In vitro and in vivo studies of ocular topically administered NLC for the treatment of uveal melanoma.

Uveal melanoma is one of the most common and aggressive intraocular malignancies, and, due to its great capability of metastasize, it constitutes the most incident intraocular tumor in adults. However, to date there is no effective treatment since achieving the inner ocular tissues still constitutes one of the greatest challenges in actual medicine, because of the complex structure and barriers. Uncoated and PEGylated nanostructured lipid carriers were developed to achieve physico-chemical properties (mean particle size, homogeneity, zeta potential, pH and osmolality) compatible for the ophthalmic administration of (S)-(-)-MRJF22, a new custom-synthetized prodrug for the potential treatment of uveal melanoma. The colloidal physical stability was investigated at different temperatures by Turbiscan® Ageing Station. Morphology analysis and mucoadhesive studies highlighted the presence of small particles suitable to be topically administered on the ocular surface. In vitro release studies performed using Franz diffusion cells demonstrated that the systems were able to provide a slow and prolonged prodrug release. In vitro cytotoxicity test on Human Corneal Epithelium and Human Uveal Melanoma cell lines and Hen's egg-chorioallantoic membrane test showed a dose-dependent cytotoxic effect of the free prodrug on corneal cells, whose cytocompatibility improved when encapsulated into nanoparticles, as also confirmed by in vivo studies on New Zealand albino rabbits. Antiangiogenic capability and preventive anti-inflammatory properties were also investigated on embryonated eggs and rabbits, respectively. Furthermore, preliminary in vivo biodistribution images of fluorescent nanoparticles after topical instillation in rabbits' eyes, suggested their ability to reach the posterior segment of the eye, as a promising strategy for the treatment of choroidal uveal melanoma.

Ocular Delivery of Bimatoprost-Loaded Solid Lipid Nanoparticles for Effective Management of Glaucoma.

Glaucoma is a progressive optic neuropathy characterized by a rise in the intraocular pressure (IOP) leading to optic nerve damage. Bimatoprost is a prostaglandin analogue used to reduce the elevated IOP in patients with glaucoma. The currently available dosage forms for Bimatoprost suffer from relatively low ocular bioavailability. The objective of this study was to fabricate and optimize solid lipid nanoparticles (SLNs) containing Bimatoprost for ocular administration for the management of glaucoma. Bimatoprost-loaded SLNs were fabricated by solvent evaporation/ultrasonication technique. Glyceryl Monostearate (GMS) was adopted as solid lipid and poloxamer 407 as surfactant. Optimization of SLNs was conducted by central composite design. The optimized formulation was assessed for average particle size, entrapment efficiency (%), zeta potential, surface morphology, drug release study, sterility test, isotonicity test, Hen's egg test-chorioallantoic membrane (HET-CAM) test and histopathology studies. The optimized Bimatoprost-loaded SLNs formulation had an average size of 183.3 ± 13.3 nm, zeta potential of -9.96 ± 1.2 mV, and encapsulation efficiency percentage of 71.8 ± 1.1%. Transmission electron microscopy (TEM) study revealed the nearly smooth surface of formulated particles with a nano-scale size range. In addition, SLNs significantly sustained Bimatoprost release for up to 12 h, compared to free drug (p < 005). Most importantly, HET-CAM test nullified the irritancy of the formulation was verified its tolerability upon ocular use, as manifested by a significant reduction in mean irritation score, compared to positive control (1% sodium dodecyl sulfate; p < 0.001). Histopathology study inferred the absence of any signs of cornea tissue damage upon treatment with Bimatoprost optimized formulation. Collectively, it was concluded that SLNs might represent a viable vehicle for enhancing the corneal permeation and ocular bioavailability of Bimatoprost for the management of glaucoma.

In-vitro and in-vivo evaluation of biocompatible polymeric microgels for pH- driven delivery of Ketorolac tromethamine.

