Reconstituted epithelial tissues and native cornea: A comparison of the influence of surfactants on ocular permeability

The aim of this study was to prepare an artificial rabbit corneal epithelium (RRCE) to compare with a human corneal epithelial model and excised rabbit cornea through permeation studies to investigate differences of surfactants influence on ocular permeability of a lipophilic compound. First solubility assays with different surfactants were performed and the integrity of the RRCE was assessed by measuring transepithelial electrical resistance (TEER). The permeation parameters showed that the RRCE was more sensitive than native cornea and human cornal epithelial model to the effect of permeation enhancers

Polyvinyl alcohol/cellulose hydrogel as possible corneal stroma substitute in drug permeation tests

The permeation studies of active compounds and formulations are necessary to verify the capability of molecules to pass through the physiological barrier of our body. In order to develop a 3D model of the cornea, preparation and characterization of different hydrogels as corneal stroma substitutes were tested

Development of ophthalmic formulations and design of cell-based assays to evaluate their tolerability and efficacy

In this project three different topics were studied: possible formulations for the treatment of the Dry Eye Syndrome (DES) and Glaucoma and development of hydrogels to mimic corneal stroma in permeation studies. For the DES section, three different formulations were developed and evaluated: 1) oleuropein (OLE) complexed in hydroxypropyl-β-cyclodextrin (HP-β-CD) and encapsulated into a liposomal vesicular system in order to improve its stability. This formulation was tested for its protective activity from hyperosmotic stress and antioxidant activity on rabbit corneal epithelial cells. 2) Artificial eye-drops containing three ingredients : phospholipids, to restore the lipid layer of tear fluid; trehalose, as osmoprotectant molecule, and hyaluronic acid (HA), to keep the ocular surface moist. The aim of this project was to investigate the contribution of the individual components and any synergistic effects on dry eye protection and therefore an in vitro assay based on immortalized rabbit corneal epithelial cells was developed. 3) Cyclosporine-A (CyA) loaded nanomicellar formulation inside in situ gelling system to prolong the corneal contact time and improve the corneal penetration of the drug. The Glaucoma section was focused on an inhibitor of monoacylglycerol lipase (MAGL) which is the main hydrolytic enzyme of 2-arachidonoylglycerol (2-AG) as possible anti-glaucoma molecule. In fact, the modulation of the 2-AG levels is a promising pharmacological strategy to activate the endocannabinoid system leading to a reduction of the intraocular pressure (IOP) and therefore a possible treatment in glaucoma, that is strictly related to an increment in IOP. The section III is focused on the ethical problem of the large use of animals to test drugs and formulations. Hence, the study of possible artificial structures that simulate the cornea in permeation studies where the main barrier is the epithelium, but also the stroma plays an important role. For the native composition of the stroma, hydrogels seem promising structures as its substitutes in the development of an entire cornea construct. Therefore, this part of the project was focused on the preparation and characterization of different blends of hydrogels: polyvinyl alcohol/gelatin, polyvinyl alcohol/cellulose and chitosan hydrogels

Proprietà nutraceutiche del pomodoro arricchito con sostanze ad attività antiossidante

Sono ormai note le proprietà benefiche delle sostanze ad attività antiossidante nella prevenzione e terapia di numerose patologie, grazie alla loro azione nel contrastare gli effetti dannosi dei radicali liberi. Il pomodoro è un alimento importante dal momento che contiene notevoli quantità e diversità di prodotti antiossidanti quali carotenoidi, tra cui il più importante è il licopene, β-carotene (pro-vitamina A), vitamina C ed E, oltre alla presenza di polifenoli. Il contenuto in sostanze antiossidanti varia in base a numerosi fattori tra cui gli stress artificiali, che possono avvenire durante la raccolta e stoccaggio del frutto, ed aggressioni batteriche e fungine, che ne compromettono la qualità. Negli ultimi anni, alla scopo di favorire la conservazione degli alimenti, sono stati studiati rivestimenti edibili con azione sia di barriera protettiva che di veicoli per composti bioattivi, migliorando le proprietà funzionali del prodotto e promuovendone la loro azione nutraceutica. A tal scopo nella seguente tesi è stato impiegato un rivestimento edibile a base di chitosano, di cui erano già stati dimostrati gli effetti positivi in termini di conservabilità e regressione dello sviluppo di specie patogene sul prodotto trattato. Tale veicolo è stato addizionato di tirosolo, composto fenolico ad attività antiossidante, e valutata la capacità di questa molecola di permeare attraverso la cuticola di pomodoro al fine di aumentare le proprietà nutraceutiche del frutto. Nella presente tesi, in primo luogo, sono stati effettuati studi di permeazione in vitro attraverso la cuticola di pomodoro di dispersioni a base di chitosano con concentrazioni differenti di tirosolo per verificare la capacità di questo composto di attraversare tale barriera e penetrare nel frutto. Successivamente, la formulazione, risultata maggiormente promettente dagli studi in vitro, è stata applicata sui pomodori dopo la raccolta e lasciata in contatto con il pomodoro per differenti tempi fino a 7 giorni (0, 3, 5 e 7 giorni). La quantità di tirosolo presente nei pomodori a diversi tempi di contatto era determinata dopo aver sottoposto il materiale a liofilizzazione, estrazione ed analisi quantitativa mediante HPLC. Inoltre l’attività antiossidante del pomodoro arricchito è stata valutata in confronto con i pomodori non trattati usati come controllo a ciascun tempo in esame

Formulations based on natural ingredients for treatment of nail diseases

The 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 (i.e. onchomycosis) are the most common and responsible of 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

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-β-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

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®

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®

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 L 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