Rabbit Eyes Research Articles

Liposome‐Based Permeable Eyedrops for Effective Posterior Segment Drug Delivery

AbstractTopical eyedrop administration is identified as an ideal non‐invasive strategy for ocular drug delivery. However, multiple complex ocular barriers greatly restrict their effectiveness in the treatment of posterior ocular disease. Herein, a liposome‐based permeable eyedrops (pDrops) capable of overcoming multiple ocular barriers and achieving efficient posterior drug delivery is presented. pDrops have a core‐shell structure in which drugs are encapsulated inside the liposome core with a chitosan shell. This chitosan coating significantly enhances the pDrops’ binding to mucin in tears and facilitates the temporary opening of tight junctions in cornea/conjunctive epithelial cells, thereby achieving prolonged preocular retention and enhanced posterior segment drug delivery. In this study, hydrophilic ganciclovir (GCV) and hydrophobic curcumin (CUR) are employed as model drugs. Upon topical instillation, pDrops effectively overcome ocular barriers and delivered GCV to the posterior segment in both rat and rabbit eyes. Notably, CUR delivery by pDrops demonstrates significantly enhanced therapeutic efficacy in light‐damaged retina of mice. Considering that pDrops can deliver both hydrophobic and hydrophilic drugs to the posterior segment of the eye, it can potentially become a feasible platform for the non‐invasive delivery of various drug molecules and improve the treatment efficiency of posterior ocular diseases.

Aqueous VEGF-A Levels as a Liquid Biopsy Biomarker of Retinoblastoma Vitreous Seed Response to Therapy.

Regression of retinoblastoma vitreous seeds (VS) during intravitreal chemotherapy can be delayed, resulting in supernumerary injections. Similarly, VS relapse may not be clinically evident at first. A predictive biomarker of tumor regression and relapse could help guide real-time clinical decision making. Retinoblastoma is an oxygen-sensitive tumor; paradoxically, VS survive in the hypoxic vitreous. We hypothesized that VS elaborate pro-angiogenic cytokines. The purpose was to determine if pro-angiogenic cytokine signatures from aqueous humor could serve as a biomarker of VS response to treatment. Multiplex ELISA was performed on aqueous from rabbit eyes with human retinoblastoma VS xenografts to identify expressed proangiogenic cytokines and changes in aqueous cytokine levels during intravitreal treatment were determined. Confirmatory RNAscope in situ hybridization for VEGF-A was performed on human retinoblastoma tumor sections and VS xenografts from rabbits. For human eyes undergoing intravitreal chemotherapy, serial aqueous VEGF-A levels measured via VEGF-A-specific ELISA were compared to clinical response. VEGF-A was highly expressed in human retinoblastoma VS in the xenograft model, and was the only proangiogenic cytokine that correlated with VS disease burden. In rabbits, aqueous VEGF-A levels decreased in response to therapy, consistent with quantitative VS reduction. In patients, aqueous VEGF-A levels associated with clinical changes in disease burden (regression, stability, or relapse), with changes in VEGF-A levels correlating with clinical response. Aqueous VEGF-A levels correlate with extent of retinoblastoma VS, suggesting that aqueous VEGF-A may serve as a predictive molecular biomarker of treatment response.

LC-MS/MS of Dexamethasone in Rabbit Vitreous Liquid Delivered through Novel Self-nanoemulsifying Drug Delivery Systems

