Hen's Egg Test-chorioallantoic Membrane Assay Research Articles

Etoposide-Loaded Poly(Lactic-co-Glycolic Acid) Intravitreal Implants: In Vitro and In Vivo Evaluation.

Etoposide-loaded poly(lactic-co-glycolic acid) implants were developed for intravitreal application. Implants were prepared by a solvent-casting method and characterized in terms of content uniformity, morphology, drug-polymer interaction, stability, and sterility. In vitro drug release was investigated and the implant degradation was monitored by the percent of mass loss. Implants were inserted into the vitreous cavity of rabbits' eye and the in vivo etoposide release profile was determined. Clinical examination and the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method were performed to evaluate the implant tolerance. The original chemical structure of the etoposide was preserved after incorporation in the polymeric matrix, which the drug was dispersed uniformly. In vitro, implants promoted sustained release of the drug and approximately 57% of the etoposide was released in 50days. In vivo, devices released approximately 63% of the loaded drug in 42days. Ophthalmic examination and HET-CAM assay revealed no evidence of toxic effects of implants. These results tend to show that etoposide-loaded implants could be potentially useful as an intraocular etoposide delivery system in the future.

Bone marrow-derived mesenchymal stem cells induced by inflammatory cytokines produce angiogenetic factors and promote prostate cancer growth

BackgroundProstate is susceptible to infection and pro-inflammatory agents in a man’s whole life. Chronic inflammation might play important roles in the development and progression of prostate cancer. Mesenchymal stem cells (MSCs) are often recruited to the tumor microenvironment due to local inflammation. We have asked whether stimulation of MSCs by pro-inflammatory cytokines could promote prostate tumor growth. The current study investigated the possible involvement of MSCs stimulated by pro-inflammatory cytokines in promotion and angiogenesis of prostate cancer through relative pathway in vitro and in vivo.MethodsA syngeneic mouse model of C57 was established. The murine prostate cancer cells (RM-1) mixing with MSCs treated with tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) or vehicle were subcutaneously injected into C57 mice. Tumor volume of C57 mouse model was estimated and serum level of platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) was test by Enzyme-linked Immunosorbent Assay (ELISA). A hen egg test-chorioallantoic membrane (HET-CAM) assay was applied to test the effect of conditioned media of stimulated MSCs in chorioallantoic membrane angiogenesis. Short interfering RNA (siRNA) knocked down either hypoxia-inducible factor-1alpha (HIF-1α) or nuclear factor-erythroid-2-related factor 2 (NRF2) were employed. mRNA of PDGF and VEGF in MSCs, as well as NRF2 and HIF-1α was test by Real time polymerase chain reaction (PCR) analyses. Protein expression levels of PDGF and VEGF from conditioned medium, NRF2, HIF-1α, as well as PDGF and VEGF in MSCs were detected by Western blot analysis.ResultsMSCs treated with TNF-α and IFN-γ promote tumor growth in C57 syngeneic mouse model, correlating with increased serum level of PDGF, VEGF. HET-CAM assay shows the angiogenic effect of conditioned medium of MSCs pre-treated with the pro-inflammatory cytokines. mRNA and protein levels of two pro-angiogenic factors (PDGF and VEGF) and key hypoxia regulators (HIF-1α and NRF2) in MSCs were induced after MSCs’ pretreatment. siRNA knockdown either HIF-1α or NRF2 results reduction of PDGF and VEGF expression.ConclusionsMSCs stimulated by pro-inflammatory cytokines increase the expression of PDGF and VEGF via the NRF2-HIF-1α pathway and accelerate prostate cancer growth in mice.

Evaluation of Ocular Irritation and Bioavailability of Voriconazole Loaded Microemulsion.

Voriconazole (VCZ), a second-generation antifungal with excellent attributes like, broad-spectrum activity, targeted delivery, and tolerability. VCZ loaded microemulsion could be an effective strategy for efficient ocular delivery of the drug. To perform corneal irritation studies and in vivo delivery of VCZ microemulsion to establish its potential as an efficient ocular delivery system. Ocular irritancy was performed by HETCAM (Hen's Egg Test Chorio Allantoic Membrane) assay, corneal histopathology and Draize test. Ex vivo and in vivo studies were performed to determine permeation efficiency of VCZ microemulsion. The irritation studies suggested the non-irritant nature of the microemulsion. The ex vivo studies performed on excised cornea displayed significant enhancement in drug permeation/penetration from microemulsion in contrast to the drug suspension. Further, the in vivo study confirmed the higher availability of VCZ (from microemulsion) in aqueous humor with minimal nasolacrimal drainage (lower plasma drug content) when compared with the drug suspension. The non-irritant nature and high corneal permeation of VCZ encourages the role of microemulsion as a potential ocular delivery system.

