Eye Irritation Test Research Articles

Multifunctional Nanocomposites Based on Liposomes and Layered Double Hydroxides Conjugated with Glycylsarcosine for Efficient Topical Drug Delivery to the Posterior Segment of the Eye.

To achieve efficient drug delivery to the posterior segment of the eye via topical instillation, novel multifunctional nanocomposites were prepared by hybridizing dexamethasone disodium phosphate (DEXP)-loaded liposome (LP) glycylsarcosine (GS)-anchored layered double hydroxides (named DEXP-HSPC@LDH-GS) and then fully characterized. The nanocomposites exhibited sustained-release performance as well as prolonged precorneal retention ability. MTT assays showed that the nanocomposites were not cytotoxic to both human corneal epithelial cells (HCEpiC) and human conjunctival epithelial cells (HConEpiC) at an LDH concentration of 100 μg/mL. The DEXP-HSPC@LDH-GS nanocomposites showed superior in vitro permeability on the HConEpiC-cell-based model. In the case of HConEpiC cells, both clathrin-mediated endocytosis and active transport by the peptide transporter-1 (PepT-1) were involved in the internalization of the nanocomposites. Fluorescent images of frozen sections of ocular tissues suggested that the possible route for the delivery of doxorubicin hydrochloride (DOX)-labeled nanocomposites from the ocular surface to the back of the eye was a non-corneal pathway. Furthermore, in rabbit eyes, the hybrid nanocomposites displayed markedly higher drug concentration in choroid-retina tissue than other single nanocarriers, such as LPs and LDH. Besides, the results of the eye irritancy test showed that nanocomposite eye drops can be classified as nonirritant, which are suitable to be used as eye drops. In a word, multifunctional nanocomposites based on LPs and LDH could be used as promising vehicles for efficient noninvasive drug delivery to the posterior segment of the eye.

Investigating the potential of carboxymethyl pullulan for protecting the rabbit eye from systematically induced precorneal tear film damage

The present investigation was aimed to develop a rabbit model for protecting the rabbit eye from systematically induced precorneal tear film (PTF) damage, evaluation of carboxymethyl pullulan for its protective action against PTF damage and its curative potential. For the same, pullulan was modified by carboxymethylation and structural modification was confirmed by spectral attributes. Further, the carboxymethyl pullulan (CMP) solutions (0.1–2.0%, w/v) were evaluated for their physical properties and its concentration 1.5% (w/v) was found to fit the criterion to prepare an eye solution. The safety and non-toxicity of CMP (1.5%, w/v) eye solution was confirmed by HET-CAM method and rabbit eye irritation test. Further, a systematic rabbit eye model was developed that mimic PTF damage in day to day life. Therefore, three levels of PTF damage were developed equating symptoms of damage due to high temperature (level I) or long term mobile use (level II) or heavy air pollution (level III). Thus, a representative model with benzalkonium chloride (BAC, 0.1% v/v, 0.2% v/v and 0.3% v/v), administered two drops twice a day for two days to develop level I, level II and level III eye damage. The CMP (1.5%, w/v) eye solution possessed a protective potential against level I and II PTF damage. The rabbit eyes remained unharmed and comparable with the normal control during the complete experimental period. Additionally, CMP (1.5%, w/v) eye solution has shown early fast recovery (8 days) from PTF damage induced by instillation of PTF damage agent (BAC). Carboxymethyl pullulan eye protective solution has normalized the tear film stability in rabbit eye model. It is established from the present work that, carboxymethyl pullulan has protective action against precorneal tear film damage and it potentiates the early recovery too.

Preparation and Evaluation of Topically Applied Azithromycin Based on Sodium Hyaluronate in Treatment of Conjunctivitis.