The aim of the current study was to prepare glutamic acid crosslinked poly(itaconic acid/methacrylic acid) microgels for pH-responsive delivery of ketorolac tromethamine, using aqueous free radical polymerization technique. The polymerization of polymer with monomers was carried out by a crosslinking agent N', N'-methylene bisacrylamide in the presence of initiator ammonium persulfate. The prepared microgels were characterized for structure, surface morphology, thermal stability, and crystallinity. Similarly, studies such as sol-gel analysis, drug loading, and polymer volume fraction were performed for the fabricated microgels. The pH-sensitivity of the developed microgels was investigated at three different pH values i.e., pH 1.2, 4.6, and 7.4 by swelling and in-vitro drug release studies. Maximum swelling and drug release were found at pH 7.4 as compared to pH 1.2 and 4.6, which indicated the pH-sensitive nature of the prepared microgels. The toxicity of the prepared microgels was evaluated by cell line and HET-CAM test, which demonstrated no toxic effect of the prepared microgels. In-vivo study was carried out on rabbits and high plasma concentration was reported for the drug loaded microgels as compared to drug solution and commercial product Keten. Hence, the prepared microgel system could be employed as an excellent carrier for the controlled drug delivery system.

Preclinical assessment of nanostructured liquid crystalline particles for the management of bacterial keratitis: in vivo and pharmacokinetics study.

The research work was driven to develop, optimize, and characterize novel nanostructured liquid crystalline particles as carriers for the ocular delivery of vancomycin. The formulations were developed by fragmenting the cubic crystalline phase of glycerol monooleate, water, and poloxamer 407. A four-factor, three-level Taguchi statistical experimental design was constructed to optimize the formulation. Formulations exhibited internal-cubic structure of the vesicles with particle size in the range of 51.11 ± 0.96 nm to 158.73 ± 0.46 nm and negative zeta potential. Ex vivo transcorneal permeation studies demonstrated that the optimized cubosomes had a 2.4-fold increase in apparent permeability co-efficient as compared to vancomycin solution, whereas in vivo studies in rabbits demonstrated that the severity of keratitis was considerably lowered on day 3 with optimized cubosomes. Ocular pharmacokinetic studies evaluated the level of drug in aqueous humor, and results revealed that the time to peak concentration (Tmax) of vancomycin-loaded cubosomal formulation was about 1.9-fold higher and mean residence time was 2.2-fold greater than vancomycin solution. Furthermore, histological examination revealed that the corneal layers displayed well-maintained morphology without any stromal swelling, consequently indicating the safety of formulation. It could be concluded that the developed nanostructured liquid crystalline particles of vancomycin demonstrated improved pre-ocular residence time, increased permeability, reduced dosing frequency, controlled drug release, and reduced systemic side-effects. Results manifested that the developed vancomycin-loaded cubosomes could be a promising novel ocular carrier and an ideal substitute for conventional eye drops for the management of bacterial-keratitis.

In vitro evaluation of the cytotoxicity and eye irritation potential of preservatives widely used in cosmetics

Abstract The consumption of cosmetics has been increasing every year and is expected to reach $675 billion by 2020 at an estimated growth rate of 6.4% per year. Exposure to skin irritants is the major cause of non-immunological inflammation of the skin. Therefore, the safety evaluation of cosmetic preservatives should be increased. Thus, the present work aimed to evaluate the cytotoxicity as the viability endpoint and the eye irritation potential of preservatives used in cosmetics. Cytotoxicity assays were performed using MTT and NRU in human keratinocytes (HaCaT), human dermal fibroblasts, adult (HDFa), and human hepatoma cells (HepG2). The eye irritation potential was evaluated using the Hen's Egg Test-chorioallantoic membrane (HET-CAM). The evaluated preservatives were methylparaben (MP), propylparaben (PP), phenoxyethanol (PE), and a mixture of methylchloroisothiazolinone and methylisothiazolinone (CMI/MI). All preservatives showed cytotoxic potential within the permitted concentrations for use in cosmetic products. In the HET-CAM test, PE and CMI/MI, MP, and PP were classified as severe, moderate, and poor irritants, respectively. Our results indicate that proper safety evaluations are required to ensure the beneficial properties of preservatives on cosmetic products without exceeding exposure levels that would result in adverse health effects for consumers.

Comparative evaluation of HET-CAM and ICE methods for objective assessment of ocular irritation caused by selected pesticide products.

