Draize Test Research Articles

Human relevance of in vivo and in vitro skin irritation tests for hazard classification of pesticides

Background: Test methods to inform hazard characterization and labeling of pesticides to protect human health are typically conducted using laboratory animals, and for skin irritation/corrosion the rabbit Draize test is currently required by many regulatory agencies. Although the Draize test is generally regarded to provide protective classifications for human health, new approach methodologies (NAMs) have been developed that offer more human relevant models that circumvent the uncertainty associated with species differences that exist between rabbits and humans. Despite wide applicability and use of these test methods across a broad range of chemicals, they have not been widely adopted for testing pesticides and pesticidal formulations. One of the barriers to adoption of these methods in this sector is low concordance with results from the Draize rabbit test, particularly for chemicals within the mild to moderate irritation spectrum. Methods: This review compares and contrasts the extent to which available models used in skin irritation testing mimic the anatomy and physiology of human skin, and how each aligns with the known key events leading to chemically-induced adverse skin irritation and corrosion. Doing so fully characterizes the human relevance of each method. Results: As alternatives to the rabbit Draize test, several protocols using ex vivo, in chemico, and in vitro skin models are available as internationally harmonized test guidelines. These methods rely on a variety of models of human skin, including excised rodent skin, synthetic biochemical models of barrier function, cell culture systems, and reconstructed human tissue models. We find these models exhibit biological and mechanistic relevance aligned with human skin irritation responses. Further, recent retrospective analyses have shown that the reproducibility of the Draize test is less than 50% for mild and moderate responses, with many of the replicate predictions spanning more than one category (e.g., a moderate response reported in one study followed by a non-irritant response reported in another study). Conclusions: Based on this comparative evaluation, we recommend top-down and bottom-up testing strategies that use the most human relevant in vitro test methods for skin irritation and corrosion classification of pesticides and pesticide formulations. To further discriminate among mild and non-irritant formulations, optimization of a cytokine release protocol and subsequent analyses of reference formulation test results is recommended.

Polymeric Mixed Micelle-Loaded Hydrogel for the Ocular Delivery of Fexofenadine for Treating Allergic Conjunctivitis.

This study was designed to formulate a polymeric mixed micelle (PMM) formulation to sustainably release fexofenadine (FEX) to treat allergic conjunctivitis effectively. A 32 factorial design was employed where the studied factors were PL90G amount (X1) and Pluronic (F127 and P123) mixture ratio (X2), and the dependent variables were entrapment efficacy (EE, Y1, %), particle size (PS, Y2, nm), zeta potential (ZP, Y3, mV), and the percent of drug released after 6 h (Q6h, Y4, %). The optimized formula was blended with a hydrogel base to develop an FEX-PMM hydrogel, where the safety and efficiency of this hydrogel were evaluated using in vivo studies. The EE% of FEX-PMM ranged from 62.15 ± 2.75 to 90.25 ± 1.48%, the PS from 291.35 ± 6.43 to 467.95 ± 3.60 nm, the ZP from -5.41 ± 0.12 to -9.23 ± 0.23 mV, and the Q6h from 50.27 ± 1.11 to 95.38 ± 0.92%. The Draize test results confirmed the safety of the FEX-PMM hydrogel. Furthermore, the FEX-PMM hydrogel showed rapid recovery in animals with induced allergic conjunctivitis compared to the free drug hydrogel. These results assure PMM's capability to deliver FEX to the conjunctival surface in a sustained pattern, consequently achieving better therapeutic outcomes.

Transdermal application of diacerin loaded-terpene enriched invasomes: an approach to augment anti-edema and nociception inhibition activity

This study aimed to formulate diacerein loaded terpene-enriched invasomes (DCN-TINV) to fulfill a fruitful management of osteoarthritis. A 23 factorial design was adopted, including A: cholesterol concentration (%w/v), B: ethanol volume (mL) and C: phosphatidylcholine: drug ratio as the studied factors. Invasomes were constructed using the thin film hydration technique. Herein, percent entrapment efficiency (EE%), particle size (PS), poly-dispersity index (PDI) and zeta potential (ZP) were statistically analyzed using Design-Expert® software to select the optimum formula. The selected criteria for detecting the optimum formula were restricting PS (<350 nm), dismissing PDI, magnifying ZP (as absolute value) and EE%. The selected formula was further scrutinized through multiple in-vitro studies, including Fourier-transform infrared spectroscopy, differential scanning calorimetry, pH measurement, stability study, release profile and transmission electron microscopy. Furthermore, the ex-vivo performance was evaluated through ex-vivo skin permeation and deposition. Finally, it was subjected to an array of in-vivo tests, namely Draize test, histopathology, In-vivo skin penetration, edema size, and nociception inhibition measurements. The optimum formula with desirability (0.913) demonstrated EE% (89.21% ± 2.12%), PS (319.75 ± 10.11 nm), ZP (−55 ± 3.96 mV) and a prolonged release profile. Intriguingly, revamped skin permeation (1143 ± 32.11 µg/cm2), nociception inhibition (77%) and In-vivo skin penetration (144 µm) compared to DCN suspension (285 ± 21.25 µg/cm2, 26% and 48 µm, respectively) were displayed. The optimum DCN-TINV exhibited plausible safety and stability profiles consolidated with auspicious efficacy for better management of osteoarthritis.