Azithromycin (AZI) eye drops containing sodium hyaluronate (SH) were developed to improve the bioavailability of AZI. Interaction between AZI and SH in the AZI-SH formulation was investigated by differential scanning calorimetry, X-ray diffraction, and 1H-nuclear magnetic resonance spectroscopy analyses. Moreover, advantages of using SH as an excipient were investigated by comparing physiological properties and pharmacokinetic behaviors of SH-containing AZI eye drops with that of hydroxypropyl methylcellulose (HPMC)-containing formulation. In addition, safety of the developed AZI-SH eye drops was evaluated by in vitro 3-(4,5-dimethyl-2-Thiazyl)-2, 5-diphenyl-2H-tetrazolium bromide assay (MTT assay) and neutral red uptake assay as well as in vivo eye irritation test and acute toxicity test. The results indicated that AZI formed a complex with SH under a slightly acidic condition. The area under the curve (AUC) of AZI in SH-containing formulation was 1.58-fold higher (P < 0.01) than that in HPMC-containing formulation due to the interaction between the amine group of AZI and the carboxyl group of SH, despite of the higher viscosity of HPMC-containing formulation. Safety evaluation showed that AZI-SH eye drops caused no obvious eye irritation and acute toxicity. In conclusion, the developed SH-containing AZI formulation possessing advantages of longer retention time and higher drug availability was a promising drug formulation for topical ocular therapy.

Microfluidic Devices for Eye Irritation Tests of Cosmetics and Cosmetic Ingredients

In this paper, we describe the development and application of a simple microfluidic device for in vitro irritation tests of cosmetics. The device was fabricated with a three-compartment diffusion system to mimic a hen’s egg test-chorioallantoic membrane (HET-CAM) system. Human umbilical vein endothelial cells were cultured in the three compartments, and the tested substances were injected into the compartment, and then were drawn through the small microfluidic channels. The mortalities of cells in each compartment were monitored using a fluorescent microscope. The TS (toxicity score) values were evaluated on the basis of the mortalities of cells as a function of time when the cells were exposed to the test substances. Four kinds of cosmetic materials were tested with the microfluidic system, and the results were compared with IS (irritation score) values of a HET-CAM test to validate the in vitro irritation test system. The method developed in this study could be used for evaluation of the ocular toxicity of cosmetic ingredients, and could be widely used as an alternative test for cosmetic and medical tests or for the development of new medicines.

Self-assembled hexagonal liquid crystalline gels as novel ocular formulation with enhanced topical delivery of pilocarpine nitrate

The aim of this paper was to develop hexagonal liquid crystalline (HII) gels that can be used as a novel ocular delivery system for pilocarpine nitrate (PN). HII gels were prepared by a vortex method using phytantriol/triglyceride/water (71.15: 3.85: 26, w/w) ternary system. The gels were characterized by crossed polarized light microscopy, small-angle X-ray scattering, differential scanning calorimetry and rheology. And, in vitro drug release behavior and ex vivo corneal permeation were investigated. Finally, preocular residence time evaluation, eye irritation test, histological examination and miotic tests were studied in vivo and compared with carbopol gel. Based on various characterization techniques, the inner structure of the gels were HII mesophase and exhibited a pseudoplastic fluid behaviour. In vitro release results revealed that PN could be released continuously from HII gel over a period of 24 h. The ex vivo apparent permeability coefficient of HII gel was 3.15-fold (P < 0.01) higher than that of the Carbopol gel. Compared with Carbopol gel, HII gel displayed longer residence time on the eyeballs surface using fluorescent labeling technology. Furthermore, the HII gel caused no ocular irritation was estimated by corneal hydration levels, Draize test and histological inspection. Additionally, in vivo miotic study showed that HII gel had a remarkably long-lasting decrease in the pupil diameter of rabbits. In conclusion, HII gels would be a promising sustained-release formulation for ocular drug delivery.

