MAGL inhibitor NanoMicellar formulation (MAGL-NanoMicellar) for the development of an antiglaucoma eye drop.

The ocular endocannabinoid system (ECS) including enzymes and CB1/CB2 receptors determines various substantial effects, such as anti-inflammatory activity and reduction of the intraocular pressure (IOP). The modulation of 2-arachidonoylglycerol (2-AG) levels obtained via MAGL inhibition is considered as a promising pharmacological strategy to activate the ECS. Within the scope of this study, the effect of a selective monoacylglycerol lipase (MAGL) inhibitor (MAGL17b) was investigated by measuring the IOP reduction in normotensive rabbits after performing a solubilisation process of the molecule with non-ionic surfactants, to produce suitable eye drops containing the highest possible concentration of the drug. Furthermore, the study involved the evaluation of cytotoxicity and of in vitro/ex vivo corneal permeation of MAG17b of selected formulations based on polyoxyl(35)castor oil (C-EL) and polyethylene glycol (80) sorbitan monolaurate (TW80). The solubilisation of 0.5 mM MAGL17b with 3 % w/w TW80 (TW80/3-17b), through the formation of NanoMicellar structures (diameter of 12.3 nm), determined a significant permeation of MAGL17b, both through excised rabbits corneas and reconstituted corneal epithelium, with a limited corneal epithelial cells death. The blockade of MAGL activity induced a IOP reduction up to 4 mmHg in albino and pigmented rabbits after topical instillation, thus confirming the potential efficacy of the MAGL inhibition approach in the treatment of ocular pathologies.

Hydrogels as Corneal Stroma Substitutes for In Vitro Evaluation of Drug Ocular Permeation.

Hydrogels are complex hydrophilic structures, consisting of crosslinked homopolymers or copolymers insoluble in water. Due to their controllable bio-physicochemical properties mimicking the morphology of the native extracellular matrix, they are a key part of a lot of research fields, including medicine, pharmaceutics, and tissue engineering. This paper was focused on the preparation and characterization of hydrogels from different blends of polyvinyl alcohol (PVA) with microcrystalline cellulose (MCC) and gelatin (GEL) at various ratios, and from gelatin and chitosan alone to understand their feasibility of utilizing as corneal stroma substitutes in permeability tests for drug candidate molecules in early stages of their development. The characterization was carried out by differential scanning calorimetry, electron microscopy (SEM), water content, mass loss, water permeability, wettability, and tensile stress-strain tests. After the physicochemical characterization, PVA/MCC blend and chitosan proved to be the most promising constructs, showing negligible mass loss after immersion in aqueous medium for two weeks and low hydrodynamic permeability. They were then employed in drug molecules permeation studies and these data were compared to that obtained through excised tissues. The results obtained showed that PVA/MCC hydrogels have similar mechanical and permeability properties to corneal stroma.

A hybrid ocular delivery system of cyclosporine-A comprising nanomicelle-laden polymeric inserts with improved efficacy and tolerability.

We report on hybrid nanomicelle-polymer inserts for improved delivery of cyclosporine A (CyA) to the surface of the eye. Hybrid inserts containing a nanomicellar formulation were prepared by the solvent casting method; their characteristics, in vitro release of CyA, eye irritation potential, nanomicelle distribution inside the insert, and in vivo pharmacokinetics of the most promising solid formulation (F3) were investigated. Nanomicelles capable of accommodating a therapeutically relevant amount of CyA (57.22 ± 5.90-68.52 ± 1.4 µg) were incorporated into five different polymeric formulations (F1-F5). The developed inserts displayed promising characteristics (size, weight, surface pH, and contact angle) that fulfill ocular tolerability requirements. Considering the technological properties and CyA in vitro release, F3 and F5 were the most promising formulations. SEM analysis suggested the F3 formulation as the potential prototype for CyA ocular delivery. The F3 formulation (CyA: 60.08 ± 2.85 µg) did not induce conjunctival irritation when HET-CAM assay was performed and was hence considered suitable for further study in a rabbit eye. The AUC value for CyA loaded in the F3 insert was about 2-fold greater than that obtained with the Ikervis® used as a control formulation. F3 produced a significant reduction (of about 7-folds) in the rate of CyA elimination from the tear fluid relative to Ikervis® and about 4-fold greater reduction than Nano-CyA (p = 0.0187). The ability of F3 to delay the elimination of the drug from the precorneal area is particularly desirable when treating dry eye syndrome. Furthermore, F3 did not induce ocular discomfort, a typical characteristic of solid ocular inserts, including commercially available ones.

