OPHTHALMIC FORMULATIONS PISA
OPHTHALMIC FORMULATIONS: CHALLENGES AND ADVANCES
Poster session
Set up of an in vitro dry eye model for medical device testing
Bray G., Galgani G., Federighi F., Sansò M., Citi V., Calderone V.
Dipartimento di Farmacia, Università di Pisa
Dry eye disease (DED) leads to ocular symptoms like discomfort, pain, redness, and corneal inflammatio Topical formulations (drugs and medical devices (MD)), represent the main strategies to treat DED. In th work, human corneal epithelial 3D tissues (HCE) were used for the set-up of DED in vitro model: tissues were maintained in low humidity and high temperature (Rh 40%; T 40°C) (HYP-DRY) and were treated with: 1) Sorbitol 0.6M in basolateral culture medium for 8h and dexamethasone [1mg/ml] (DEX) and sodium hyaluronate 0.2% + TS-polysaccharide 0.2% (HA + TSP) were added on the apical side, maintain the tissues in HYP-DRY conditions for further 16h. 2) Sorbitol 0.6M added in basolateral culture medium and on the apical side for 8h and the treatments were added on the apical side after washing procedure maintaining the tissues in HYP-DRY conditions for further 16h. At the end of the treatment, the analysis demonstrated that HA + TSP and DEX induced an appreciable reduction in IL-1β release. The barrier eff of HA + TSP was also evaluated as a marker of tissue permeability. HA + TSP was applied on the apical s for 15 min; then a solution of caffeine 5 mg/ml was added. The caffeine permeation was monitored for 2 hours and quantified using HPLC analysis. Caffeine permeability through CT resulted 10.7% after 15 minutes and 37.8% after 2 hours, referring to the initial dose; HA + TSP decreased caffeine permeability nearly 35% during the first 15 minutes and 20% after 2 hours, compared to CT. References 1 Zemanová M. DRY EYE DISEASE. A REVIEW. Cesk Slov Oftalmol. 2021 Winter;77(3):107–119. English. d 10.31348/2020/29. PMID: 34107689.
Pyrroloquinoline quinone exerts neuroprotective effects on retinal ganglion cell degeneration
Alessio Canovai1,2, James R Tribble1, Melissa Jöe1, Rosario Amato2, Maurizio Cammalleri2, Massimo Dal Monte2, Pete A Williams1
1. Department of Clinical Neuroscience, Division of Eye and Vision, St. Erik Eye Hospital, Karolinska Institutet, Stockholm, Sweden.
2. Department of Biology, University of Pisa, Pisa, Italy.
Glaucoma is the leading cause of irreversible blindness, and it has been estimated to affect 80 million patients worldwide. The disease comprises the progressive loss of function and the consequent death o retinal ganglion cells (RGCs). Metabolic and mitochondrial dysfunctions have been recently described a key pathophysiological events occurring in the early stages of glaucoma. In this respect, pyrroloquinolin quinone (PQQ) seems to have the potential as a neuroprotective factor acting on metabolic and mitochondrial dynamics. Here, we investigate the neuroprotective potential of PQQ against RGC degeneration in glaucoma and its related mechanisms. We used biochemical assays to evaluate if PQQ was rapidly used by RGC-related tissues by measuring ATP levels in dissociated retina and isolated opti nerves. We tested the ability of PQQ to induce ATP production in vivo and provide neuroprotection in ex vivo and in vivo models of glaucoma, using biochemical and immunofluorescence techniques. Metabolomics and molecular analyses were performed to assess PQQ effects on metabolism, mitochondrial function and content. The treatment with PQQ rapidly increased ATP levels in dissociated retinal cells and isolated optic nerves. Systemic delivery of PQQ increased ATP production, altered the metabolic profiles of visual system tissues in vivo and protected RGCs from degeneration in animal mod of glaucoma. The long-term administration of PQQ resulted in molecular variations of mitochondrial markers in RGC-related tissues in absence of morphological changes. Taken together, these results suggest that the administration of PQQ might be an effective strategy to protect RGCs from degenerat by altering metabolism, thus offering a safe option to treat glaucoma and other RGC-related neurodegenerative diseases.