Background: Dexamethasone (DEX), delivered through newly developed selfnanoemulsifying drug delivery systems, which is administered to rabbit eyes, has been presented. The instrumental parameters of the multiple LC-MS/MS methods in the literature are modified according to rabbit vitreous liquid and current laboratory conditions. Methods: Quick optimization of the chromatographic and mass spectrometric parameters was done by inspecting the available literature for the analysis of DEX by LC-MS/MS from biological matrices. Chromatographic separation was achieved in the reverse mode using C18 (50x2.1mm, 5μm) as a stationary phase and acetonitrile and water with ammonium acetate as a mobile phase in gradient elution. Quantitation was done in multiple reaction monitoring (MRM) mode by following the transition of m/z 393 > m/z 373 in positive ion mode. The method was also validated in terms of selectivity, within-day accuracy and precision, linearity, and limit of detection (LOD). The extraction of DEX from rabbit vitreous liquid samples was carried out by protein precipitation using acetonitrile: water (70:30, v/v). Results: DEX and beclomethasone (IS) were successfully separated and detected under optimized experimental settings. The method was selective for DEX and linear in the range of 0.5 and 250 ng/ml. The lower limit of quantification (LLOQ) was determined to be 0.238 ng/ml. The percent relative standard deviation (RSD) and recovery (%) of the low, medium, and high calibration levels were below 10% and within the range of 111%-114%, respectively. RSD (%) and recovery (%) of the LLOQ were below 17% and 82%, respectively. The validated method was successfully applied for the determination of pharmacokinetic properties of newly formulated dexamethasone selfnanoemulsifying drug delivery systems (DEXSNEDDS) used to administer DEX intravitreally to the rabbit. Conclusion: LC-MS/MS conditions for the analysis of LC-MS/MS were determined by examining relevant literature for accomplishing simple and practical optimization of the experimental parameters, followed by method validation and analysis of rabbit vitreous liqiud. As conclusion, pharmacokinetic data of DEXSNEDDS has been obtained in the most accurate, sensitive, economical and rational way possible.

Sustained release of brimonidine from conjunctival sac insert to reduce intraocular pressure for glaucoma treatment

ABSTRACT Background Glaucoma is one of the major irreversible blinding eye diseases in the world. Reducing intraocular pressure (IOP) is the primary treatment option, and taking eye drops daily is the common method. However, short drug duration and poor bioavailability of eye drops may lead to unsatisfied therapeutic effects and inadequate patient compliance. Methods A brimonidine-loaded silicone rubber insert (BRI@SR@PT) was prepared by loading brimonidine into a surface-modified silicone rubber ring, followed by polydopamine/thermoplastic polyurethane coatings. The physical properties, in vitro cytocompatibility and drug release of BRI@SR@PT were investigated. The BRI@SR@PT was administrated in the conjunctival sac of rabbit eyes, and its in vivo drug release, IOP-lowering efficacy and biosafety were assessed. Results The BRI@SR@PT presented great thermal stability and excellent elasticity. The BRI@SR@PT was able to release BRI sustainably for 28 days with little toxicity in vitro. Compared to BRI eye drops, the BRI@SR@PT effectively lowered IOP for 21 days based on the sustained BRI release with great biosafety when administrated in conjunctival sac of rabbit eyes in a noninvasive fashion. Conclusions The conjunctival sac insert (BRI@SR@PT), as a promising drug-delivery platform, may provide a sustained IOP-lowering treatment for patients with ocular hypertension or glaucoma, without the need for invasive procedures.

Two novel Archaeorhizomyces species isolated from ericoid mycorrhizal roots and their association with ericaceous plants in vitro

Archaeorhizomyces is a diverse and ubiquitous genus of the subphylum Taphrinomycotina, which contains soil-inhabiting/root-associated fungi. Although ecological importance and root-associating lifestyles of Archaeorhizomyces can be postulated, morphological aspects of fungal body and root colonization are largely unknown due to the scarcity of cultures. We obtained three unidentified Archaeorhizomyces isolates from ericoid mycorrhizal (ErM) roots of Rhododendron scabrum and Rhododendron × obtusum collected in Japan. To advance our understanding of lifestyle of the genus, we investigated their general morphology, phylogeny, and in vitro root-colonizing ability in ericoid mycorrhizal hosts, Vaccinium virgatum and Rhododendron kaempferi. Some morphological characteristics, such as slow glowing white-to-creamy-colored colonies and formation of yeast-like or chlamydospore-like cells, were shared between our strains and two described species, Archaeorhizomycesfinlayi and Archaeorhizomyces borealis, but they were phylogenetically distant. Our strains were clearly distinguished as two undescribed species based on morphology and phylogenetic relationship. As seen in typical ErM fungi, both species frequently formed hyphal coils within vital rhizodermal cells of ErM plants in vitro. The morphology of hyphal coils was also different between species. Consequently, two novel species, Archaeorhizomyces notokirishimae sp. nov. and Archaeorhizomyces ryukyuensis sp. nov., were described.