Limonene-enriched ultra-structural cubosomes to augment ocular delivery of a poorly water soluble anti-fungal drug: Fabrication, characterization, statistical optimization, in vivo corneal uptake and histopathological evaluation in rabbits

The target of this investigation was to obtain the composition of the optimized Fenticonazole nitrate (FCN)-loaded cubosomal formula that can prolong precorneal retention, increase corneal absorption, enhance corneal permeation, and anti-fungal efficacy of FCN. Cubosomes are bi-continuous structural vesicles fabricated using the hot emulsification technique in accordance with a 23-full factorial design. The selected fabrication factors were the concentration of the Lipid phase (X1), glyceryl monooleate: lipid phase stabilizer ratio (X2), and aqueous phase stabilizer types (X3). Whereas, the encapsulation efficiency percent (EE%), Mean particle size (PS), Polydispersity index (PDI), and Zeta potential (ZP) were chosen as measured responses. The numerical optimization criterion of the design expert software was utilized to select the optimized formula (Op) for extra investigations. The Op formula had EE% of 87.94 ± 0.7 %, PS of 249.75 ± 1.48 nm, and ZP of −34.25 ± 0.64 mV. The in vitro release study of the Op formula in comparison with FCN suspension showed a zero-order and controlled kinetic release behavior. Moreover, the results of the pH, refractive index, surface tension, and mucoadhesion study confirmed the superiority and the safety of the Op formula. Further, the ex vivo permeation study demonstrated a significant enhancement in the transcorneal permeation of the Op formula than FCN suspension by 2.69-fold. Besides, the in vivo corneal uptake confirmed the higher permeation of the Op formula in comparison with FCN suspension. Draize test and histopathological examination ensured the in vivo safety and tolerability of the Op formula. Overall, the previous findings ensured the efficacy of cubosomes in enhancing ocular retention, absorption, permeation, and bioavailability of FCN.

Preparation and Evaluation of Aloe-Vera and Vitamin E Anti-aging cream

Aging is a natural progressive process that leads to aesthetic and functional changes in the skin. Premature aging is also observed at young age around 20 due to pollution, cigarette smoke, sunlight, effects of an unhealthy lifestyle, insufficient nutritional intake, and prolonged illness. The aim of this research work is to formulate and evaluate an herbal antiaging face cream using Aloe-Vera and Vitamin E. Radical scavenging activity of prepared cream was studied using DPPH method and standard ascorbic acid was used. The evaluation test like, Organoleptic Test, pH, viscosity, spreadability, phase separation, homogeneity test, cream type, Draize test and ease to removal test were carried out. The stability study over three week was also executed.

Development of nanobigel system combining apoptosis-inducing daidzein and flaxseed oil for the synergistic treatment of skin cancer

Daidzein (DDZ), an isoflavone containing phytoestrogen, and flaxseed oil (FSO), an omega-3-polyunsaturated fatty acid rich dietary lipid, are known to exert anticancer effects via inducing apoptosis in cancer cells. However, oral administration of DDZ and FSO is associated with biopharmaceutical issues. This study aimed to develop a stable topical bigel (BG) system containing DDZ and FSO to exert synergistic effects in the treatment of skin cancer. High speed homogenization method was used for the preparation of DDZ-loaded bigel (DDZ@BG). In vitro investigations and morphological studies were performed to characterize the developed formulation. MTT cytotoxicity tests were conducted to evaluate the effect of the combination of DDZ and FSO on A431human skin cancer cell lines. To assess the toxicity, skin irritation studies using the Draize test was performed followed by histopathological analysis. The optimized DDZ@BG exhibited uniform texture, nanometric size and high drug content with sustained release profile and improved drug permeation. The MTT cytotoxicity studies revealed an IC50 value of 110.2 μg/mL for the optimized formulation. The developed nanobigel formulation exhibited high potential in the effective topical treatment of skin cancer with improved safety profile.