United States regulatory requirements for skin and eye irritation testing

Purpose: Eye and skin irritation test data are required or considered by chemical regulation authorities in the United States to develop product hazard labelling and/or to assess risks for exposure to skin- and eye-irritating chemicals. The combination of animal welfare concerns and interest in implementing methods with greater human relevance has led to the development of non-animal skin- and eye-irritation test methods. To identify opportunities for regulatory uses of non-animal replacements for skin and eye irritation tests, the needs and uses for these types of test data at U.S. regulatory and research agencies must first be clarified.Methods: We surveyed regulatory and non-regulatory testing needs of U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) agencies for skin and eye irritation testing data. Information reviewed includes the type of skin and eye irritation data required by each agency and the associated decision context: hazard classification, potency classification, or risk assessment; the preferred tests; and whether alternative or non-animal tests are acceptable. Information on the specific information needed from non-animal test methods also was collected.Results: A common theme across U.S. agencies is the willingness to consider non-animal or alternative test methods. Sponsors are encouraged to consult with the relevant agency in designing their testing program to discuss the use and acceptance of alternative methods for local skin and eye irritation testing.Conclusions: To advance the implementation of alternative testing methods, a dialog on the confidence of these methods to protect public health and the environment must be undertaken at all levels.

Me-too validation study for in vitro eye irritation test with 3D-reconstructed human cornea epithelium, MCTT HCETM

The need for in vitro eye irritation test replacing in vivo is steadily increasing. The MCTT HCE™ eye irritation test (EIT) using 3D reconstructed human cornea-like epithelium, was developed to identify ocular irritants from non-irritants those that are not requiring classification and labelling for eye irritation. Here, we report the results of me-too validation study, which was conducted to evaluate the reliability and relevance of the MCTT HCETM EIT, according to performance standards (PS) of OECD TG 492. The optimal cutoff to determine irritation in the prediction model was preliminarily established at 45% with the receiver operation characteristics (ROC) curve for 141 reference substances. To demonstrate the reproducibility of within- and between-laboratory (WLR and BLR), a set of 30 PS reference chemicals were tested in three laboratories three times. The WLR and BLR concordance with the binary decision of whether non-irritant or irritant was estimated to be 90–100% and 90%, respectively, and both met the PS requirements. The predictive capacity of the respective laboratories for the 30 reference chemicals were evaluated based on three different estimation methods, and the results were comparable, with sensitivity ranging from 89.6 to 93.3%, the specificity ranging from 62.2 to 66.7%, and the accuracy ranging from 75.9 to 80.0%. Additional test with the new set of 30 PS substances in the revised OECD GD 216 yielded a performance of sensitivity ranging from 92.6–93.3%, the specificity 62.2–66.7% and the accuracy 77.4–80.0%. 95.0% sensitivity, 67.2% specificity, and 83.0% accuracy were obtained for 141 reference substances in total. Furthermore, separate cutoffs for liquids and solids, 35% and 60%, respectively, produced better predictivity, which was established as a final prediction model. Collectively, our study demonstrated that MCTT HCETM EIT meets the reproducibility and predictivity criteria stated in OECD TG 492 PS

Safety and Tolerability of Overdosed Artificial Tears by Abraded Rabbit Corneas.

Purpose: Preservative-free cationic emulsion-based artificial tear (AT) is an innovative eye drop based on the Novasorb® technology with cetalkonium chloride (CKC) as the cationic agent. The cationic emulsion Cationorm is designed for the management of mild-to-moderate dry eye disease (DED) patients that present cornea epithelium alterations. The aim of the present study was to evaluate the safety and tolerability of overdosed ATs by altered corneal epithelium in vivo and assess the usefulness of the ex vivo eye irritation test (EVEIT) as a predictive alternate toxicity test method.Methods: The experimental procedure, treatment duration, and instillation frequency closely mimic in vivo the ex vivo protocol described by Pinheiro et al. and discussed in the Discussion and Conclusion section of this article. Two to 3-month-old female New Zealand white rabbits, n = 6 per group, were treated with ATs (21 instillations/day over 3 days) following corneal abrasion. Corneal fluorescein staining, in vivo confocal microscopy (IVCM), and slit lamp examinations were performed to assess corneal epithelium recovery and the ocular tolerability of the overdosed ATs.Results: All abraded eyes experienced almost complete epithelium recovery within 3 days following treatments with Cationorm, Optive, Vismed, and Saline. Benzalkonium chloride (BAK, 0.02%) treatment resulted in 82.4% reepithelialization. IVCM data illustrated corneal epithelium normal recovery. Acute local tolerability of the overdosed ATs was confirmed using Draize and McDonald–Shadduck's test scales.Conclusions: The different ATs were demonstrated to be well tolerated by abraded corneas in vivo, and the extreme overdosing regimen did not hamper the wound healing process of the rabbit eye in comparison to saline. These data did not confirm the ones obtained with the nonvalidated ex vivo eye irritation test.