Topical Solution for Retinal Delivery: Bevacizumab and Ranibizumab Eye Drops in Anti-Aggregation Formula (AAF) in Rabbits

PurposeWet age-related macular degeneration (AMD) is a blinding retinal disease. Monthly intravitreal anti-VEGF antibody injections of bevacizumab (off-label) and ranibizumab (FDA approved) are the standard of care. Antibody aggregation may interfere with ocular absorption/distribution. This study assessed topical delivery of dilute antibodies to the posterior segment of rabbit eyes using a novel anti-aggregation formula (AAF).MethodsBevacizumab, or biosimilar ranibizumab was diluted to 5 mg/ml in AAF. All rabbits were dosed twice daily. Substudy 1 rabbits (bevacizumab, 100 µl eye drops): Group 1 (bevacizumab/AAF, n = 6); Group 2 (bevacizumab/PBS, n = 7) and Vehicle control (AAF, n = 1). Substudy 2 rabbits (ranibizumab biosimilar/AAF, 50 µl eye drops): (ranibizumab biosimilar/AAF, n = 8). At 14.5 days, serum was drawn from rabbits. Aqueous, vitreous and retina samples were recovered from eyes and placed into AAF aliquots. Tissue analyzed using AAF as diluent.ResultsBevacizumab in AAF permeated/accumulated in rabbit aqueous, vitreous and retina 10 times more, than when diluted in PBS. AAF/0.1% hyaluronic acid eye drops, dosed twice daily, provided mean tissue concentrations (ng/g) in retina (29.50), aqueous (12.34), vitreous (3.46), and serum (0.28 ng/ml). Additionally, the highest concentration (ng/g) of ranibizumab biosimilar was present in the retina (18.0), followed by aqueous (7.82) and vitreous (1.47). Serum concentration was negligible (< 0.04 ng/ml). No irritation was observed throughout the studies.ConclusionsBevacizumab and ranibizumab, in an AAF diluent eye drop, can be delivered to the retina, by the twice daily dosing of a low concentration mAb formulation. This may prove to be an adjunct to intravitreal injections.

Enhanced ophthalmic bioavailability and stability of atropine sulfate via sustained release particles using polystyrene sulfonate resin

Atropine sulfate (ATS) eye drops at low concentrations constitute a limited selection for myopia treatment, with challenges such as low ophthalmic bioavailability and inadequate stability. This study proposes a novel strategy by synthesizing ophthalmic sodium polystyrene sulfonate resin (SPSR) characterized by a spherical shape and uniform size for cationic exchange with ATS. The formulation of ATS@SPSR suspension eye drops incorporates xanthan gum and hydroxypropyl methylcellulose (HPMC) as suspending agents. In vitro studies demonstrated that ATS@SPSR suspension eye drops exhibited sustained release characteristics, and tropic acid, its degradation product, remained undetected for 30 days at 40 °C. The ATS levels in the tear fluids and aqueous humor of New Zealand rabbits indicated a significant increase in mean residence time (MRT) and area under the drug concentration–time curve (AUC0-12h) for ATS@SPSR suspension eye drops compared to conventional ATS eye drops. Moreover, safety assessment confirmed the non-irritating nature of ATS@SPSR suspension eye drops in rabbit eyes. In conclusion, the cation-responsive sustained-release ATS@SPSR suspension eye drops enhanced the bioavailability and stability of ATS, offering a promising avenue for myopia treatment.

Prolonged Release Niosomes For Ocular Delivery of Loteprednol: Ocular Distribution Assessment on Dry Eye Disease Induced Rabbit Model.

