Anxo Fernández-Ferreiro

In vitro and in vivo ocular safety and eye surface permanence determination by direct and Magnetic Resonance Imaging of ion-sensitive hydrogels based on gellan gum and kappa-carrageenan

Gellan gum, kappa-carrageenan and alginates are natural polysaccharides able to interact with different cations that can be used to elaborate ion-activated in situ gelling systems for different uses. The interaction between fluid solutions of these polysaccharides and cations presents into the tear made these biopolymers very interesting to elaborate ophthalmic drug delivery systems. The main purpose of this study is to evaluate the ability of mixtures of these polymers to obtain ion-activated ophthalmic in situ gelling systems with optimal properties for ocular use. To achieve this purpose different proportion of the biopolymers were analyzed using a mixture experimental design evaluating their transparency, mechanical properties and bioadhesion in the absence and presence of simulated tear fluid. Tear induces a rapid sol-to-gel phase transition in the mixtures forming a consistent hydrogel. The solution composed by 80% of gellan gum and 20% kappa-carrageenan showed the best mechanical and mucoadhesive properties. This mixture was evaluated for rheological behavior, microstructure, cytotoxicity, acute corneal irritancy, ex-vivo and in vivo ocular toxicity and in vivo corneal contact time using Magnetic Resonance Images (MRI) techniques. Result indicates that the system is safe at ophthalmic level and produces an extensive ocular permanence higher than 6h.

Cyclodextrin–polysaccharide-based, in situ-gelled system for ocular antifungal delivery

Summary Fluconazole was studied with two different hydrophilic cyclodextrins (hydroxypropyl-β-cyclodextrin (HPBCD) and sulfobutyl ether-β-cyclodextrin (SBECD)) for the formation of inclusion complexes. HPBCD and SBECD showed low cell cytotoxicity in human keratocytes as assessed by the label-free xCELLigence system for real-time monitoring. The fluconazole–HPBCD complex was incorporated into an ion-sensitive ophthalmic gel composed of the natural polysaccharides gellan gum and κ-carrageenan. This system showed good bioadhesive properties and effective control of fluconazole release.

PET/CT imaging of 3D printed devices in the gastrointestinal tract of rodents

Fused deposition modelling (FDM) 3D printing (3DP) is a revolutionary technology with the potential to transform drug product design in both the pre-clinical and clinical arena. The objective of this pilot study was to explore the intestinal behaviour of four different polymer-based devices fabricated using FDM 3DP technology in rats. Small capsular devices of 8.6 mm in length and 2.65 mm in diameter were printed from polyvinyl alcohol-polyethylene glycol graft-copolymer (PVA-PEG copolymer, Kollicoat IR), hydroxypropylcellulose (HPC, Klucel), ethylcellulose (EC, Aqualon N7) and hypromellose acetate succinate (HPMCAS, Aquasolve-LG). A smaller sized device, 3.2 mm in length and 2.65 mm in diameter, was also prepared with HPMCAS to evaluate the cut off size of gastric emptying of solid formulations in rats. The devices were radiolabelled with Fluorodeoxyglucose (18F-FDG) and small animal positron emission tomography/computed tomography (microPET/CT) was used to track the movement and disintegration of the fabricated devices in the rats. The PVA-PEG copolymer and HPC devices disintegrated after 60min following oral administration. The EC structures did not disintegrate in the gastrointestinal tracts of the rats, whereas the HPMCAS-based systems disintegrated after 420 min. Interestingly, it was noted that the devices which remained intact over the course of the study had not emptied from the stomach of the rats. This was also the case with the smaller sized device. In summary, we report for the first time, the use of a microPET/CT imaging technique to evaluate the in vivo behaviour of 3D printed formulations. The manipulation of the 3D printed device design could be used to fabricate dosage forms of varying sizes and geometries with better gastric emptying characteristics suitable for rodent administration. The increased understanding of the capabilities of 3DP in dosage form design could, henceforth, accelerate pre-clinical testing of new drug candidates in animal models.

Chitosan and Kappa-Carrageenan Vaginal Acyclovir Formulations for Prevention of Genital Herpes. In Vitro and Ex Vivo Evaluation.