Evaluation of anti-inflammatory effect of traditional medicinal plants, Gynura segetum

Gynura segetum, family Asteraceae is a cultivated species and can be found growing in the tropical regions of Indonesia and Malaysia. The plant is known for its use for the treatment of cancer, inflammation, diabetes, hypertension and skin afflictions. In the current study, anti-inflammatory effect of Gynura segetum leaf has been investigated. The present study investigated the in vivo anti-inflammatory effect of Gynura segetum leaf by using hen's egg test-chorioallantoic membrane (HET-CAM) assay. Different solvent extract of Gynura segetum leaf were tested, the most active methanol extract was further fractionated and tested. Among the extracts tested, the methanol extract showed a significant good anti-inflammatory effect (76.8% inhibition at 50 <TEX>${\mu}g/disc$</TEX>) on the HET-CAM assay as compared with the anti-inflammatory drug indomethacin (82.1% inhibition). However, the fractionated sample exhibited a significantly lower activity in comparison to crude methanol extract. The results demonstrated that Gynura segetum leaf displayed remarkable anti-inflammatory effects, which support the folkloric uses of this plant for treatment of inflammation.

Characterization and Evaluation of Poly(ε-caprolactone) Nanoparticles Containing 2-Ethylhexyl-p-Methoxycinnamate, Octocrylene, and Benzophenone-3 in Anti-Solar Preparations

Ultraviolet radiation can bring both harm and benefits to human health. Among those harms are erythemas, photosensitivity, photoaging, and the most worrying, skin cancer. Nanoencapsulation of sunscreen agents (SA) by using a biocompatible and biodegradable polymer such as poly(epsilon-caprolactone) (PCL) is advantageous as it increases the retention of UV absorbers in the skin, avoids systemic absorption, and consequently, improves water resistance and stability of the preparation. The aim of this work is to develop, characterize, and study the encapsulation of 3 different SA: 2-ethylhexyl-p-methoxycinnamate, benzophenone-3, and octocrylene in PCL nanoparticles (Nps). Nps were prepared by the solvent emulsification and evaporation method. The process yield was calculated, and the Nps were characterized in terms of size, polydispersity index (PI), morphology, zeta potential (ZP), encapsulation efficiency (EE) (%), and sunscreen agent content (SAC). The final formulations were submitted to the hen's egg test-chorioallantoic membrane (HET-CAM), chorioallantoic membrane-trypan blue staining (CAM-TBS), red blood cell (RBC), Draize tests, in vitro release, in vitro sun protection factor (SPF), UVA protection factor (PF-UVA), and photostability. All the Nps were in the nanometric scale. PI showed monodisperse systems. ZP became more negative as the Np were lyophilized and were added to the formulations. EE varied from 84 to 90%. The SAC went from 44 to 65 microg of sunscreen agents by milligram of Np. The process yield went from 60 to 76%. Nps were predominantly spherical and elliptical forms. The addition of Np diminished the release of the SA. The SPF increased with Np presence and helped to maintain the PF-UVA after irradiation. The HET-CAM assay evaluated the formulation as slightly irritant, CAM-TBS and RBC tests as non irritant, and the Draize test as moderately irritant.

Experience with the HET-CAM Method in the Routine Testing of a Broad Variety of Chemicals and Formulations