Loteprednol etabonate (LE) is a topical corticosteroid for the symptomatic management of ocular conditions, encompassing both allergic and infectious etiologies. Owing to the dynamic and static barriers of the eye, LE exhibits significantly low bioavailability, necessitating an increase in the frequency of drug administration. The objective of this study is to overcome the limitations by developing niosomal systems loaded with LE. Design of Experiments (DoE) approach was used for the development of optimal niosome formulation. The optimal formulation was characterized using DLS, FT-IR, and DSC analysis. In vitro and ex vivo release studies were performed to demonstrate drug release patterns. After that HET-CAM evaluation was conducted to determine safety profile. Then, in vivo studies were carried out to determine therapeutic activity of niosomes. Zeta potential (ZP), particle size, polydispersity index (PI), and encapsulation efficacy (EE) were -33.8mV, 89.22nm, 0.192, and 89.6%, respectively. Medicated niosomes had a broad distribution within rabbit eye tissues and was absorbed by the aqueous humor of the bovine eye for up to 6h after treatment. Cumulative permeated drug in the bovine eye and rabbit eye were recorded 52.45% and 54.8%, respectively. No irritation or hemorrhagic situation was observed according to the results of HET-CAM study. Thus, novel LE-loaded niosomal formulations could be considered as a promising treatment option for the dry-eye-disease (DED) due to enhanced bioavailability and decreased side effects.

Developing a model for aqueous deficient dry eye secondary to periglandular cicatrizing conjunctivitis

PurposeThe current study used various techniques to develop a rabbit animal model of lacrimal gland damage caused by scarring conjunctivitis in the periglandular area. MethodsLeft eyes of New Zealand white rabbits were injected with 0.1 ml of 1M NaOH subconjunctivally around superior and inferior lacrimal gland orifices (Group 1, n = 4), touched with 1M NaOH for 100 s to the superior and inferior fornices with conjunctival denuding (Group 2; n = 4), and electrocauterization to the ductal opening area (Group 3; n = 4). The ocular surface staining, Schirmer I, lacrimal gland, and conjunctival changes were observed at baseline,1, 4, 8, and 12 weeks. The degree of glandular inflammation, conjunctival fibrosis (Masson Trichrome), and goblet cell density (PAS) were also assessed. ResultsAt 12 weeks, the lacrimal glands of group 1 rabbits with periglandular injection showed severe inflammation with mean four foci/10HPF and a significant mean reduction in the Schirmer values by 7.6 mm (P = 0.007). Lacrimal glands had diffuse acinar atrophy, loss of myoepithelial cells, and ductular dilatation. The overlying conjunctiva showed fibrosis, goblet cell loss, and corneal vascularization in the inferotemporal quadrant. No lacrimal gland or ocular surface changes were observed in groups 2 and 3 at 12 weeks, except for localized subconjunctival fibrosis. ConclusionPeriglandular injection of 0.1 ml of 1M NaOH induced extensive lacrimal gland damage with reduced secretion and scarring in the subconjunctival plane compared to direct cauterization or direct NaOH contact to the ductal orifices of the rabbit lacrimal gland.

Design and Biocompatibility of a Novel, Flexible Artificial Cornea.

We sought to introduce the materials, design, and biocompatibility of a flexible and suturable artificial corneal device. Single-piece, fully synthetic, optic-skirt design devices were made from compact perfluoroalkoxy alkane. The skirt and the optic wall surfaces were lined with a porous tissue ingrowth material using expanded polytetrafluoroethylene. Full-thickness macroapertures around the skirt perimeter were placed to facilitate nutrition of the recipient cornea. Material properties including the skirt's modulus of elasticity and bending stiffness, optic light transmission, wetting behavior, topical drug penetrance, and degradation profile were evaluated. The final prototype suitable for human use has a transparent optic with a diameter of 4.60mm anteriorly, 4.28mm posteriorly, and a skirt outer diameter of 6.8mm. The biomechanical and optical properties of the device closely align with the native human cornea with an average normalized device skirt-bending stiffness of 4.7kPa·mm4 and light transmission in the visible spectrum ranging between 92% and 96%. No optical damage was seen in the 36 devices tested in fouling experiments. No significant difference was observed in topical drug penetrance into the anterior chamber of the device implanted eye compared with the naïve rabbit eye. The flexibility and biocompatibility of our artificial cornea device may offer enhanced tissue integration and decreased inflammation, leading to improved retention compared with rigid keratoprosthesis designs. We have developed a fully synthetic, flexible, suturable, optic-skirt design prototype artificial cornea that is ready to be tested in early human feasibility studies.