SkinEthic™ HCE Eye Irritation Test: Similar performance demonstrated after long distance shipment and extended storage conditions

Assessment of ocular irritation risk is an international regulatory requirement in the safety evaluation of products. In response to this need, L’Oréal developed the SkinEthic™ Human Corneal Epithelium (HCE) Eye Irritation Test (EIT) that has been included in OECD Test Guideline 492. SkinEthic™ HCE EIT is able to correctly and reliably identify chemicals not requiring classification versus labelling for eye irritation or serious eye damage according to UN GHS. In an effort to promote its global use, the performance of the method was evaluated after long-distance shipment and compared to European shipment conditions. Results obtained by Cosmos Technical Center (Japan) after extended tissues transit were compared to results obtained in L’Oréal (France). Thirty-nine out of 40 blinded chemicals, representing different functional chemical classes, were consistently classified in both laboratories. The SkinEthic™ HCE EIT test method was also evaluated for its performance after extended storage of the tissues. The performance was in agreement with the values reported in OECD TG 492, with an overall accuracy of 87.1% (based on 119 chemicals), sensitivity of 95.5% and specificity of 73.5%. The reliability and relevance of SkinEthic™ HCE EIT test method after long-distance shipment and extended storage remain in agreement with regulatory validation criteria.

A simplified numerical scoring system to quantify skin and eye irritation/corrosion

In 1944, Draize et al., published a paper entitled “Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes”. The Organization for Economic Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.

Acute toxicity study of a polysaccharide based hydrogel from linseed for potential use in drug delivery system

ABSTRACT Linseed hydrogel (LSH) was evaluated by acute toxicity for its potential application in oral drug delivery design. White albino mice and rabbits were divided in four groups (I-IV) and different doses of LSH (1, 2 and 5 g/kg body weight) were given except to the control group (I) that was left untreated. Rabbits were monitored for eye irritation, acute dermal toxicity and primary dermal irritation, whereas, body weight, food and water consumption, hematology and clinical biochemistry, gross necropsy and histopathology of vital organs were scrutinized in mice. LSH was considered safe after eye irritation test as no adverse signs or symptoms were seen in the eye. In dermal toxicity and irritation study, skin of treated rabbits was found normal in color without any edema or erythema. After oral administration, there was no sign of any abnormalities in treated group animals (II-IV). The hematology and clinical biochemistry of treated group animals was comparable with the control group. Histopathology of vital organs has not shown any lesion or abnormalities. In the light of these outcomes, it can be concluded that LSH is not a hazardous biomaterial and could be incorporated as an excipient in oral and dermal preparations.

Development and evaluation of extended release ciprofloxacin HCl ocular inserts employing natural and synthetic film forming agents