Formulation of hesperidin-loaded in situ gel for ocular drug delivery: a comprehensive study.

Allergic conjunctivitis is one of the most common eye disorders. Different drugs are used for its treatment. Hesperidin is an active substance isolated from Citrus sinensis L. (Rutaceae) fruit peels, with known anti-inflammatory activity but low solubility. It was complexed with cyclodextrin and encapsulated in situ gel to extend its duration in the eye. The optimized formulation comprised 1% hesperidin, 1.5% hydroxyethyl cellulose, and 16% poloxamer 407. The viscosity at 25 °C was 492 ± 82 cP, and at 35 °C it was 8875 ± 248 cP, the pH was 7.01 ± 0.03, gelation temperature was 34 ± 1.3 °C, and gelation time was 33 ± 1.2 s. There was a 66% in vitro release in the initial 2 h, with a burst effect. A lipoxygenase (LOX) inhibition test determined that hesperidin was active at high doses on leukotyrens seen in the body in allergic diseases. In cell-culture studies, the hesperidin cyclodextrin complex loaded in situ gel, BRN9-CD (poloxamer 16%, hydroxy ethyl cellulose (HEC) 1.5%), enhanced cell viability in comparison with the hesperidin solution. It was determined that BRN9-CD did not cause any irritation in the ocular tissues in the Draize test. The findings of this study demonstrate the potential of the in situ gel formulation of hesperidin in terms of ease of application and residence time on the ocular surface. Due to its notable LOX inhibition activity and positive outcomes in the in vivo Draize test, it appears promising for incorporation into pharmaceutical formulations. © 2024 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Glycyrrhizae radix et rhizoma processing ameliorates adverse reactions of polygalae radix in zebra fish and rabbit models

Ethnopharmacological relevancePolygala tenuifilia Willd (Polygalaceae), a traditional Chinese medicine, has been used for a long time to treat various illnesses with serious adverse reactions. Glycyrrhizae radix et rhizoma processing is generally used to reduce the adverse reactions. Aim of the studyThe aim of this study was to validate the irritation caused by raw Polygalaceae (RPA), to investigate whether processed Polygalaceae (PGA) was less irritating, and to screen and validate irritant properties of virgaureagenin G (polygala acid, PA), 3,6′-disinapoylsucrose (DSS), Tenuifolia (TEN) and polygalaxanthone III (POL), which had pharmacologically active in Polygalaceae. Zebrafish model, Draize test and High-Performance Liquid Chromatography (HPLC) were utilized to achieve the aim. Materials and methodsScanning Electron Microscopy (SEM) and optical microscope were used to determine the presence of calcium oxalate needle crystal in RPA and PGA. Zebrafish egg spinning changes and zebrafish embryo behavior were used for irritation validation, irritation comparison and irritant screening. For additional evidence, the Draize test, HE staining of rabbit eyes and ELISA kit were used. Finally, changes in the composition of RPA and PGA were investigated using HPLC. ResultsSEM and optical microscopy revealed no calcium oxalate needle crystals in Polygalaceae. RPA, PGA, PA and DSS were able to accelerate the spinning of zebrafish eggs and the movement of embryos, while TEN and POL were not. RPA, PGA, DSS and PA may cause rabbit eyes to become hyperemic and swollen, resulting in damage to the iris, cornea and conjunctiva and increased levels of interleukin-6 (IL-6) and interleukin-10 (IL-10). Comparatively, the effects caused by PGA were less severe than those caused by RPA. In addition, compared to RPA, PGA had lower levels of DSS and PA. ConclusionsRPA, PGA, DSS, and PA were irritating. However, processing and curing could reduce the irritation by reducing the levels of DSS and PA. DSS and PA could be two potential irritants of Polygalaceae.

The Therapeutic Role and Mechanism of Glabridin Under Aspergillus fumigatus Infection.