Retention time of different ophthalmic viscosurgical devices during phacoemulsification in rabbit ocular model: A comparative analysis.

To compare retention times of various ophthalmic viscosurgical devices (OVDs) and soft-shell combinations. Experimental study. Eighteen rabbit eyes were divided into six groups of three eyes, based on OVDs tested. A: Endocoat, B: HealonPro, C: Viscoat, D: Provisc, E: Endocoat and HealonPro and F: Viscoat and Provisc. OVDs were stained with 10% fluorescein dye before being injected into the anterior chamber. Phacoemulsification, using fixed parameters, was performed. If OVD persisted after 60 s, a standardised irrigation and aspiration technique replaced phacoemulsification. The time until central and complete clearance of the OVD were video-recorded and measured. Mean central retention times (CRT) were found to be: HealonPro-5.33 ± 2.56 s, Provisc-3.33 ± 1.11 s, Endocoat-75.0 ± 3.26 s, Viscoat-62.33 ± 5.19 s, combining HealonPro with Endocoat-22.67 ± 4.75 s and Provisc with Viscoat-11.0 ± 0.82 s. The mean total retention time (TRT) was: Endocoat-80.0 ± 8.17 s, Viscoat-81.67 ± 2.09 s, HealonPro with Endocoat-81.33 ± 3.35 s, and Provisc with Viscoat-71.0 ± 2.94 s. For HealonPro and Provisc, CRT and TRT remained identical across all trials. Retention times varied, with cohesive OVDs exhibiting shorter durations than dispersive OVDs. Among dispersive OVDs, TRTs were comparable; however, Endocoat displayed an extended CRT. In soft shell trials, the combination of HealonPro and Endocoat exhibited prolonged CRT and TRT, suggesting enhanced corneal protection.

Ethylene promotes fruit ripening initiation by downregulating photosynthesis, enhancing abscisic acid and suppressing jasmonic acid in blueberry (Vaccinium ashei).

Blueberry fruit exhibit atypical climacteric ripening with a non-auto-catalytic increase in ethylene coincident with initiation of ripening. Further, application of ethephon, an ethylene-releasing plant growth regulator, accelerates ripening by increasing the proportion of ripe (blue) fruit as compared to the control treatment. To investigate the mechanistic role of ethylene in regulating blueberry ripening, we performed transcriptome analysis on fruit treated with ethephon, an ethylene-releasing plant growth regulator. RNA-Sequencing was performed on two sets of rabbiteye blueberry ('Powderblue') fruit: (1) fruit from divergent developmental stages; and (2) fruit treated with ethephon, an ethylene-releasing compound. Differentially expressed genes (DEGs) from divergent developmental stages clustered into nine groups, among which cluster 1 displayed reduction in expression during ripening initiation and was enriched with photosynthesis related genes, while cluster 7 displayed increased expression during ripening and was enriched with aromatic-amino acid family catabolism genes, suggesting stimulation of anthocyanin biosynthesis. More DEGs were apparent at 1 day after ethephon treatment suggesting its early influence during ripening initiation. Overall, a higher number of genes were downregulated in response to ethylene. Many of these overlapped with cluster 1 genes, indicating that ethylene-mediated downregulation of photosynthesis is an important developmental event during the ripening transition. Analyses of DEGs in response to ethylene also indicated interplay among phytohormones. Ethylene positively regulated abscisic acid (ABA), negatively regulated jasmonates (JAs), and influenced auxin (IAA) metabolism and signaling genes. Phytohormone quantification supported these effects of ethylene, indicating coordination of blueberry fruit ripening by ethylene. This study provides insights into the role of ethylene in blueberry fruit ripening. Ethylene initiates blueberry ripening by downregulating photosynthesis-related genes. Also, ethylene regulates phytohormone-metabolism and signaling related genes, increases ABA, and decreases JA concentrations. Together, these results indicate that interplay among multiple phytohormones regulates the progression of ripening, and that ethylene is an important coordinator of such interactions during blueberry fruit ripening.