Ocular Application of Oleuropein in Dry Eye Treatment: Formulation Studies and Biological Evaluation.

BACKGROUND: Oleuropein is already known for its numerous pharmacological properties, but its activity in the ocular field has not yet been investigated. The study aims to verify a possible use of oleuropein (OLE)-based eye drops both in terms of efficacy in dry eye syndrome and stability in aqueous solution. METHODS: OLE was co-precipitated with HP-ß-cyclodextrin, and the obtained complex was encapsulated into liposomes prepared by hydration of a lipid film composed of Lipoid S100 and cholesterol with different pH buffer solutions. The hydrated vesicles were shrunk by ultrasonication or extrusion. The preparations were characterized from the physicochemical point of view by subjecting them to differential scanning calorimetry, ATR-FTIR, dynamic light scattering analysis, and microscopy. Subsequently, OLE protective activity against hyperosmotic and oxidative stress on rabbit corneal epithelial cells (RCE) was evaluated. RESULTS: The liposomal vesicles obtained after extrusion showed a tendency towards greater encapsulation efficiency (up to 80.77%) compared to that obtained by sonication, and the liposomes hydrated in pH 5.5 solution tended to incapsulate more than the neutral ones. Ultrasonication produced two-dimensional populations of liposomes, the largest of which reached 2149 nm. On the contrary, the extruded liposomes showed homogeneous diameters of about 250 nm. Complexation with cyclodextrin and subsequent encapsulation in liposomes greatly increased the OLE stability in aqueous solution, especially at 4 °C and for the extruded formulations. OLE aqueous solution (OLE7.4-sol, reference) and neutral extruded liposomes (F7.4-e) were well tolerated on RCE cells. Moreover, OLE was able to control the effects of hyperosmolarity on ocular surface cells and to prevent oxidative stress-induced loss of cell viability.

Hyaluronic-Coated Albumin Nanoparticles for the Non-Invasive Delivery of Apatinib in Diabetic Retinopathy.

PURPOSE: Apatinib (Apa) is a novel anti-vascular endothelial growth factor with the potential to treat diabetic retinopathy (DR); a serious condition leading to visual impairment and blindness. DR treatment relies on invasive techniques associated with various complications. Investigating topical routes for Apa delivery to the posterior eye segment is thus promising but also challenging due to ocular barriers. Hence, the study objective was to develop Apa-loaded bovine serum albumin nanoparticles (Apa-BSA-NPs) coated with hyaluronic acid (HA); a natural polymer possessing unique mucoadhesive and viscoelastic features with the capacity to actively target CD44 positive retinal cells, for topical administration in DR. METHODS: Apa-BSA-NPs were prepared by desolvation using glutaraldehyde for cross-linking. HA-coated BSA-NPs were also prepared and HA: NPs ratio optimized. Nanoparticles were characterized for colloidal properties, entrapment efficiency (EE%), in vitro drug release and mucoadhesive potential. In vitro cytotoxicity on rabbit corneal epithelial cells (RCE) was assessed using MTT assay, while efficacy was evaluated in vivo in a diabetic rat model by histopathological examination of the retina by light and transmission electron microscopy. Retinal accumulation of fluorescently labeled BSA-NP and HA-BSA-NP was assessed using confocal microscope scanning. RESULTS: Apa-HA-BSA-NPs prepared under optimal conditions showed size, PdI and zeta potential: 222.2±3.56 nm, 0.221±0.02 and -37.3±1.8 mV, respectively. High EE% (69±1%), biphasic sustained release profile with an initial burst effect and mucoadhesion was attained. No evidence of cytotoxicity was observed on RCE cells. In vivo histopathological studies on DR rat model revealed alleviated retinal micro- and ultrastructural changes in the topical HA-Apa-BSA-NP treated eyes with normal basement membrane and retinal thickness comparable to normal control and intravitreally injected nanoparticles. Improved retinal accumulation for HA-BSA-NP was also observed by confocal microscopy. CONCLUSION: Findings present HA-Apa-BSA-NPs as a platform for enhanced topical therapy of DR overcoming the devastating ocular complications of the intravitreal route.