Purpose: To characterize the efficiency of glabridin alone and in combination with clinical antifungals in Aspergillus fumigatus keratitis. Methods: The broth microdilution method was performed to investigate whether glabridin exerted an antifungal role on planktonic cells and immature and mature biofilm. Antifungal mechanism was evaluated by Sorbitol and Ergosterol Assays. The synergistic effect of glabridin and antifungals was assessed through the checkerboard microdilution method and time-killing test. Regarding anti-inflammatory role, inflammatory substances induced by A. fumigatus were assessed by real-time quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Drug toxicity was assessed by Draize test in vivo. Macrophage phenotypes were examined by flow cytometry. Results: Regarding antifungal activity, glabridin destroyed fungal cell wall and membrane on planktonic cells and suppressed immature and mature biofilm formation. After combining with natamycin or amphotericin B, glabridin possessed a potent synergistic effect against A. fumigatus. Regarding anti-inflammatory aspects, Dectin-1, toll‑like receptor (TLR)-2 and TLR-4 expression of human corneal epithelial cells were significantly elevated after A. fumigatus challenge and reduced by glabridin. The elevated expression of interleukin-1β and tumor necrosis factor-alpha induced by A. fumigatus or corresponding agonists were reversed by glabridin, equivalent to the effect of corresponding inhibitors. Glabridin could also contribute to anti-inflammation by downregulating inflammatory mediator expression to suppress macrophage infiltration. Conclusions: Glabridin contributed to fungal clearance by destroying fungal cell wall and membrane, and disrupting biofilm. Combining glabridin with clinical antifungals was superior in reducing A. fumigatus growth. Glabridin exerted an anti-inflammatory effect by downregulating proinflammatory substance expression and inhibiting macrophage infiltration, which provide a potential agent and treatment strategies for fungal keratitis.

Liposomes versus microemulsions: Two novel nanosystems achieved an increased latanoprost hypotensive effect compared to traded medication

Aims/Purpose: The design of new topical ophthalmic nanosystems is linked to obtain non‐harmful carriers capable of increasing drug bioavailability and reduce the frequency of its instillations. Hypotensive actives such as latanoprost (L) are used to decrease intraocular pressure (IOP) in glaucoma patients. Two novel formulations, L‐loaded liposomes (LL) and L‐loaded microemulsions (LME), both including hyaluronic acid for its mucoadhesive properties were elaborated. The efficacy in reducing IOP of both formulations (48 h) was compared to a traded L‐ophthalmic solution (TM).Methods: LL were manufactured by the lipid film hydration method using synthetic phospholipids and cholesterol. LME were produced according to the self‐emulsification method by employing well‐tolerated oils and co‐solvents. Both LL and LME were characterized in terms of pH, osmolarity, size distribution, viscosity, and surface tension. Their hypotensive effect was evaluated up to 48 h in normotensive male albino New Zealand rabbits (n = 6, 12 eyes) using a tonometer following a single 25 μL topical administration and related to TM (n = 6). Draize test was performed before and 4 h after the instillation to establish LL, LME and TM tolerance.Results: Both LL and LME displayed proper physicochemical properties for the parameters evaluated. According to Draize test, both formulations resulted well‐tolerated without any sign of conjunctival hyperaemia, chemosis or discharge. Both LL and LME showed at least a 24‐h longer IOP reduction compared to TM, with the higher decrease at 3 h for LL and TM (24.02 ± 8.26% LL; 21.94 ± 10.14% TM) and 28 h for LME (30.40 ± 6.50%). Whilst at 24 h animals treated with TM (101.68 ± 5.27%) recuperated basal values, LL (91.08 ± 7.89%) and LME (80.14 ± 7.19%) rabbits still showed IOP decrease effect.Conclusions: Liposomes and microemulsions showed a longer hypotensive effect compared to traded medication, which makes them encouraging novel compliance‐friendly eye drops for glaucoma treatment.Acknowledgments: PID2020‐113281RB‐C21 funded by MCIN/AEI/10.13039/501100011033; 813440‐ORBITAL‐H2020‐MSCA‐ITN‐2018.

Schaftoside reduces inflammation in Aspergillus fumigatus keratitis through the inhibition of the TLR4/MyD88 pathway