In this study, solvent casting technique was employed in fabricating ocular inserts using Pullulan as a natural film forming agent and Hydroxy propyl methyl cellulose as a synthetic film forming agent. Ciprofloxacin hydrochloride—an effective ocular antibiotic- was employed as a model drug in this study. Further modification of ciprofloxacin release from the ocuserts is obtained via coating the dual sides of the ocuserts by hydroxy ethyl cellulose as release controlling membrane. The prepared ocuserts were evaluated for various physiochemical parameters, and the in-vitro release pattern. Furthermore, accelerated stability test, sterility test, microbiological studies as well as eye irritancy test were appraised for a selected formula. All the developed ocular inserts showed that as the rate controlling membrane increased in percentage, the thickness and weight of sealed ocuserts increased. The drug content varied between 85.4 ± 16.5–98.2 ± 27.4% and the drug release followed zero-order pattern. The selected formula showed the highest sustainment effect indicated by the least release rate (16.3%/h) containing pullulan (2.0%) and dual coat of hydroxy ethyl cellulose (6.0%). Pullulan based ocular insert remained stable and intact at accelerated conditions for 30 days with overall degradation of the drug less than 2.5%. Non-irritant response in rabbits with primary irritation indices score 0 on Peyman Scale and excellent microbiological activity represented by a greater zone of inhibition (5.6 cm) compared to an ocuserts with no drug (0.8 cm) against Staphylococcus aureus. This study showed the suitability of dual sided coated Pullulan ocusert in attaining the desired sustainment effect with excellent stability.

Discovery of Ezrin Expression as a Potential Biomarker for Chemically Induced Ocular Irritation Using Human Corneal Epithelium Cell Line and a Reconstructed Human Cornea-like Epithelium Model.

Numerous studies have attempted to develop a new in vitro eye irritation test (EIT). To obtain more reliable results from EIT, potential new biomarkers that reflect eye irritation by chemicals must be identified. We investigated candidate biomarkers for eye irritation, using a proteomics approach. Sodium lauryl sulfate (SLS) or benzalkonium chloride (BAC) was applied on a reconstructed human cornea-like epithelium model, MCTT HCE, and corneal protein expression was examined by two-dimensional gel electrophoresis. We found that ezrin (EZR) was significantly upregulated by SLS or BAC. In addition, upregulation of EZR in immortalized human corneal cells treated with SLS or BAC was confirmed by quantitative reverse transcription-PCR and western blot analysis. Furthermore, other well-known eye irritants such as cetylpyridinium bromide, Triton X-100, cyclohexanol, ethanol, 2-methyl-1-pentanol, and sodium hydroxide significantly increased EZR expression in immortalized human corneal cells. Induction of EZR promoter activity in irritant-treated human corneal cells was confirmed by a luciferase gene reporter assay. In conclusion, EZR expression may be a potential biomarker for detecting eye irritation, which may substantially improve the performance of in vitro EIT.

The Ex vivo Eye Irritation Test (EVEIT) model as a mean of improving venom ophthalmia understanding

Snakes belonging to the genus Naja (Elapid family), also known as “spitting cobras”, can spit venom towards the eyes of the predator as a defensive strategy, causing painful and potentially blinding ocular envenoming. Venom ophthalmia is characterized by pain, hyperemia, blepharitis, blepharospasm and corneal erosions. Elapid venom ophthalmia is not well documented and no specific treatment exists. Furthermore, accidental ejection of venom by non-spitting vipers, as Bothrops, also occurs. The Ex vivo Eye Irritation Test model (EVEIT) has enabled important progress in the knowledge of chemical ocular burns. Considering the lack of experimental animal model, we adapted the EVEIT to study venom ophthalmia mechanisms. Ex vivo rabbit corneas were exposed to venoms from spitting (Naja mossambica, Naja nigricollis) and non-spitting (Naja naja, Bothrops jararaca and Bothrops lanceolatus) snakes, and rinsed or not with water. The corneal thickness and the depth of damage were assessed using high-resolution optical coherence tomography (HR-OCT) imaging and histological analysis. All Naja venoms induced significant corneal edema, collagen structure disorganization and epithelial necrosis. Corneas envenomed by African N. mossambica and N. nigricollis venoms were completely opaque. Opacification was not observed in corneas treated with venoms from non-spitting snakes, such as the Asian cobra, N. naja, and the vipers, B. jararaca and B. lanceolatus. Moreover, Bothrops venoms were able to damage the epithelium and cause collagen structure disorganization, but not edema. Immediate water rinsing improved corneal status, though damage and edema could still be observed. In conclusion, the present study shows that the EVEIT model was successfully adapted to set a new experimental ex vivo animal model of ophthalmia, caused by snake venoms, which will enable to explore new therapies for venom ophthalmia.