Eye irritation potency of pesticides (fungicides, herbicides, insecticides) was comparatively tested by HET-CAM and ICE method. Based on the results of the tests the statistical analysis of agreement between classification using individual methods was done by Goodman-Kruskal's rank correlation and determination (calculation) of Cohen's kappa coefficient. Statistical analysis of agreement between classification revealed significant correlation between results of in vivo and in HET-CAM assays (76%). There was no significant correlation between result of in vivo and in ICE methods (64%). Weakest correlation was found between the data from in vitro HET-CAM and ICE tests. The percentage of agreement between two in vitro data was 48%. They may be recommended as a part of a battery of tests to reduce experimentation on mammals and to limit or eliminate pain and injury inflicted on experimental animals. The HET-CAM test is a useful tool for studying in vivo the potential conjunctival irritation, while the ICE test can be used to study corneal irritant effects in detail.

Stimulus Responsive Ocular Gentamycin-Ferrying Chitosan Nanoparticles Hydrogel: Formulation Optimization, Ocular Safety and Antibacterial Assessment.

PURPOSE: The present study was designed to study the gentamycin (GTM)-loaded stimulus-responsive chitosan nanoparticles to treat bacterial conjunctivitis. METHODS: GTM-loaded chitosan nanoparticles (GTM-CHNPs) were prepared by ionotropic gelation method and further optimized by 3-factor and 3-level Box-Behnken design. Chitosan (A), sodium tripolyphosphate (B), and stirring speed (C) were selected as independent variables. Their effects were observed on particle size (PS as Y1), entrapment efficiency (EE as Y2), and loading capacity (LC as Y3). RESULTS: The optimized formulation showed the particle size, entrapment efficiency, and loading capacity of 135.2±3.24 nm, 60.18±1.65%, and 34.19±1.17%, respectively. The optimized gentamycin-loaded chitosan nanoparticle (GTM-CHNPopt) was further converted to the stimulus-responsive sol-gel system (using pH-sensitive carbopol 974P). GTM-CHNPopt sol-gel (NSG5) exhibited good gelling strength and sustained release (58.99±1.28% in 12h). The corneal hydration and histopathology of excised goat cornea revealed safe to the cornea. It also exhibited significant (p<0.05) higher ZOI than the marketed eye drop. CONCLUSION: The finding suggests that GTM-CHNP-based sol-gel is suitable for ocular delivery to enhance the corneal contact time and improved patient compliance.

Investigating The Retention Potential of Chitosan Nanoparticulate Gel: Design, Development, In Vitro & Ex Vivo Characterization.

INTRODUCTION: The main purpose of the research was to develop, optimize and characterize tobramycin sulphate loaded chitosan nanoparticles based gel in order to ameliorate its therapeutic efficacy, precorneal residence time, stability, targeting and to provide controlled release of the drug. METHODS: Box-Behnken design was used to optimize formulation by 3-factors (chitosan, STPP and tween 80) and 3-levels. Developed formulation was subjected for characterizations such as shape and surface morphology, zeta potential, particle size, in vitro drug release studies, entrapment efficiency of drug, visual inspection, pH, viscosity, spreadability, drug content, ex vivo transcorneal permeation studies, ocular tolerance test, antimicrobial studies, isotonicity evaluation and histopathology studies. RESULTS: Based on the evaluation parameters, the optimized formulation showed a particle size of 43.85 ± 0.86 nm and entrapment efficiency 91.56% ± 1.04, PDI 0.254. Cumulative in vitro drug release was up to 92.21% ± 1.71 for 12 hours and drug content was found between 95.36% ± 1.25 to 98.8% ± 1.34. TEM analysis unfolded spherical shape of nanoparticles. TS loaded nanoparticulate gel exhibited significantly higher transcorneal permeation as well as bioadhesion when compared with marketed formulation. Ocular tolerance was evaluated by HET-CAM test and formulation was non-irritant and well-tolerated. Histopathology studies revealed that there was no evidence of damage to the normal structure of the goat cornea. As per ICH guidelines, stability studies were conducted and were subjected for 6 months. CONCLUSION: Results revealed that the developed formulation could be an ideal substitute for conventional eye drops for the treatment of bacterial keratitis.