PurposeThe purpose of this research study was to investigate the impact of schaftoside on Aspergillus fumigatus (A. fumigatus) keratitis and elucidate its underlying mechanisms. MethodsIn order to establish safe experimental concentrations of schaftoside in human corneal epithelial cells (HCECs), RAW264.7 cells, and mouse models, various techniques were employed including cytotoxicity assay (CCK-8) assay, cell scratch assay, and Draize test. The therapeutic effect of schaftoside was assessed using slit-lamp biomicroscopy, clinical scores, as well as determination of neutrophil infiltration through hematoxylin and eosin (HE) staining, immunofluorescence (IF) staining, and myeloperoxidase (MPO) assay. The levels of Toll-like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), pro-inflammatory mediators interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were determined using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and IF techniques. ResultsSchaftoside at a concentration of 160 μM displayed no harmful side effects on HCECs, RAW cells, and mouse corneas, rendering it suitable for further experiments. In a murine fungal keratitis model, schaftoside mitigated the severity of fungal keratitis by inhibiting neutrophil infiltration and reducing MPO activity. Both in vitro and in vivo experiments demonstrated that schaftoside treatment suppressed the upregulation of IL-1β, TNF-α, and IL-6 expression, while also downregulating the expressions of TLR4 as well as MyD88 at both mRNA and protein levels. ConclusionsSchaftoside demonstrated a protective effect against A. fumigatus keratitis by reducing corneal damage through inhibition of neutrophil recruitment and downstream inflammatory cytokines. The anti-inflammatory properties of schaftoside in A. fumigatus keratitis may involve modulation of the TLR4/MyD88 pathway.

Evaluation of Anti-nociceptive and Anti-Inflammatory Effects of Markhamia tomentosa Cream Co-Formulated with Eucalyptus Oil

Background: Pain is a noxious, sensory, and emotional experience due to intense stimuli. Pain is intimately linked with nociception. The clinical therapies available at the moment for pain are tricyclic antidepressants, non -steroidal anti-inflammatory drugs, and opioids. There is a need to develop and investigate safer alternatives that minimise the chances of psychological or physical drug dependence and drug dose tolerance. The purpose of this research is to develop an anti-inflammatory and anti-nociceptive formulation (Markhamia tomentosa cream co-formulated with Eucalyptus oil as a permeation enhancer) and evaluate its efficacy. Methods: In this study, Markhamia tomentosa was extracted from its leaves using maceration technique. A qualitative screening of the Markhamia tomentosa ethanolic extract (EEMT) was carried out. Five emulsions (A1-A5) containing varying compositions of Markhamia tomentosa were developed, and a negative control (an emulsion that did not contain Markhamia tomentosa or Eucalyptus oil) was also developed using fusion method. The anti-inflammatory and anti-nociceptive properties of the topical creams were evaluated using mice paw-licking and xylene-induced ear oedema tests. Diclofenac and aspirin administered at 20 mg/kg and 50mg/kg orally, respectively, were used as standards. All experiments were carried out in triplicates. Results: The phytochemical investigation of the Markhamia tomentosa extract showed a marked presence of key phyto-constituents such as saponins, flavonoids, alkaloids, and terpenoids. A1 and A2 had significantly higher percentage of nociception inhibition at phase I—neurogenic and phase II—inflammatory stages compared to Diclofenac 20 mg/kg. A1 and A3 had anti-inflammatory properties more potent than Aspirin 50mg/kg. In both tests, the negative controls did not show any level of potency. All results in this investigation were expressed as mean ± S.D (n=3) and (p&lt;0.05) for all data sets. Formulation A1 had the highest concentration of EEMT. No rash, erythema, or oedema was observed in the Draize test implying that the formulations are safe for topical application. Conclusion: Markhamia tomentosa and eucalyptus oil cream showed potent anti-nociceptive and anti-inflammatory activity. Markhamia tomentosa cream co-formulated with Eucalyptus oil as a permeation enhancer can be used effectively as a non-opioid, anti-nociceptive, and anti-inflammatory cream topically for pain and its symptoms

Engineering a novel water-in-oil biocompatible microemulsion system for the ocular delivery of dexamethasone sodium phosphate in the treatment of acute uveitis

Due to their unique characteristics, microemulsions (ME) represent one of the most promising delivery systems which can conquer poor ocular drug bioavailability providing long residence time. Development of a ME system, relying on the use of a safe and non-irritant surfactant combination derived from sustainable resources and which can consolidate the small ME droplets, is the goal of this work. Herein, we report the design and characterization of a novel biocompatible, eco-friendly ME system loaded with the hydrophilic dexamethasone sodium phosphate (DEXP) using a novel surfactant mixture composed of D-α-tocopherol polyethylene glycol succinate (TPGS) and Plantacare® (coco-Glycosides). Capryol™ PGMC and double-distilled water were used as the respective oil and aqueous phases and the MEs were prepared by the water titration method, suitable for scaling up. Optimization of ME formulae was conducted by varying Plantacare® grades, TPGS to Plantacare® mass ratios and drug loading. The formulae were characterized in terms of physical appearance, droplet size (PS), size distribution (PDI), zeta potential (ZP), and stability. The optimized DEXP-loaded ME formula attained acceptable PS, PDI, and ZP values of 43 ± 5 nm, 0.35 ± 0.07, −12 ± 4 mV, respectively. TEM images confirmed a small PS ≤ 100 nm. The in vivo safety of ME was proved by the Draize test. The ME formula prompted excellent mucoadhesion and transcorneal permeation. The confocal studies showed deep penetration into the rabbits’ corneas. In vivo studies using endotoxin-induced uveitis showed high ocular efficacy and a significant reduction in inflammatory cells, including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). The obtained results elect the novel engineered ME system as a promising tool for the ocular delivery of hydrophilic moieties in the management of various ophthalmic diseases.