Data on eye irritation are generally needed for the hazard identification of chemicals. For the routine testing of a broad variety of chemicals and formulations, we have used the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM) method. In the course of a tiered-testing strategy, and due to the lack of global regulatory acceptance of the HET-CAM method, we have also performed the Rabbit Eye Irritation test according to OECD Test Guideline 405. Of the 145 substances tested, 76% were classified as non-irritant/mild irritant and 13% were identified as irritant in vivo, according to the EU classification system (61% and 28%, respectively, with the GHS classification). The remaining 11% were severe irritants in vivo, based on the irreversibility of the effects and not due to sufficiently high irritation scores in the three days following application. The retrospective analysis revealed that the overall accuracy of the HET-CAM assay was 65% and the overall rates of false-negatives (FN) and false-positives (FP) were 50% and 33%, respectively. The HET-CAM assay was sufficiently specific (few FP) for water-soluble substances, but failed to identify nearly all the severe irritants within this group. In contrast, it was highly sensitive (no FN) for non-soluble and oil-soluble substances, but the specificity for this group was rather low. Therefore, we conclude that the HET-CAM assay is not useful in our tiered-testing strategy for eye irritation testing. However, for water-insoluble substances, it might be applicable in combination with another in vitro method, provided that regulatory acceptance is gained.

Sparfloxacin-loaded PLGA nanoparticles for sustained ocular drug delivery

Poor ocular bioavailability of drugs (<1%) from conventional eye drops (ie, solution, suspension, and ointments) is mainly due to the physiologic barriers of the eye. In general, ocular efficacy is closely related to ocular drug bioavailability, which may be enhanced by increasing corneal drug penetration and prolonging precorneal drug residence time. In our current work, we develop and evaluate a new colloidal system, that is, poly(dl-lactide- co-glycolide) (PLGA) nanoparticles for sparfloxacin ophthalmic delivery, to improve precorneal residence time and ocular penetration. Nanoparticles were prepared by nanoprecipitation technique and characterized for various properties such as particle size, zeta potential, in vitro drug release, statistical model fitting, stability, and so forth. Microbiological assay was carried out against Pseudomonas aeruginosa using the cup-plate method. Precorneal residence time was studied in albino rabbits by gamma scintigraphy after radiolabeling of sparfloxacin by Tc-99m. Ocular tolerance of the developed nanosuspension was also studied by the Hen Egg Test-Chorioallantoic Membrane (HET-CAM) method. The developed nanosuspension showed a mean particle size in the range of 180 to 190 nm, suitable for ophthalmic application with zeta potential of –22 mV. In vitro release from the developed nanosuspension showed an extended release profile of sparfloxacin according to the Peppas model. Acquired gamma camera images showed good retention over the entire precorneal area for the developed nanosuspension compared with that of a marketed formulation. The marketed drug formulation cleared very rapidly from the corneal region and reached the systemic circulation through the nasolacrimal drainage system, as significant radioactivity was recorded in kidney and bladder after 6 hours of ocular administration, whereas the developed nanosuspension cleared at a very slow rate ( P < .05) and remained at the corneal surface for longer duration, as no radioactivity was observed in the systemic circulation. HET-CAM assay with 0 score in 8 hours indicates the nonirritant property of the developed nanosuspension. The developed lyophilized nanosuspension was found to be stable for a longer duration of time than the conventional marketed formulation with a good shelf life. From the Clinical Editor Poor ocular bioavailability of drugs (<1%) from conventional eye drops is mainly due to the eye physiological barriers. In this study, a new colloidal system, PLGA nanoparticle for sparfloxacin ophthalmic delivery was demonstrated to improve precorneal residence time and ocular penetration. The developed lyophilized nanosuspension was found to be stable for longer duration of time than conventional marketed formulations.

Antibacterial Activity and Irritation Potential of Selected Essential Oil Components – Structure-Activity Relationship

A selection of structurally diverse components of essential oils was subjected to an antibacterial screening against the Gram-positive bacteria Staphylococcus aureus and Streptococcus pneumoniae as well as the Gram-negative bacteria Escherichia coli and Haemophilus influenzae using a broth microdilution method. In addition, the irritation potential of these substances to mucous membranes was evaluated by a modified HET-CAM (Hen's Egg Test-Chorioallantoic Membrane) assay. Minimum inhibitory concentrations (MIC) and irritation threshold concentrations (ITC) were compared in order to identify substances with a low mucous membrane irritation potential at the antibacterially effective concentration. Regarding the antibacterial activity, the most active substances were cinnamaldehyde, phenols, monoterpene alcohols and aldehydes. Oxygenated monoterpenes and phenylpropanoids without either alcohol or aldehyde function (ethers, ketones) showed moderate activity. Mono- and sesquiterpene hydrocarbons, as well as sesquiterpene alcohols, were more or less inactive against most of the bacteria tested, with the exception of Streptococcus pneumoniae whose growth was inhibited by sesquiterpenes at exceptionally low concentrations. In most cases, the irritation potential of the test compounds could also be traced back to their structural features. 2-Phenylethanol, monoterpene alcohols, phenols and aromatic aldehydes had the lowest ITC-values, followed by monoterpene aldehydes, ketones and ethers. In contrast, monoterpene hydrocarbons caused irritation only at higher doses. Of special interest was the fact that all the sesquiterpenes tested were not irritant even when applied undiluted to the chorioallantoic membrane (CAM).