Microemulsions and solid lipid nanoparticles as a novel approach in the treatment of macular degeneration: preliminary in vivo studies
G. Chindamo, M. Gallarate, S. Sapino, E. Peira, D. Chirio, G. C. Martinez Navarrete, A. E. Parra Sanchez and E. Fernandez
Dipartimento di Scienza e Tecnologia del Farmaco - UniTO
Treating retinal diseases with repeated anti-VEGF intravitreal (IVT) injections is a common practice in th clinic to treat some diseases such as age-related macular degeneration and diabetic macular edema. Currently, bevacizumab (BVZ), is one of the major anti-VEGF drugs used off-label in ophthalmic practice However, IVT injections can lead to complications, such as endophthalmitis, retinal detachment and vitreous hemorrhage; moreover, they are uncomfortable and therefore can cause low patient complianc The development of novel nanosystems able to extend in-situ permanence of BVZ after IVT injection an to allow its topical administration could provide a solution. The aim of this work was to investigate the efficacy of previously developed BVZ-loaded solid lipid nanoparticles (BVZ-SLN) or microemulsion (BVZ μE) composed of a thermoreversible polymer solution as aqueous phase on Brown Norway Rats from Envigo Laboratories used as animal models. 10 rats divided into 3 groups were taken to perform the IVT injection of SLN and were inoculated with BVZ-SLN, drug-free SLN, or undiluted Avastin®, respectively. Then, lesions were created on the retina level, which will serve to mimic the degenerative ocular patholo Regarding μE, a total of 9 rats divided into groups of 3 rats each which received one drop in each eye of different formulation (drug-free μE, BVZ-μE or a BVZ solution) for at least 20 days. Optical coherence tomographies (OCTs) were performed at scheduled times to verify rats’ conditions. OCT results reveale that 7 days after the IV injection of BVZ-SLN, the depth of the lesion is evidently reduced compared to it dimensions at day 3. Even for the BVZ-μE, a reduction of lesion depth is clearly visible through the OCT after 7 days, comparable with results obtained with BVZ solution. Future tests will further confirm data obtained with the proposed systems repeating these experiments on a larger number of animals also evaluating different conditions.
NS-anti-miR-155-5p formulation slow down retinal degeneration in rd10 mice
Francesca Corsi, Ilaria Piano, Beatrice Polini, Sara Carpi, Giovanni Petrarolo, Luca Quattrini, Maria Cristina Gamberini, Grazia Chiellini, Concettina La Motta, Paola Nieri and Claudia Gargini
Department of Pharmacy - University of Pisa
MicroRNAs (miRNAs) represent interesting therapeutic targets that can regulate common dysregulated pathways underlying retinal damage, and miR-155-5p has emerged as one of the most relevant. Retinitis pigmentosa (RP) refers to a group of inherited eye diseases affecting the retina; therefore, targeting miR 155-5p with a specific inhibitor could be a novel strategy for the treatment of RP. In this study, we investigated the efficacy of an eye drop containing the anti-miR-155-5p antisense oligonucleotide loaded onto a nanometer colloidal vector (NS). Initially, the ability of NS to transport and deliver anti-miR-155-5p into retinal tissue, was demonstrated, C57Bl/6J mice using a fluorescently labeled vector. Then, the efficacy of the NS-anti-miR-155-5p formulation was studied in an animal model of autosomal recessive RP (rd10), starting treatment from postnatal day (P) 18 until P30 or P60. The results show a decrease in miR-155 levels after chronic administration in the treated eye by real-tim PCR compared with the control eye. Functionally, electroretinogram recording showed improvement in the treated eye, suggesting a reduct in degenerative processes. Real-time PCR data and from western blotting analysis in retinal tissues confirm that this improvement could be due to the reduction of inflammatory processes. Overall, we can say that the NS-anti-miR-155-5p formulation could be a viable therapeutic approach for topical treatment of RP.