Microscopic and Biopharmaceutical Evaluation of Emulsion and Self-Emulsifying Oil with Cyclosporine.

Among the currently available commercial eye drops with cyclosporine A (Cs) there is a lack of long-acting dosage forms and products with a concentration of the drug substance higher than 0.1%, although Cs is widely used in ophthalmology. The aim of the research was to conduct the microscopic and biopharmaceutical evaluation of two formulations, an emulsion (EM) and a self-emulsifying oil (SEO), both with 0.5% of Cs, proposed for use in eye drops, and the comparison of both. SEO eye drops with Cs or any other drug substance are currently not available as marketed products, and the highest concentration of Cs in the ocular emulsion is only 0.1%. The microscopic evaluation of the emulsion and the SEO after emulsification with water was carried out using a high-resolution digital microscopy. The properties of both preparations were compared using the high dynamic range function or optical shadow effect mode. Images in the 3D composition mode were also recorded. The in vivo study of the Cs formulations was performed on male albino rabbits. The eye tolerance of the preparations was assessed using the ocular irritation test, which is a modified Draize test. Placebo carriers (without the drug substance) were also subjected to irritation testing. The concentration of Cs in the tissues (cornea and conjunctiva) and fluids (tear fluid and aqueous humor) of the rabbit eye was determined after multiple instillations of Cs-EM or Cs-SEO. The tested preparations were compared using the digital microscopy technique, which highlights the features of the formulations and eliminates the risk of unnoticeable properties that are difficult to observe in classical optical microscopy. Both tested Cs-loaded formulations are classified as practically non-irritating. There were also no significant differences when testing the placebo carriers. After a topical administration, Cs was widely distributed in all tissues (e.g., in cornea 1.3 ng/mg and 1.0 ng/mg) and fluids of the eye (e.g., in tear fluid 11.6 µg/mL and 4.3 µg/mL), after the administration of Cs-SEO and Cs-EM, respectively. The obtained results allow us to recognize both tested formulations, the emulsion and the self-emulsifying oil with 0.5% Cs content, as carriers safe for ophthalmic use and effective in delivering the drug substance to the structures of the eye.

Ocular antibacterial chitosan-maleic acid hydrogels: In vitro and in vivo studies for a promising approach with enhanced mucoadhesion

The aim was to design and evaluate a chitosan-based conjugate providing high mucoadhesiveness and antibacterial activity for ocular infections treatment. Chitosan was conjugated with maleic acid via amide bond formation and infrared spectroscopy. Furthermore, 2,4,6-Trinitrobenzene sulfonic acid (TNBS) allowed characterization and quantification of conjugated groups, respectively. Biocompatibility was tested via hemolysis assay and Hen's Egg-Chorioallantoic membrane test. Characterization of the pH and osmolarity of hydrogels was followed by mucoadhesion assessment utilizing rheology. In addition, antibacterial studies were carried out towards Escherichia coli by broth microdilution test and agar-disk diffusion assay. In vivo studies were carried out following the already established Draize test and determining pharmacokinetic profile of dexamethasone in aqueous humour. The conjugate exhibited a degree of modification of 50.05 % and no toxicity or irritability. Moreover, mucoadhesive properties were enhanced in 2.68-fold and 1.81-fold for elastic and viscous modulus, respectively. Furthermore, rheological synergism revealed the presence of a gel-like structure. Additionally, broth microdilution and agar disk diffusion studies exhibited enhancement in antibacterial activity. Finally, in vivo studies manifested that hydrogels were highly tolerated, evidencing promising characteristics of the developed conjugate. The conjugate presented promising antimicrobial, long lasting mucoadhesive features and highly improved pharmacokinetics, leading to a revolutionizing approach in the treatment of ocular bacterial infections.

Cationic Dextran Stearate (Dex-St-GTMAC): Synthesis and Evaluation as Polymeric Micelles for Indomethacin Corneal Penetration.