Nanohydroxyapatite Coating Attenuates Fibrotic and Immune Responses to Promote Keratoprosthesis Biointegration in Advanced Ocular Surface Disorders.

Keratoprosthesis (KPro) implantation is frequently the only recourse for patients with severe corneal disease. However, problems arise due to inadequate biointegration of the KPro, particularly the PMMA optical cylinder, such as tissue detachment, tissue melting, or eye-threatening infection in the interface. Here, using the AuroKPro as a model prosthesis, a surface functionalization approach─coating the optical cylinder with nanohydroxyapatite (nHAp)─was trialed in rabbit eyes with and without a proceeding chemical injury. In chemically injured eyes, which simulated total limbal epithelial stem cell deficiency, clear benefits were conferred by the coating. The total modified Hackett-McDonald score and area of tissue apposition differences 12 weeks after implantation were 5.0 and 22.5%, respectively. Mechanical push-in tests revealed that 31.8% greater work was required to detach the tissues. These differences were less marked in uninjured eyes, which showed total score and tissue apposition differences of 2.5 and 11.5%, respectively, and a work difference of 23.5%. The improved biointegration could be contributed by the attenuated expression of fibronectin (p = 0.036), collagen 3A1 (p = 0.033), and α-smooth muscle actin (p = 0.045)─proteins typically upregulated during nonadherent fibrous capsule envelopment of bioinert material─adjacent to the optical cylinders. The coating also appeared to induce a less immunogenic milieu in the ocular surface tissue, evidenced by the markedly lower expression of tear proteins associated with immune and stimulus responses. Collectively, the level of these tear proteins in eyes with coated prostheses was 1.1 ± 13.0% of naïve eyes: substantially lower than with noncoated KPros (246.5 ± 79.3% of naïve, p = 0.038). Together, our results indicated that nHAp coating may reduce the risk of prosthesis failure in severely injured eyes, which are representative of the cohort of KPro patients.

Controlled release of vitamin A palmitate from crosslinked cyclodextrin organic framework for dry eye disease therapy

Controlled release drug delivery systems of eye drops are a promising ophthalmic therapy with advantages of good patient compliance and low irritation. However, the lack of a suitable drug carrier for ophthalmic use limits the development of the aforementioned system. Herein, the crosslinked cyclodextrin organic framework (COF) with a cubic porous structure and a uniform particle size was synthesized and applied to solidify vitamin A palmitate (VAP) by using the solvent-free method. The VAP@COF suspension eye drops were formulated by screening co-solvents, suspending agents, and stabilizing agents to achieve a homogeneous state and improve stability. According to the in vitro release study, the VAP@COF suspension exhibited a controlled release of VAP within 12 h. Both the ex vivo corneal contact angle and in vivo fluorescence tracking indicated that the VAP@COF suspension prolonged the VAP residence time on the ocular surface. This suspension accelerated the recovery of the dry eye disease (DED) model in New Zealand rabbits. Furthermore, the suspension was non-cytotoxic to human corneal epithelial cells and non-irritation to rabbit eyes. In summary, the particulate COF is an eye-acceptable novel carrier that sustains release and prolongs the VAP residence time on the ocular surface for DED treatment.

Studies on cationic ocular emulsions containing bipartitioned oil droplets to codeliver cyclosporin A and etodolac.

Background: To prepare ocular emulsions containing bipartitioned oil droplets to entrap cyclosporin A (0.05% w/w) and etodolac (0.2% w/w) by using castor, olive and silicon oils. Methods: The physicochemical characterizations of prepared emulsions were performed. The drug's biodistribution profiles and pharmacokinetic parameters from emulsions were checked using the ultraperformance liquid chromatography-tandemmass spectrometry method in the ocular tissues of the healthy rabbit eye model. Results: The emulsions displayed 365.13±7.21nm size and 26.45±2.09mV zeta potential. The ferrying of two drugs after releasing from emulsions occurred across corneal/conjunctival tissues to enter the vitreous and sclera following a single drop administration into the rabbit's eyes. Conclusion: The dual drug-loaded emulsions weremore likely to produce synergistic anti-inflammatory activity for managing moderate-to-severe dry eye disease.