Combination of Nanomicellar Technology and In Situ Gelling Polymer as Ocular Drug Delivery System (ODDS) for Cyclosporine-A.

A combination of in situ gelling systems and a loaded drug self-assembling nanomicellar carrier was chosen in this study as a new potential Ocular Drug Delivery System (ODDS) for Cyclosporine-A (CyA), a poorly water-soluble drug. Two non-ionic surfactants (d-α-tocopherol polyethylene glycol succinate, VitE-TPGS and polyoxyl 40 hydrogenated castor oil, RH-40) were used to produce the nanomicelles. The physical-chemical characterization of the nanomicelles in terms of CyA entrapment (EE%) and loading efficiency (LE%), cloud point (CP), regeneration time (RT), size and polydispersity index (PI) allowed us to select the best combination of surfactant mixture, which showed appropriate stability, high CyA-EE (99.07%), very small and homogeneous dimensions and favored the solubilization of an amount of CyA (0.144% w/w) comparable to that contained in marketed emulsion Ikervis®. The selected nanomicellar formulation incorporated into optimized ion-sensitive polymeric dispersions of gellan gum (GG-LA: 0.10, 0.15 and 0.20% w/w) able to trigger the sol-gel transition after instillation was characterized from technological (osmolality, pH, gelling capacity, rheological behavior, wettability, TEM and storage stability at 4 and 20 °C) and biopharmaceutical points of view. This new combined approach allowed us to obtain clear aqueous dispersions that were easy to instill and able to form a viscous gel when in contact with the tear fluid, improving CyA ocular bioavailability. Furthermore, this new ODDS prevented CyA transcorneal permeation, exhibited low cytotoxicity and prolonged the CyA resident time in the precorneal area compared to Ikervis®.

Tyrosol-Enriched Tomatoes by Diffusion across the Fruit Peel from a Chitosan Coating: A Proposal of Functional Food.

Chitosan is receiving increasing attention from the food industry for being a biodegradable, non-toxic, antimicrobial biopolymer able to extend the shelf life of, and preserve the quality of, fresh food. However, few studies have investigated the ability of chitosan-based coatings to allow the diffusion of bioactive compounds into the food matrix to improve its nutraceutical quality. This research is aimed at testing whether a hydrophilic molecule (tyrosol) could diffuse from the chitosan-tyrosol coating and cross the tomato peel. To this end, in vitro permeation tests using excised tomato peel and an in vivo application of chitosan-tyrosol coating on tomato fruit, followed by tyrosol quantification in intact fruit, peel and flesh during a seven-day storage at room temperature, were performed. Both approaches demonstrated the ability of tyrosol to permeate across the fruit peel. Along with a decreased tyrosol content in the peel, its concentration within the flesh was increased, indicating an active transfer of tyrosol into this tissue. This finding, together with the maintenance of constant tyrosol levels during the seven-day storage period, is very promising for the use of chitosan formulations to produce functional tomato fruit.

Development and Characterization of a Novel Peptide-Loaded Antimicrobial Ocular Insert.

Infectious ocular keratitis is the leading cause of blindness worldwide. Bacterial resistance to classical pharmacological treatments raised the interest of researchers towards antimicrobial peptide (AMP)-based therapy. hLF 1-11, a synthetic antimicrobial peptide derived from the N-terminus of human lactoferrin, proved effective against different bacteria and yeast but, like all proteinaceous materials, it is unstable from chemical, physical, and biological points of view. In this study, new freeze-dried solid matrices containing mucoadhesive polymers were prepared and characterized in terms of rheology, hydration time, bioadhesion, drug content, and in vitro release. The formulation HPMC/T2/HA/hLF 1-11fd was selected for the delivery of hLF 1-11, since it showed good drug recovery and no chemical degradation up to at least 6 months (long-term stability). Furthermore, the HPMC/T2/HA/hLF 1-11fd matrix allowed for the release of the drug in a simulated physiological environment, linked to an optimal hydration time, and the peptide antimicrobial activity was preserved for up to 15 months of storage, a very promising result considering the chemical liability of proteinaceous material. For its properties, the freeze-dried matrix developed in this study could be a good platform for the delivery of antimicrobial peptides in the precorneal area to treat infectious phenomena of the ocular surface.