Background Indomethacin as a non-steroidal anti-inflammatory drug (NSAID) is commonly used to treat some ocular inflammatory disorders. Unfortunately, indomethacin is a drug that is poorly soluble in water; therefore, it has low efficacy. An attractive approach is the targeted delivery of indomethacin to the cornea using cationic dextran stearate as a polymeric micelle drug carrier. Methods A dextran stearate-glycidyl trimethylammonium chloride (Dex-St-GTMAC) copolymer was prepared through the reaction of GTMAC, stearoyl chloride, and dextran. Then, Dex-St-GTMAC was characterized by Fourier transform infrared (FT-IR) spectroscopy and 1H NMR spectroscopy. Dex-St-GTMAC forms micelles in the presence of indomethacin. The prepared polymeric micelles were characterized for size, ζ-potential, drug loading, particle morphology, critical micelle concentration, and encapsulation efficiency. To study the irritation potential of the indomethacin-loaded Dex-St-GTMAC, Het-Cam and Draize tests have been performed. Prepared cationic micelles were subjected to the in vitro drug release and ex vivotrans-corneal permeation test. Results The dialysis method was used for the preparation of indomethacin-loaded micelles (10, 20, and 30%). Measurement of the particle size showed a mean diameter of 122.1 and 150.9 nm for the drug-loaded micelles. Scanning electron microscopy (SEM) images showed that the morphology of the particles is spherical. 10% formulation was chosen as the best formulation due to more surface charge and reasonable drug loading. ζ-potential measurement for the 10% drug-containing micelles showed a value of +39.1 mV. Drug loading efficiency and the encapsulation efficiency for 10% drug-containing micelles were 6.36 and 63.61%, respectively. The results of the Het-Cam and Draize tests indicated that the indomethacin-loaded Dex-St-GTMAC formulation had no toxicity to eye tissues. Based on our results, the prepared micelles (indomethacin-loaded Dex-St-GTMAC) exhibited a sustained drug release pattern compared to the control group. Indomethacin penetration from the micelles to the excised bovine cornea was 1.75-fold greater than the control (indomethacin 0.1% in phosphate-buffered saline (PBS)). Conclusions Data from the ζ-potential, SEM, drug loading capacity, and in vitro drug release studies indicated that cationic dextran stearate polymeric micelles are an appropriate carrier for the efficient penetration of indomethacin into cornea tissues.

Flexosomes as a promising nanoplatform for enhancing tolnaftate ocular delivery: Formulation, in vitro characterization, statistical optimization, ex vivo and microbial in vivo studies

The eye is a complex organ with a unique physiology and anatomy. Using novel nanosystems is expected to enhance ocular drug permeation and retention. Hence, this work aimed to study the potential of flexosomes as an ocular delivery system to enhance the corneal permeation and antifungal activity of Tolnaftate (TOL). Different flexosomes formulae were formulated using ethanol injection method, employing a 31.22 full factorial design. The studied formulation variables were: X1: amount of stearyl amine, X2: hydration volume and X3: type of edge activator. Encapsulation efficiency, particle size and zeta potential were selected as dependent variables. FX5 was selected as the optimal TOL flexosomes and showed encapsulation efficiency of 66.08 ± 11.38%, particle size of 154.99 ± 29.11 nm and zeta potential of 42.95 ± 0.64 mV. FX5 was subjected to further ex vivo and in vivo studies which showed that TOL flux was significantly increased through FX5 compared to TOL suspension. Draize test and histopatholoigal tests assured that FX5 is safe to be used for eye.. The in vivo fungal susceptibility testing using Aspergillus niger demonstrated the superior and more durable antifungal activity of FX5 than TOL suspension. Hence, FX5 can be considered as promising nanocarrier for safe and efficient ocular TOL delivery.

Baricitinib Lipid-Based Nanosystems as a Topical Alternative for Atopic Dermatitis Treatment.