Nanoassembling systems for the ocular delivery of therapeutic proteins
M. Di Gangi, P. Chetoni, S.Burgalassi, D. Monti, S. Tampucci
Dipartimento di Farmacia, Università di Pisa
The eye is a complex organ with protective mechanisms that can reduce the drugs bioavailability to less than 5% of topically applied dose. Recently, a great attention is being focused on the development of effective drug delivery systems for t treatment of ocular disorders. Nanotechnologies seem promising especially for therapeutic proteins delivery for several advantages: lower toxicity, improved drugs solubility and bioavailability, higher drug accumulation in targeted ocular tissues, reduced dosing frequency. The aim of this research is the development of ophthalmic formulations able to guarantee pharmacolog levels of drug into the back of eyes following topical/periocular administration. Self-assembling nanomicelles based on a single poloxamers, (P188, P407 and P338), or on binary mixture of a poloxame and VitE-TPGS, encapsulating Bovine Serum Albumin (BSA), chosen as model protein, have been prepared. The nanomicelles were prepared by dissolution of different amounts of polymers and differe time of stirring, and were characterized in terms of hydrodynamic diameter, polydispersity and CMC. The increase of poloxamers concentration produced a reduction of mean size and CMC, while the incre of stirring time, from 30 min to 16 hours, determined a size reduction depending on the type of polyme stirring time of 30 minutes has been selected due to the more reproducible results. The size of the nanomicelles increased slightly in presence of appropriate concentrations of BSA (5 mM confirming their absorption capability. A significant reduction in size has been obtained by preparing the nanomicelles with a binary polymer blends containing Vit E -TPGS. Further studies will be performed to verify the interaction of the mixed nanomicelles with BSA.
We acknowledge the support of the European Union by the Next Generation EU project ECS00000017 ‘Ecosistema dell’Innovazione’ Tuscany Health Ecosystem (THE, PNRR, Spoke 4: Nanotechnologies for diagnosis and therapy)
Identification of new rhodopsin-stabilizing compounds through computational and biological investigations
Di Stefano Miriana, Demontis Gian Carlo, Galati Salvatore, Poli Giulio, Macchia Marco and Tuccinardi Tiziano
University of Pisa
Rhodopsin, a G-protein coupled receptor (GPCR) protein present in the rod cells of the retina, comprise two primary constituents: an opsin protein and a light-sensitive molecule known as 11-cis retinal. When light enters the eye, the phototransduction mechanism starts: 11-cis retinal isomerizes to all-trans-retina causing a conformational change in the opsin and triggering a series of biochemical reactions that eventually lead to the generation of electrical signals that are transmitted and processed by the brain to generate a visual perception. Mutations in RHO, the gene responsible for encoding rhodopsin, can resu various eye disorders, including retinitis pigmentosa (RP). Some RHO mutations contribute to the disappearance of rod cells by inducing misfolding of opsin or compromising its stability. In vitro studies have indicated that small chaperones based on retinoids can partially restore the proper folding and transportation of P23H RHO, a mutant form of rhodopsin. However, these chaperones have limitations d to photoinduced isomerization during protein synthesis in the endoplasmic reticulum, leading to protein instability. For this reason, the aim of this work was to identify new non-retinoid compounds capable of stabilizing wild-type rhodopsin (wt). A receptor-based virtual screening (VS) study, incorporating pharmacophore modeling, consensus docking, and molecular dynamics (MD) simulations, was conduct to achieve this objective. Preliminary biological studies suggest that some compounds identified in the study bind to rhodopsin, compete with 9-cis retinal, and reduce rhodopsin regeneration, similar to the effects of beta-ionone, a known ligand for this receptor. The potential stabilizing effect will also be assessed in the mutated form of rhodopsin to evaluate its impact on misfolding, which represents a primary objective in the development of a potential pharmacological strategy for treating RP.