Formulations Based on Natural Ingredients for the Treatment of Nail Diseases.

Nail is a strong and resistant structure, characterized by a low permeability to foreign molecules. Nails can be subjected to many diseases, among which fungal infections (e.g. onchomycosis) are the most common and responsible for nail structure alteration. Many formulations have been produced for the delivery of active ingredients to treat nail disorders, based on newly synthesized active molecules or containing chemical enhancers or chemically-modified polymers able to improve the drug transungual penetration. To avoid permanent alterations of the nail structure due to the use of chemical compounds or organic solvent-based formulation, researchers have developed novel formulations focusing on the use of new natural-based compounds. The purpose of this review is to provide information on the outcoming of natural ingredients-based formulations that have been developed in the last years as potential alternative to chemical-based formulations.

Effect of 5-Oxo-2-Pyrrolidinecarboxylic Acid (PCA) as a New Topically Applied Agent for Dry Eye Syndrome Treatment.

The aim of the study was the evaluation of the suitability of 5-oxo-2-pyrrolidinecarboxylic acid (PCA), also in combination with hyaluronic acid (HA), as artificial tears for treatment of dry eye syndrome (DES). Different aqueous formulations containing 0.10% w/w of PCA were used to determine: (i) ex vivo permeation profile of PCA in isolated rabbit corneas; (ii) in vivo residence time of PCA in the precorneal area of rabbits; and (iii) in vivo ability of PCA to counteract the reduction of tear production in an experimental model of DES induced in rabbits. The pharmacokinetic profile of PCA in tear fluid was characterized by high concentrations immediately after application, followed by a rapid decrease, with half-life values of 17.16 and 22.27 min for solutions containing PCA alone and in combination with HA, respectively, when 100 µL of solutions were instilled. The addition of HA almost doubled the PCA bioavailability minimizing the ex vivo apparent corneal permeability of PCA. A positive Shirmer Test Score (STS) was observed for PCA compared to contralateral eyes at all days of treatment for PCA/HA formulation. PCA provides protection from desiccation probably for its osmoprotective activity and high water⁻binding capability, and this behaviour was enhanced by HA.

Effect of Tumor Relevant Acidic Environment in the Interaction of a N-hydroxyindole-2-Carboxylic Derivative with the Phospholipid Bilayer.

PURPOSE: The inhibitors of the human isoform 5 of lactate dehydrogenase (hLDH5) have attracted growing interest as efficient anti-cancer agents. In the present paper, the interactions between an efficient hLDH5 inhibitor (N-hydroxyindole-2-carboxylic derivative) and lipid bilayers based on dipalmitoylphosphatidylcholine (DPPC) were investigated. Additionally, since interstitial acidification plays a key role in tumor pathogenesis and tumor drug therapy, the effect of acidic pH was assessed and correlated to DPPC/drug interaction. METHODS: Four different techniques were used: differential scanning calorimetry, dynamic light scattering, UV-VIS second derivative spectrometry and attenuated total reflection Fourier transformed infrared spectroscopy. RESULTS: All techniques concur in highlighting a structural change of lipid assembly, susceptible both to pH change and to the presence of the antitumor compound. Lipid vesicles appeared more compact at the lower pH, since the thermal pre-transition from the lamellar gel phase to the ripple gel phase was absent at pH 7.4 and the infrared analysis revealed a stronger acyl chain packing as well as a different hydration degree. Drug interaction was mainly detected in the lipid region including the ester linkages and the first portion of the acyl chains. Furthermore, a lower drug partitioning was recorded at pH 6.6. CONCLUSIONS: The investigated antitumor agent possesses a stable negative charge at the investigated pH values, thus the lower interaction at the acidic pH is mainly ascribable to an environmental effect on lipid assembly. Therefore, drug efficacy under tumor acid conditions may be hampered by the observed lipid membrane constraints, and suggest for the development of suitable prodrugs.