Atopic dermatitis (AD) is a chronic autoimmune inflammatory skin disorder which causes a significant clinical problem due to its prevalence. The ongoing treatment for AD is aimed at improving the patient's quality of life. Additionally, glucocorticoids or immunosuppressants are being used in systemic therapy. Baricitinib (BNB) is a reversible Janus-associated kinase (JAK)-inhibitor; JAK is an important kinase involved in different immune responses. We aimed at developing and evaluating new topical liposomal formulations loaded with BNB for the treatment of flare ups. Three liposomal formulations were elaborated using POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine), CHOL (Cholesterol) and CER (Ceramide) in different proportions: (i) POPC, (ii) POPC:CHOL (8:2, mol/mol) and (iii) POPC:CHOL:CER (3.6:2.4:4.0 mol/mol/mol). They were physiochemically characterized over time. In addition, an in vitro release study, ex vivo permeation and retention studies in altered human skin (AHS) were also performed. Histological analysis was used to study the tolerance of the formulations on the skin. Lastly, the HET-CAM test was also performed to evaluate the irritancy capacity of the formulations, and the modified Draize test was performed to evaluate the erythema and edema capacity of the formulations on the altered skin. All liposomes showed good physicochemical properties and were stable for at least one month. POPC:CHOL:CER had the highest flux and permeation, and the retention in the skin was equal to that of POPC:CHOL. The formulations exhibited no harmful or irritating effects, and the histological examination revealed no changes in structure. The three liposomes have shown promising results for the aim of the study.

A Comparison of Type IV Hypersensitivity Reaction to Synthetic Polyisoprene Gloves versus Control Gloves Using Modified Draize-95 Test in Normal Individual

Background: The emergence of epidemics in recent years, such as COVID-19 and monkeypox, has increased the need for protective gloves. Objectives: This study was performed to provide clinical evidence that no residual chemical additives are present in the powder-free, non-latex, with and without pigment, sterile and non-sterile nitrile, and polyisoprene gloves at a level that may induce type IV allergy in the unsensitized general user population. Methods and Materials: We conducted the modified Draize-95 test on the GAMMEX® Non-Latex PI, sterile powder-free synthetic polyisoprene surgical gloves (S01) and the GAMMEX® Non-Latex PI Green, sterile powder free synthetic polyisoprene surgical gloves (S02). A quasi-experimental non-equivalent study consisted of 180 healthy unsensitized adult volunteers at the Islamic University of Gaza. Participants were divided into two groups: 150 white subjects and 30 black subjects. Each group divided into two groups, each one contained approximately the same numbers of males and females. We used two sets of different test materials as 2x2 cm pieces of the glove's materials from the inner and outer surfaces mounted on an adhesive patch. For the control groups, we used Nitrile Skin Examination Powder-Free Disposable Gloves (C1) and Aegis Examination Gloves (C2). Results: None of the participants showed positive reactions to the test materials of equal or greater than 1.5 in the challenge or rechallenge phase, both on the inner and outer surfaces of the GAMMEX® Non-Latex PI, sterile powder-free synthetic polyisoprene surgical gloves (S01), and the GAMMEX® Non-Latex PI Green, sterile powder-free synthetic polyisoprene surgical gloves (S02). Only one white woman showed a reaction to the inner surface of the S02 material in the induction phase. Three participants showed skin reactions to the second control material, the Aegis Nitrile Examination Gloves (C2). Those three were a white female in the induction phase and two white males in the challenge phase. However, none of them showed any reaction later. Conclusion: The modified Draize test negative results for the GAMMEX Non-Latex PI, sterile powder-free, synthetic polyisoprene surgical gloves (white and green) support the claim that the chemical sensitization potential of these materials is low and that no residual chemical additives are present in these gloves at a level that could cause type IV hypersensitivity reactions.

In Silico Prediction of Eye Irritation Using Hansen Solubility Parameters and Predicted pKa Values.

An in silico method has been developed that permits the binary differentiation between pure liquids causing serious eye damage or eye irritation, and pure liquids with no need for such classification, according to the UN GHS system. The method is based on the finding that the Hansen Solubility Parameters (HSP) of a liquid are collectively important predictors for eye irritation. Thus, by applying a two-tier approach in which in silico predicted pKa values (firstly) and a trained model based solely on in silico-predicted HSP data (secondly) were used, we have developed, and validated, a fully in silico approach for predicting the outcome of a Draize test (in terms of UN GHS Cat. 1/Cat. 2A/Cat. 2B or UN GHS No Cat.) with high validation set performance (sensitivity = 0.846, specificity = 0.818, balanced accuracy = 0.832) using SMILES only. The method is applicable to pure non-ionic liquids with molecular weight below 500 g/mol, fewer than six hydrogen bond donors (e.g. nitrogen-hydrogen or oxygen-hydrogen bonds) and fewer than eleven hydrogen bond acceptors (e.g. nitrogen or oxygen atoms). Due to its fully in silico characteristics, this method can be applied to pure liquids that are still at the desktop design stage and not yet in production.