This research was supported by :
Progetto NET-2016-02363765
Ministero della Salute
Regione Toscana
An in-vitro retinal model to test ophthalmic drugs
Alessio Esposito, Gabriele Maria Fortunato, Carmelo De Maria, Giovanni Vozzi
Department of Information Engineering and Research Center Enrico Piaggio, University of Pisa, Italy
The eye, a complex sensory organ, allows us to see and consists of two segments: the front (cornea, conjunctiva, etc.) and the back (vitreous humour, retina, etc.). It serves as a pathway for delivering drugs systemically and locally in the body, with various methods of administration. However, drug passage is impeded by biological barriers, including the Blood-Retinal-Barrier (BRB) that controls the exchange between blood and the retina. The BRB has two distinct regions: the inner BRB, composed of tight junctions in retinal capillary cells, prevents free molecule passage through retinal vessels, and the outer BRB, formed by a tight layer of retinal pigment epithelial cells, blocks substances from entering the neu retina. To address this issue, this project aims to develop an in vitro device that replicates the eye's biological barriers, facilitating the testing of ophthalmic drugs while adhering to the 3R principles. To recreate the Blood-Retinal Barrier (BRB) a circuit that imitates the capillary network of the retina, with a membrane (Bruch’s membrane) placed on top to cultivate a layer of retinal pigment epithelial (RPE) cell was designed and realized with advanced biofabrication techniques. To ensure stability and ease of use the entire structure will be enclosed in a case, holding the components in place.
Citrus fruit polyphenols for ocular antisepsis
Diletta Mazzantini, Mariacristina Massimino, Marco Calvigioni, Francesco Celandroni, Emilia Ghelardi
Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy
Endophthalmitis, a severe microbial infection of the eye that can lead to blindness, represents one of th most critical complication of intraocular surgery. Prophylaxis for eye surgery includes topical antisepsis the adoption of general hygiene measures, and the application of topical antibiotics. Nevertheless, in th era of antibiotic resistance, research is progressively focusing on the use of antimicrobial compounds (e antiseptics) that can prevent and control microbial infections of the eye. To this regard, a new liposomal ocular solution (named OS) was recently introduced in the market as ocular antiseptic. OS contains a mixture of polyphenols extracted from citrus fruits (named B) as active ingredient with antimicrobial activity. This study aimed at investigating the activity of OS and B toward Gram-positive and Gram-negative bacteria, including antibiotic resistant strains. Minimal inhibitory (MICs) and Minimal Bactericid (MBCs) concentrations of OS and B were determined. For B, time kill tests were additionally performed result, OS and B showed bactericidal effect and a wide-spectrum antimicrobial activity, also extended to multidrug resistant bacteria. As evidenced by the killing assay, most of the reduction in bacterial viabilit occurred within the first minute of exposure to B and no residual living cells were obtained starting from minutes of incubation. Overall, our results support the use of OS and B as prophylactic approach in ophthalmology to prevent microbial infections.
This research was supported by PNRR THE - Tuscany Health Ecosystem - Spoke 7 – Innovating Translational Medicine - Sub-project 5- Innovative models for management of infections caused by antibiotic-resistant bacteria (Project code: ECS00000017; CUP: I53C22000780001).