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.

Safety profile of silver sulfadiazine-bFGF-loaded hydrogel for partial thickness burn wounds

In the present investigation, the safety of novel combinational silver sulfadiazine-bFGF-loaded hydrogel was assured by performing acute skin irritation, sensitization, acute dermal toxicity, and eye irritation in compliance with the Organization for Economic Co-operation and Development guidelines. In the skin irritation study, placebo, test, and positive control (0.8% w/v aqueous solution of formaldehyde) were applied on New Zealand rabbits and monitored for abnormal skin responses including erythema and edema. The placebo and test formulation did not induce any adverse reactions and were classified as nonirritating materials. In the skin sensitization test, guinea pigs were sensitized by positive control (0.1% w/v 1-chloro-2,4-dinitrobenzene in 10% of propylene glycol as a standard skin sensitizing agent), placebo, and test formulations. Weak sensitization was observed in the placebo and test formulation treated groups. Additionally, acute dermal toxicity test was performed in Wistar rats, where no signs of toxicity were observed in biochemical, hematological, and histopathological studies. Moreover, the acute eye irritation test was carried out in rabbits and no abnormal clinical signs were evident in the cornea or iris. As a whole, these findings suggest that the hydrogel formulation does not cause any skin irritation, skin sensitizationand dermal toxic effects, and eye irritation following dermal and ocular applications, respectively. Therefore, all the findings obtained from this preclinical study indicated that this hydrogel formulation is nontoxic and safe for use in animal models.

Ezrin as a complementary marker in ocular toxicity assessment using a three-dimensional reconstructed human corneal-like epithelium model, EpiOcular™

IntroductionA variety of in vitro tests to replace the Draize test have been developed; however, there is no available method for assessing the full spectrum of Globally Harmonized System (GHS) categories. Human cornea-like three-dimensional (3D) reconstructed tissue models are the most promising in vitro systems. The objective of this study was to evaluate the ocular toxicity of 11 test substances using the EpiOcular™ model after performing proficiency tests. We further evaluated the effectiveness of ezrin staining as a complementary marker in histological analysis to overcome the limitation of eye irritation tests using 3D reconstructed human corneal epithelium models. MethodsThe assessment of ocular toxicity was performed by the suggested OECD TG 492 procedure. After treatment with proficiency test chemicals and 10 test substances, EpiOcular™ tissue models were stained with hematoxylin and eosin and ezrin, and the histological changes were observed by immunofluorescence microscopy. ResultsThe ocular toxicity assessment of 10 test chemicals using the EpiOcular™ eye irritation test were in accordance with the UN GHS classification of test chemicals. Histological analysis of ezrin staining showed that the cell membranes of models treated with 10 out of 11 non-irritant chemicals were maintained, whereas those of models treated with 14 eye irritant substances resulted in the apparent translocation of ezrins from the cell membrane to the cytoplasm or nucleus by destruction of cell membrane. DiscussionEzrin may be used as a complementary marker to more accurately assess ocular toxicity using 3D reconstructed human cornea-like epithelium models.

Sustained release timolol maleate loaded ocusert based on biopolymer composite

In the present investigation, the effect of timolol maleate loaded ocuserts was studied as an alternative for conventional anti-glaucoma formulation. Ocuserts were prepared using natural polymer sodium alginate and ethyl cellulose. Physico-chemical properties along with drug entrapment efficiency (94–98%), content uniformity (93.1% ± 0.264–98.00% ± 0.321), in vitro drug release (83.42% ± 0.35 at end of 12 h), ex vivo permeation all showed satisfactory results, which was found to follow zero order kinetics. Ex vivo permeation studies showed better results, revealed that the permeability coefficient was dependent on polymer type. The sterility test accelerated stability studies and in vivo studies such as eye irritancy test, in vivo drug release of the optimized ocusert was determined. The anti-glaucoma activity was measured using Schiotz tonometer at different time interval. Significant reduction in Intra ocular pressure (IOP) within 3 days was observed in case of rabbits treated with ocusert in comparison to the rabbit treated with marketed eye drop formulation. Hence timolol maleate loaded ocuserts proved to be a promising and viable alternative over conventional eye formulation for the sustained and controlled ophthalmic drug delivery, targeting the drug within the ocular globe thus improving patient compliance for the treatment of glaucoma.