تحضير وتقييم جل العين الموضعي بآلية تحفيز مزدوجة لتوصيل جاتيفلوكساسين وبيتاميثازون فوسفات الصوديوم

Background: Bacterial infections of the eye are treated by administering ophthalmic solutions containing corticosteroids and antibacterial agents. The main challenges faced when used for topical instillation are precorneal fast clearance and multiple applications, particularly with gatifloxacin. Objectives: To develop an ocular gel that utilizes both ion-induced and thermal-sensitive mechanisms to achieve gelation. Methods: We prepared and compared formulations containing different percentages of poloxamer 407 and gellan gum (F1–F24) in terms of gelation temperature, gelling capacity, gelation time, and permeation. We tested the optimum formulation for isotonicity and irritation in rabbits. Results: The formulations' pH varied from 6.7 to 7.3. Formulations that passed the gelation temperature test successfully were F6, F7, F9, and F10. For both drugs (F6, F7, F9, and F10), the drug content percentages ranged from 98.64% to 99.95%. In situ, gels (F6, F7, F9, and F10) showed pseudoplastic shear-thinning rheological behavior, which means that their viscosity decreased as the angular velocity went up. F7, which contains 17% poloxamer and 0.5% gellan gum, had 15 seconds of gelation time at 34oC and remained in gel form for 270 min. It was isotonic and did not change the size or shape of RBCs when topically applied. The rabbit's eyes did not experience irritation due to the extended release of both drugs. Conclusions: The new in situ gel formulation may be a superior alternative to the traditional eye drops of gatifloxacin and betamethasone for ocular infections.

Developing a quantitative method of mangiferin in rabbit lacrimal fluid as a tool for investigating pre-cornea drug residence

Mangiferin (MGF) has been evidenced to effectively treat infectious diseases caused by Herpes simplex virus, especially on the acyclovir-resistant strains. For ophthalmic disease treatment, prolonging drug retention in the eyes plays an essential role in effectively delivering drugs, which are prone to elimination by eye blinking and tear fluid secretion. The objective of this study was to develop a method for analysis of MGF in tear fluid that can be further utilised to evaluate the kinetics of precornea drug concentration. For sample collection, lacrimal tear fluid was absorbed into pre-weighed absorbing paper strips, which were then immediately weighed again right after sampling to calculate the sample mass. Subsequently, MGF was extracted from the paper strips using a mixture of methanol : acetic acid 0.6% (35:65, v/v). The extracted solution was finally injected into an HPLC system for MGF analysis (RP-column C18 - 4.6 x 250 mm, 5 μm was stabilised at 40oC; moblie phase methanol : acetic acid 0.6%; 35 : 65 (v/v) at a flow rate of 1.0 mL/min; autosampler, injection volume 10 µL; detection wavelength: 258 nm). The developed methodology was validated following the U.S. FDA and ICH M10 guidances for bioanalytical method validation, with respect to system suitability, linearity, recovery, accuracy and precision, dilution integrity and stability. The method was also successfully employed to determine MGF concentration in the tear fluid collected from rabbit eyes at one hour after instilling with MGF suspension. The methodology herein provides a practical tool to investigate kinetics of MGF in tear fluid that can be applied in ophthalmic drug development as well as drug quality evaluation activities.

