. All GHS class 2 materials showed damage to the stroma (1-11%), whereas GHS corrosives caused significantly greater damage to the stroma (38-100%; P < 0.001). Additionally, one corrosive material, which produced a stromal DoI of 99% at 24 h, produced no apparent damage at 3-hours post-exposure. Based on these findings, histopathologic EPA and GHS prediction models are proposed that appear to separate and identify reversible irritants from other irritant classes. Furthermore, GHS classification appears to require stromal damage, whereas NC materials may or may not damage the corneal epithelium.

Reprint of "CON4EI: Selection of the reference chemicals for hazard identification and labelling of eye irritating chemicals".

Assessment of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. In the past, several prospective and retrospective validation studies have taken place in the area of serious eye damage/eye irritation testing. Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. A very important aspect to ensure development of successful alternative test methods and/or strategies for serious eye damage/eye irritation testing is the selection of appropriate reference chemicals. A set of 80 reference chemicals was selected for the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project, in collaboration with Cosmetics Europe, from the Draize Reference Database published by Cosmetics Europe based on key criteria that were set in their paper (e.g. balanced by important driver of classification and physical state). The most important goals of the CON4EI project were to identify the performance of eight in vitro alternative tests in terms of driver of classification and to identify similarities/differences between the methods in order the build a successful testing strategy that can discriminate between all UN GHS categories. This paper provides background on selection of the test chemicals.

CON4EI: Development of testing strategies for hazard identification and labelling for serious eye damage and eye irritation of chemicals.

Assessment of acute eye irritation potential is part of the international regulatory requirements for safety testing of chemicals. In the last decades, many efforts have been made in the search for alternative methods to replace the regulatory in vivo Draize rabbit eye test (OECD TG 405). Success in terms of complete replacement of the regulatory in vivo Draize rabbit eye test has not yet been achieved. The main objective of the CEFIC-LRI-AIMT6-VITO CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project was to develop tiered testing strategies for serious eye damage and eye irritation assessment that can lead to complete replacement of OECD TG 405. A set of 80 reference chemicals (e.g. balanced by important driver of classification and physical state), was tested with seven test methods. Based on the results of this project, three different strategies were suggested. We have provided a standalone (EpiOcular ET-50), a two-tiered and three-tiered strategy, that can be used to distinguish between Cat 1 and Cat 2 chemicals and chemicals that do not require classification (No Cat). The two-tiered and three-tiered strategies use an RhCE test method (EpiOcular EIT or SkinEthic™ EIT) at the bottom (identification No Cat) in combination with the BCOP LLBO (two-tiered strategy) or BCOP OP-KIT and SMI (three-tiered strategy) at the top (identification Cat 1). For our proposed strategies, 71.1% - 82.9% Cat 1, 64.2% - 68.5% Cat 2 and ≥80% No Cat chemicals were correctly identified. Also, similar results were obtained for the Top-Down and Bottom-Up approach.

Controllable continuous sub-tenon drug delivery of dexamethasone disodium phosphate to ocular posterior segment in rabbit.

Corticosteroids have been used for treatment of posterior segment eye diseases, but the delivery of drug to the posterior segments is still a problem to resolve. In our study, we explore the feasibility of Sub-tenon's Controllable Continuous Drug Delivery to ocular posterior segment. Controllable continuous sub-tenon drug delivery (CCSDD) system, intravenous injections (IV) and sub-conjunctival injections (SC) were used to deliver dexamethasone disodium phosphate (DEXP) in rabbits, the dexamethasone concentration was measured in the ocular posterior segment tissue by Shimadzu LC-MS 2010 system at different time points in 24 h after first dose injection. Levels of dexamethasone were significantly higher at 12, 24 h in CCSDD than two other approaches, and at 3, 6 h in CCSDD than IV in vitreous body (p < 0.01); at 6, 12, 24 h in CCSDD than two other approaches, and at 1, 3 h in CCSDD than IV in retinal/choroidal compound (p < 0.01); at 3, 6, 12, 24 h in CCSDD than two other approaches, and at 1 h in CCSDD than IV in sclera (p < 0.05). The AUC0-24 in CCSDD group is higher than two other groups in all ocular posterior segment tissue. Our results demonstrated that dexamethasone concentration could be sustained moderately higher in the posterior segment by CCSDD than SC and IV, indicating that CCSDD might be a therapeutic alternative to treat a variety of intractable posterior segment diseases.

Dexamethasone distribution characteristic following controllable continuous sub-tenon drug delivery in rabbit.

Drug delivery systems are required to be safe, minimally invasive and effectively delivery drug to the target tissues. But delivery drugs to the eye has not yet satisfied this need. Here, we focused on examining the distribution of dexamethasone (DEX) in ocular and plasmic samples following controllable continuous sub-Tenon drug delivery (CCSDD) of dexamethasone disodium phosphate (DEXP) in rabbit, and to compare that with two traditional routes: subconjunctival injection and intravenous injection. The DEX concentration was analyzed by Shimadzu LC-MS 2010 system. In CCSDD group, during observed 24 h, the mean DEX level in collected samples from highest to lowest following in order: sclera, cornea, retina/choroid, iris, plasma, aqueous humor, lens and vitreous body. In ocular solid tissue, the DEX level in posterior segment is higher than in anatomic corresponding anterior segment, but it is opposite in ocular fluid tissue. High levels of DEX were maintained at 12 h in the ocular tissue immediately after the administration. Even at 24 h, the mean DEX concentration was 31.72 ng/ml and 22.40 ng/ml in aqueous and vitreous, respectively. In CCSDD group, the ocular DEX exposure (AUC0-24) is much higher and plasma exposure is much less than IV group, and it is also similar in SC group except iris. The amount of DEX levels are markedly increased in ocular tissues but it yield lower plasma levels indicating reduction of systemic absorption by CCSDD. Thus, CCSDD is an effective method of delivering DEX into anterior and posterior segment of the eye.