Polyherbal formulation containing antioxidants may serve as a prophylactic measure to diabetic cataract: Preclinical investigations in rat model

Background: Cataract is a major cause of visual impairment in diabetic patients. Due to increasing numbers of type 1 and type 2 diabetics worldwide, the incidence of diabetic cataracts steadily rises. Cataract surgery is the best possible cure for patients suffering from this ailment. However, the elucidation of pathomechanisms to delay or prevent the development of cataract in diabetic patients remains a challenge. Objective: The aim of the present study was to develop a polyherbal eyedrops containing potent antioxidant herbal extract and study the effectiveness as prophylactic treatment against galactose-induced diabetic cataract. Materials and Methods: Formulations were prepared by using extracts of Ginkgo biloba leaves, beet root (Beta vulgaris), and amla (Emblica officinalis) fruits. The viscosity enhancers were used to increase the retention time. Formulations (F1–F5) were prepared by using carboxy methyl cellulose and poly ethylene glycol-400 as thickening agents and propylene glycol as a solubilizer. Preliminary evaluation showed that formulations have passed clarity, sterility, and eye irritancy tests. Viscosity and pH of formulation were within the normal range. Diabetic cataract was induced in Wistar rats by 10% galactose drink (for 30 days) and anticataract activity was evaluated. Formulation was installed in eye as a prophylactic treatment from day 1 of galactose drinking and continued for 30 days. Results: Slit-lamp photography of eyes of rats showed clear lens of rats without any trace of opacity. On the other hand, galactose-treated rats developed dense nuclear opacity in lens as an indication of diabetic cataract. Rats which received prophylactic treatment showed less percent opacity as compared to that of cataract control group and decreased vacuoles. Conclusion: We may conclude that polyherbal formulation containing extracts of Ginkgo biloba leaves, beet root (Beta Vulgaris), and amla (Emblica officinalis) fruits may prevent the development of cataract in diabetic patients. Abbreviations used: ARI: Aldose reductase inhibitors; GPx: Glutathione peroxidase; GR: Glutathione reductase; DTNB: Dithio-bis-nitrobenzoic acid; GSH: Thiol.

Nervonoylceramide (C24:1Cer), a lipid biomarker for ocular irritants released from the 3D reconstructed human cornea-like epithelium, MCTT HCE™

Due to invasive and painful procedures during in vivo rabbit eye irritation test, in vitro alternative methods have been widely investigated. Recently, 3D reconstructed human cornea-like epitheliums (RhCEs) garner a huge attention. RhCEs employ the tissue viability as a primary endpoint to determine ocular irritancy but additional biomarkers may improve its predictive capacity. Here, we explored lipid biomarkers for ocular irritants in MCTT HCE™ RhCE model. Three irritants; sodium lauryl sulfate, benzalkonium chloride and triton X-100 were selected to represent anionic, cationic and non-ionic detergent respectively. After treating MCTT HCE™ with irritants, the alteration of lipids in the treated tissues was examined with Nile Red staining, which revealed the depletion of corneal lipids. We further quantitated the release of ceramides and free fatty acids, major lipid components of cornea, into the medium during the post-treatment incubation, employing a sensitive UPLC-MS/MS method. Among 44 lipid species, nervonoylceramide (C24:1Cer) was found to be released commonly by all three irritants in a concentration-dependent manner. Tests with 10 additional reference substances further supported that C24:1Cer release was significantly correlated with viability. Examination of the genes involved in the biosynthetic pathway for C24:1Cer revealed that stearoylCoA desaturase (SCD) and elongase1 (ELOVL1) were upregulated, suggesting that lipids and related genes may be employed as biomarkers for ocular irritants.