Evaluation of a modified HET-CAM assay as a screening test for eye irritancy

The hen's egg test-chorioallantoic membrane (HET-CAM) assay, an alternative to the Draize eye irritation test, was developed by Luepke and has been improved on by means of a microscopic examination and the use of a test substance applicator (TSA). The TSA is a double teflon ring in which a perlon mesh is locked, and has several advantages over conventional protocols, reducing subjectivity of the method and avoiding the need for rinsing after treatment. It was confirmed by statistical analysis that the HET-CAM-TSA method can reproduce potential in vivo irritant effects on the conjunctiva. The classification based on the in vitro results was compared with four in vivo classifications [MAS (maximal average score) with thresholds of 15.0 and 25.0; the Kay and Calandra method; and EC criteria]. Cooper's parameters (specificity, sensitivity and concordance with the Draize test) were calculated according to these four in vivo classifications. When the most rigorous classification (MAS threshold of 15.0) was taken into account, a sensitivity of 80%, a specificity of 81.3% and a concordance with the Draize test of 80.4% were obtained for this set of 46 compounds.

An evaluation of five potential alternatives in vitro to the rabbit eye irritation test in vivo

Five alternative techniques, each of which had been successfully used by one of the participating companies, were evaluated in the assessment of the eye-irritation potential of 32 samples. The 32 samples included chemical ingredients and preparations from household cleaning product, personal care, and cosmetic categories. Historical data from rabbit eye irritation tests in vivo existed for each sample; it was therefore not necessary to carry out any tests in vivo as part of this evaluation exercise. The five alternative methods used were the silicon microphysiometer test, the Microtox test, the neutral red uptake assay, the chorioallantoic membrane vascular assay (CAMVA) and the hen egg test-chorioallantoic membrane assay (HETCAM). Three of the assays were conducted in two laboratories, allowing an interlaboratory comparison of performance to be made. The CAMVA assay was carried out on 10-day-old as well as on 14-day-old fertile eggs. Correlations between the data sets in vivo and in vitro were determined for the five assays. The results demonstrated that for the materials tested, all of the assays show some promise as alternative methods to the rabbit eye test in vivo in the prediction of eye irritation, and that the reproducibility of results of those techniques carried out in two laboratories was very good. The results from 14-day and 10-day CAMVA assays were virtually identical. It is recommended that a larger-scale validation exercise be carried out to demonstrate the ultimate usefulness of these alternative procedures in the safety evaluation process.

A national validation project of alternative methods to the Draize rabbit eye test

In June 1988, a 2.5-yr inter-laboratory study involving 13 toxicology laboratories was started in West Germany to validate alternative methods to the Draize rabbit eye test. The aim of this collaborative study is to validate the classification of chemicals with regard to their irritation potential using the neutral red/kenacid blue (NR/KB) cytotoxicity assay and the hen's egg test-chorioallantoic membrane (HET-CAM) assay. The results should make it possible to decide whether and to what extent the NR/KB cytotoxicity test and the HET-CAM assay can replace the Draize test. After two test trials, standard testing procedures and protocols were agreed on. In addition, to facilitate management of the data and to reduce costs, personal computer (PC) software was developed for both tests, which allows storage of all data on floppy discs and statistical analysis on PCs. During the preliminary phase, the applicability of the software was tested and corrected according to the experimental conditions of the validation study. Tests on the following chemicals have so far been completed, and reproducibility and repeatability have been determined: sodium dodecyl sulphate, triethanolamine, zinc pyridinethione, dimethylsulphoxide and butoxyethanol. Only zinc pyridinethione, which is severely irritating in vivo, could not be tested in the HET-CAM test. The preliminary phase has shown that the number of chemicals that can be tested in the HET-CAM test during the validation project will be limited by costs and management problems of manpower and time.