Nutritional support with spearmint (Mentha spicata L.) extract in a rat model of hypertensive glaucoma
Alberto Melecchi1, Rosario Amato1, Alessio Canovai1, Samanta Maci2, Filipa Quintela2, Brenda A. Fonseca3 , Maurizio Cammalleri1,4, Massimo Dal Monte1,4
1. Department of Biology, University of Pisa, Pisa, Italy
2. Kemin Human Nutrition and Health a division of Kemin Foods L.C., Lisbon, Portugal
3. Kemin Foods L.C., Des Moines, IA, USA
4. Interdepartmental Research Center Nutrafood “Nutraceuticals and Food for Health”, University of Pisa Pisa, Italy
Glaucoma is characterized by progressive loss of retinal ganglion cells (RGCs) leading to blindness. Wh the role of intraocular pressure (IOP) elevation in glaucoma has been established, reduction of oxidative stress and inflammation have emerged as promising targets for neuronal tissue supporting glaucoma management. Therefore, we evaluated the effect of a proprietary spearmint extract (SPE) on RGC dens activity, and neuronal health markers in a rat model of hypertensive glaucoma. Methods: Animals were divided in four groups: untreated healthy control and 3 glaucomatous groups receiving either orally administered vehicle, SPE-low or SPE-high dose for 28 days. Ocular hypertension was induced through intracameral injection of methylcellulose (MCE) at day 15. Vehicle, or SPE were administered daily by oral gavage before and after MCE injection. At day 29, rats underwent electroretinogram (ERG) recordings, and retinas were analyzed for RGC density and markers of neural trophism, oxidative stress, and inflammation. Results: SPE exerted a dose-dependent response benefits on all markers compared to vehicle-treated MCE rats except for IOP elevation. SPE significantly improved RGC-related ERG responses, cell density neurotrophins, oxidative stress and inflammation markers. Also, in SPE-high rats most of the parameter were not statistically different from healthy controls. Conclusions: SPE, a plant-based, polyphenolic extract, could be an effective nutritional support for neuronal tissue. These results suggest that SPE may not only be a complementary approach for the nutritional management of glaucoma but may also serve as nutritional support in other eye-brain conditions where antioxidant, anti-inflammatory and neuroprotective mechanism are often disrupted.
Epigenetic approach to delay cone loss in Retinitis Pigmentosa mice
Orsini N., Carullo G., Piano I., Corsi F., Fontana A., Napoli D., Di Marco B., Galante A., Salamone G., Gargini C., Campiani G., Strettoi E.
Neuroscience Institute of CNR University of Florence
Retinitis Pigmentosa (RP) is a group of retinal dystrophies leading to progressive photoreceptor degeneration, retinal pigment epithelium (RPE) abnormalities and culminating into blindness. Usually, t primary mutation affects rods but cones suffer from a so-called bystander effect, leading to their secondary death. A promising therapy for RP is delaying the secondary death of cones to rescue daylig vision. Previous studies have demonstrated that Histone Deacetylases (HDACs), which act as epigeneti modulators by deacetylating histones, are altered in various neurodegenerative diseases, including RP. HDACs can also target non-histone proteins, further influencing gene expression and cellular functions. Thus, regulating the activity of HDACs is a promising cure to delay neurodegeneration. We investigated the effect of a novel synthesized inhibitor (called 5d) specifically targeting HDAC6. We used a human RP cell line (ARPE-19) and the rd10 mouse model of RP, mimicking an autosomal recessive form of RP. Different doses of the compound were tested in vitro to determine the less toxic dose, yet effective in inhibiting HDAC. We also tested 5d in vivo to find the therapeutical dose. Data obtained by immunohistochemistry (IHC) show that a single intravitreal injection of 5d in rd10 mouse eyes leads to a evident maintenance of cone morphology and increased survival Electroretinogram (ERG) recordings demonstrate a measurable higher preservation of cone function in rd10 treated mice. An improvement the integrity of RPE structure can also be observed. Real time PCR results indicate that a potential mechanism of action of 5d involves an upregulation of glucocorticoid receptor expression, crucial for regulating and damping the inflammatory response. In conclusion, this study highlights the capability o newly HDAC6 inhibitor to promote a morphological and functional preservation of cones, delaying their loss and extending the time window of useful vision in RP.