Photoprotective activity and increase of SPF in sunscreen formulation using lyophilized red propolis extracts from Alagoas

ABSTRACT The excessive sun exposure, coupled with lack of sun protection represents one of the biggest risks to the occurrence of skin cancer and photoaging. Recent strategies for photoprotection have included the incorporation of natural antioxidant and anti-inflammatory compounds, into sunscreens, and the oral administration of natural antioxidant extracts. In this work, we use Brazilian red propolis extract because its antioxidant and anti-inflammatory activities. The aim of this work was to evaluate the sun protection factor and antioxidant activity of different hydroalcoholic extracts of red propolis (70% and 75%) prepared from lyophilized red propolis at room and high temperatures. The sun protection factor in vitro was determined by a spectrophotometric method developed by Mansur. The hydroalcoholic extracts of red propolis incorporated with Filter UVA-UVB 5% Gel Permulem TR-1 presented absorption in the UVB region. Also, the in vitro capacity of the hydroalcoholic extracts of red propolis to increase photoprotective activity of Filter UVA-UVB 5% Gel was evaluated. The hydroalcoholic extracts of red propolis incorporated presented higher values of sun protection factor and showed synergism in the photoprotective activity of Filter UVA-UVB 5% Gel Permulem TR-1. The antioxidant activity and sun protection factor are correlated with total phenolics content of the extracts and the hydroalcoholic extract of red propolis 75% at room temperature was choosen. The formulation developed with Filter UVA-UVB 5% Gel Permulem TR-1 with this hydroalcoholic extract of red propolis showed safe to be applied on the skin according HET-CAM test. Suggests indication of hydroalcoholic extract of red propolis (75% - room temperature) associated to photoprotective formulations for use in photoprotective products.

Microemulsion-based delivery of triamcinolone acetonide to posterior segment of eye using chitosan and butter oil as permeation enhancer: an in vitro and in vivo investigation.

The aim of the study was to formulate a microemulsion (ME) using chitosan (CH) and the butter oil (BO) as a permeation enhancer for targeting drug to the posterior segment of the eye, via topical route. Triamcinolone acetonide (TA) was selected as the model drug since it undergoes extensive first-pass metabolism, leading to poor oral bioavailability of 23%. For optimisation of BO concentration, different ratios of TA:BO were prepared by simple physical mixing in the ratio of 1:9 to 9:1 and diffusion study was performed. MEs containing TA, TA:BO and TA CH ME were formulated by water titration method. Globule sizes of TA ME, TA:BO ME and TA CH ME were found to be 66.06 ± 0.32 nm, 78.52 ± 1.50 nm and 97.30 ± 2.50 nm, respectively. In ex vivo diffusion studies using goats eye, TA:BO ME (31.33 ± 0.46 and 33.98 ± 0.23) and TA CH ME (24.10 ± 0.41 and 27.00 ± 0.18) showed higher percentage of drug diffusion in comparison to TA ME (13.29 ± 0.41and 15.56 ± 0.34) and TA solution (8.20 ± 1.04 and 10.39 ± 0.22) in presence and in absence of vitreous humour. Fluorescence intensity of coumarin-6 (as a marker) loaded ME with BO and CH was found to be higher, confirming their role in altering membrane permeability and facilitating coumarin-6 diffusion to the posterior chamber. Overall, it was concluded that BO enhances the bioavailability of TA across the retina, thereby proving its potential as permeation enhancer in facilitating drug delivery to the posterior segment of the eye.

HET-CAM test. Application to shampoos in developing countries.

INTRODUCTION: The use of in vitro methods for the control of cosmetic products in developing countries must overcome cost, feasibility and an unfavourable regulatory environment. Among these products, shampoos occupy an important part of the market but present a number of risks related to their use like eye irritation. The aim of this work is to propose the HET-CAM test as a method of screening the risk of eye irritation of shampoos, adapting it to the technical and regulatory conditions of a developing country such as Algeria. MATERIAL AND METHOD: 6 locally produced shampoos (4 adults and 2 for babies) were tested at 100%, 50%, 25%, 10%, 5%, 2.5% and 1.5%. RESULTS AND DISCUSSION: The use of a 10% dilution classifies the baby and adult shampoos in two different categories of irritation. A benchmark approach can be applied using this concentration to evaluate the ocular irritative risk of adult and baby shampoos in developing countries.