Phospholipid analyses of rabbit ocular surface tissues

The tissues of the integument covering the ocular surface comprise a mucus membrane functioning as a protective physical barrier and has the ability to mount a defensive inflammatory response. Since lipid metabolism has a role in both of these functions, we studied normal membrane phospholipids (PL) of the cornea and bulbar conjunctiva to (1) determine baseline PL profiles of these tissues, (2) compare and contrast these individual PL metabolite profiles as well as groups of metabolites, and (3) describe pathway-specific metabolic interrelations among these tissues. Corneal and conjunctival tissue samples were isolated from rabbit eyes (n = 30) and extracted with chloroform-methanol using a modified Folch procedure. 31P nuclear magnetic resonance spectroscopy was used to qualitatively and quantitatively measure tissue PL profiles. The cornea and conjunctiva, respectively, have the following PL composition (mole % of total detected phospholipid): phosphatidylglycerol (PG) –, 0.4; lysophosphatidylethanolamine 1.2, –; phosphatidic acid –, 0.4; diPG (cardiolipin) 2.1, 3.5; unknown PL at the chemical shift of 0.13 δ 1.5, 0.9; ethanolamine plasmalogen 11.2, 13.0; phosphatidylethanolamine 11.5, 12.8; phosphatidylserine 8.9, 10.1; sphingomyelin 10.2, 10.7; lysophosphatidylcholine 0.9, 1.4; phosphatidylinositol 5.3, 5.3; phosphatidylcholine (PC) plasmalogen or alkylacylPC 2.2, 1.9; PC 45.1, 40.0. In addition, 28 PL metabolic indices were calculated from these data, which permitted pathway-specific lipid analyses. This study (1) establishes PL profiles of the two ocular tissues of the integument that cover the surface of the eye, (2) compares and contrasts indices comprised of ratios and combinations of PL, and (3) describes pathway-specific metabolic interrelations among these tissues to serve as baselines for studies involving the distribution of tissue phospholipids.

Evaluation of Moringa Oleifera Leaf Extract for its In vitro Antibacterial Properties, Mechanism of Action, and In vivo Corneal Ulcer Healing Effects in Rabbits' Eyes.

M. oleifera is the most adapted tree species in different medicinal eco-systems and has resilience against climate changes. This multiple-use tree provides healthy foods, snacks, honey, and fuel. Besides this, it has immense promising applicationsby offering antimicrobial and antibacterial activities for targeted uses. This validates the court of Hippocrates that let food be the medicine and medicine be the food for which moringa qualifies. In view of this, the antioxidant and in vitro antibacterial potency of the hydro-ethanolic extract of M. oleifera was evaluated on clinically isolated multidrug-resistant strains of Staphylococcus aureus. Furthermore, in vivo, the healing response of M. oleifera extract was analysed on corneal ulcers induced in rabbit eyes infected with methicillin-resistant Staphylococcus aureus. TheM. oleifera extract exhibited exponential antioxidant activity. In-vitro antibacterial activity was evaluated by agar well diffusion assay showing zone of inhibition ranging from 11.05±0.36 to 20±0.40 mm at concentrations of 20, 40, 80, and 160 mg/ml, whereas, in our finding, no zone of inhibition was observed below 20 mg/ml concentration, which indicated that there is threshold limit below which the antibacterial activity of M. oleifera extract is not observed. Furthermore, continuous application of 3% and 5% M. oleifera extract (eye drop) four times a day for 14 consecutive days showed a significant healing response of the eyes of rabbits with corneal ulcers. These results suggest that M. oleifera extract could be a viable alternative to existing antibacterial therapies for corneal ulcers. Additionally, there is a possibility of commercial formulation of M. oleifera extract in the form of deliverable pharmaceutical products; therefore, it should be explored further.

A Novel Ex Vivo Blinking Method for Comparing the Corneal Residence Time of New Shampoo Formulations.

In the cosmetics sector, many products such as shampoos have a probability of accidental ocular exposure during their routine use. One very specific safety parameter is the residence time of the substance on the corneal surface, as prolonged exposure may cause injury. In this study, we developed a system that simulates corneal exposure to blinking and tear flow, for comparing the corneal clearance times of viscous detergent formulations. The Ex Vivo Eye Irritation Test (EVEIT), which uses corneal explants from discarded rabbit eyes from an abattoir, was used as the basis for the new system. To simulate blinking, we developed a silicone wiping membrane to regularly move across the corneal surface, under conditions of constant addition and aspiration of fluid, to mimic tear flow. Six shampoo formulations were tested and were shown to differ widely in their corneal clearance time. Three groups could be identified according to the observed clearance times (fast, intermediate and slow); the reference shampoo had the shortest clearance time of all tested formulations. With this new system, it is now possible to investigate an important physicochemical parameter, i.e. corneal clearance time, for the consideration of ocular safety during the development of novel cosmetic formulations.