Funded by: Velux Foundation, Zurich, (CH) (project #1236); Next Generation EU – National Recovery and Resilience Plan (PNRR), Tuscany Health Ecosystem – THE", Spoke 8
Ocular application of oleuropein in dry eye treatment
V. Paganini, D. Monti, P. Chetoni, S. Tampucci, S. Lari, S. Burgalassi
Department of Pharmacy - University of Pisa
Oleuropein (OLE) is known for its numerous pharmacological properties, but its activity in the ocular fie has not yet been investigated. The study aims to verify a possible use of OLE-based eye drops in dry ey syndrome induced by blue light oxidative stress. OLE has a poor water solubility and stability, and a high sensitivity to light (UV-C) and temperature whic does not make it the optimal candidate for an ophthalmic formulation. To solve these problems a Drug- Cyclodextrin-in-Liposome (DCL) system was developed. OLE was co-precipitated with HP--cyclodextri and the obtained complex was encapsulated into liposomes prepared by hydration of a lipid film with phosphate buffer. DCL system greatly increased the OLE stability in aqueous solution, especially at 4° C (t50% = 156 days). DCL systems containing 0.14, 0.20 and 0.23 mg/ml of OLE were used to treat rabbit corneal epithelial (RCE) cells. The results show that OLE is able of maintaining physiological values of ROS when used in formulations at a concentration of 0.14 and 0.20 mg/ml, as the concentration increases, OLE also reduc the basal levels of ROS. Subsequently, RCE cells were irradiate with blue light (405 nm; 36.70 μW/cm2) for 30 min. After 24 h fro irradiation, ROS production was determined showing OLE-lipo20 formulation restores the values of RO to a basal level and OLE-lipo23 even decreases it up to 80%, while OLE-lipo14 is unable to counteract th production of ROS. OLE-based DCL system seem a good approach to prepare formulations for ocular application. Moreover, this experimental work is part of a circular economy framework where a nanostructured prod based on polyphenols, obtained with standardized extraction processes starting from olive waste materials has been developed and characterized.
Development of nanogels based on natural polymers loaded with naringenin-β-cyclodextrin complex for the treatment of diabetic retinopathy
G. Zucca, B. Vigani, C. Valentino, M. Ruggeri, N. Marchesi, A. Pascale, G. Sandri, S. Rossi
Department of Drug Sciences, University of Pavia, 27100 Pavia
The present work aims to develop nanogels, made of chondroitin sulfate (CS, Bioiberica, I) and low molecular weight chitosan (lCH, Sigma Aldrich, I), loaded with naringenin-β-cyclodextrin complex (NAR CD), for the treatment of diabetic retinopathy. Fifteen different formulations were prepared (from N1 to N15) and different experimental variables were considered such as total polymer concentration (1, 0.75, mg/ml), lCH:CS ratio (1.5:1, 3:1, 1:1 v/v) and pH value (5.5, 4.0, 3.0). Nanogels were characterized in term size (DLS), Z potential (ELS) (Litesizer 500, Anton Paar s.r.l. GmbH, A) and morphology (SEM, Tescan Mi XMU, Brno, CR; TEM, JEOL JEM-1200 EXIII). In vitro studies were also performed on Human Umbilical V Endothelial Cells (HUVEC) to investigate nanogels biocompatibility and cellular uptake. NAR/β-CD was prepared from two stock solutions of NAR (Sigma Aldrich, I) and β-CD (Giusto Faravelli, I), respectively i ethanol and MilliQ water (1:1 molar ratio). NAR/β-CD was characterized in terms of complexation efficie % (CE%) and morphology. Solubility studies were also performed by spectrophotometric analysis (Lamb 25 UV/VIS, PerkinElmar, USA). Finally, NAR/β-CD was loaded into the most promising nanogel. The load nanosystems were characterized in terms of size, zeta-potential, loading capacity (LC%) and cell biocompatibility. The most promising sample resulted to be N9 (0.5 mg/ml total polymer concentration lCH:CS= 1:1; pH=5.5), characterized by the lowest size (< 300 nm), a positive zeta potential, biocompatibility higher than the control and capability to be internalized into HUVEC cells. NAR/β-CD w characterized by a promising CE% (71) and an increase of water solubility (305%) as compared to free N No statistically significant difference between empty and loaded N9 in terms of size was detected. The LC% was equal to 18.4. Finally, in vitro studies on cells confirmed the loaded-nanogel biocompatibility.