Poloxamer-based thermoresponsive ketorolac tromethamine in situ gel preparations: Design, characterisation, toxicity and transcorneal permeation studies.

This study was aimed at preparing, characterising and evaluating in situ gel formulations based on a blend of two hydrophilic polymers i.e. poloxamer 407 (P407) and poloxamer 188 (P188) for a sustained ocular delivery of ketorolac tromethamine (KT). Drug-polymer interaction studies were performed using DSC and FT-IR. The gelation temperature (Tsol-gel), gelation time, rheological behaviour, mucoadhesive characteristics of these gels, transcorneal permeation and ocular irritation as well as toxicity was investigated. DSC and FT-IR studies revealed that there may be electrostatic interactions between the drug and the polymers used. P188 modified the Tsol/gel of P407 bringing it close to eye temperature (35°C) compared with the formulation containing P407 alone. Moreover, gels that comprised P407 and P188 exhibited a pseudoplastic behaviour at different concentrations. Furthermore, mucoadhesion study using mucin discs showed that in situ gel formulations have good mucoadhesive characteristics upon increasing the concentration of P407. When comparing formulations PP11 and PP12, the work of adhesion decreased significantly (P<0.001) from 377.9±7.79mNmm to 272.3±6.11mNmm. In vitro release and ex vivo permeation experiments indicated that the in situ gels were able to prolong and control KT release as only 48% of the KT released within 12h. In addition, the HET-CAM and BCOP tests confirmed the non-irritancy of KT loaded in situ gels, and HET-CAM test demonstrated the ability of ocular protection against strongly irritant substances. MTT assay on primary corneal epithelial cells revealed that in situ gel formulations loaded with KT showed reasonable and acceptable percent cell viability compared with control samples.

Biopharmaceutical evaluation of epigallocatechin gallate-loaded cationic lipid nanoparticles (EGCG-LNs): In vivo, in vitro and ex vivo studies.

Cationic lipid nanoparticles (LNs) have been tested for sustained release and site-specific targeting of epigallocatechin gallate (EGCG), a potential polyphenol with improved pharmacological profile for the treatment of ocular pathologies, such as age-related macular edema, diabetic retinopathy, and inflammatory disorders. Cationic EGCG-LNs were produced by double-emulsion technique; the in vitro release study was performed in a dialysis bag, followed by the drug assay using a previously validated RP-HPLC method. In vitro HET-CAM study was carried out using chicken embryos to determine the potential risk of irritation of the developed formulations. Ex vivo permeation profile was assessed using rabbit cornea and sclera isolated and mounted in Franz diffusion cells. The results show that the use of cationic LNs provides a prolonged EGCG release, following a Boltzmann sigmoidal profile. In addition, EGCG was successfully quantified in both tested ocular tissues, demonstrating the ability of these formulations to reach both anterior and posterior segment of the eye. The pharmacokinetic study of the corneal permeation showed a first order kinetics for both cationic formulations, while EGCG-cetyltrimethylammonium bromide (CTAB) LNs followed a Boltzmann sigmoidal profile and EGCG-dimethyldioctadecylammonium bromide (DDAB) LNs a first order profile. Our studies also proved the safety and non-irritant nature of the developed LNs. Thus, loading EGCG in cationic LNs is recognised as a promising strategy for the treatment of ocular diseases related to anti-oxidant and anti-inflammatory pathways.

The hen's egg chorioallantoic membrane (HET-CAM) test to predict the ophthalmic irritation potential of a cysteamine-containing gel: Quantification using Photoshop® and ImageJ.

A modified hen's egg chorioallantoic membrane (HET-CAM) test has been developed, combining ImageJ analysis with Adobe(®) Photoshop(®). The irritation potential of an ophthalmic medicine can be quantified using this method, by monitoring damage to blood vessels. The evaluation of cysteamine containing hyaluronate gel is reported. The results demonstrated that the novel gel formulation is non-irritant to the ocular tissues, in line with saline solution (negative control). In conclusion, the modification of the established HET-CAM test can quantify the damage to minute blood vessels. These results offer the possibility to formulate cysteamine in an ocular applicable gel formulation.