Vaginal formulations for the prevention of sexually transmitted infections are currently gaining importance in drug development. Polysaccharides, such as chitosan and carrageenan, which have good binding capacity with mucosal tissues, are now included in vaginal delivery systems. Marine polymer-based vaginal mucoadhesive solid formulations have been developed for the controlled release of acyclovir, which may prevent the sexual transmission of the herpes simplex virus. Drug release studies were carried out in two media: simulated vaginal fluid and simulated vaginal fluid/simulated seminal fluid mixture. The bioadhesive capacity and permanence time of the bioadhesion, the prepared compacts, and compacted granules were determined ex vivo using bovine vaginal mucosa as substrate. Swelling processes were quantified to confirm the release data. Biocompatibility was evaluated through in vitro cellular toxicity assays, and the results showed that acyclovir and the rest of the materials had no cytotoxicity at the maximum concentration tested. The mixture of hydroxyl-propyl-methyl-cellulose with chitosan- or kappa-carrageenan-originated mucoadhesive systems that presented a complete and sustained release of acyclovir for a period of 8–9 days in both media. Swelling data revealed the formation of optimal mixed chitosan/hydroxyl-propyl-methyl-cellulose gels which could be appropriated for the prevention of sexual transmission of HSV.

[Analysis of ocular toxicity of fluconazole and voriconazole eyedrops using HET-CAM].

PURPOSE The objective of the study is to provide toxicological information through the HET-CAM test of Fluconazole and Voriconazole eye drops prepared commonly in Pharmacy Services for the treatment of fungal keratitis. METHOD Experimental Study. The ocular toxicity of topical voriconazole 10 mg/ml and fluconazole 2 mg/ml were evaluated by the hens egg test (HET) on the chorioallantoic membrane (CAM). The effects on blood vessels were based on its behavior during 300 seconds and processes that may occur at each time, then we calculated the irritation index (is, irritation score). RESULTS AND CONCLUSIONS Both eye drops, voriconazol and fluconazole have been proven to be safe, since the IS that we obtained was zero for both samples and did not present significant signs of irritation. Therefore, these eyedrops are considered suitable for ocular use from a toxicological point of view.

Ocular safety comparison of non-steroidal anti-inflammatory eye drops used in pseudophakic cystoid macular edema prevention.

Non-steroidal anti-inflammatory drug (NSAID) eye drops are widely used to treat ocular inflammatory conditions related to ophthalmic surgical procedures, such as pseudophakic cystoid macular edema, and they have been used for off-label treatments. The most commonly used NSAIDs are diclofenac and ketorolac and the new molecules bromfenac and nepafenac have also been used. We used primary human keratocytes in cell culture in combination with a novel technology that evaluates dynamic real-time cytotoxicity through impedance analysis. This study also included classic cell viability tests (WST-1(®) and AlamarBlue(®)), wound healing assay, Hens Egg Test and an ex vivo histopathological assay. NSAIDs were shown to have important cytotoxicities and to retard the healing response. Furthermore, the new eye drops containing bromfenac and nepafenac were more cytotoxic than the more classical eye drops. Nevertheless, no immuno-histochemical changes or acute irritation processes were observed after the administration of any eye drops tested. Due to cytotoxicity and the total absence of discomfort and observable injuries after the administration of these drugs, significant corneal alterations, such as corneal melts, can develop without any previous warning signs of toxicity.

Positron Emission Tomography for the Development and Characterization of Corneal Permanence of Ophthalmic Pharmaceutical Formulations.

Purpose This work is aimed at describing the utility of positron emission tomography/computed tomography (PET/CT) as a noninvasive tool for pharmacokinetic studies of biopermanence of topical ocular formulations. Methods The corneal biopermanence of a topical ophthalmic formulation containing gellan gum and kappa carragenan (0.82% wt/vol) labeled with 18Fluorine (18F) radiotracers (18F-FDG and 18F-NaF) was evaluated by using a dedicated small-animal PET/CT, and compared with the biopermanence of an aqueous solution labeled with the same compounds. Regions of interest (ROIs) were manually drawn on the reconstructed PET images for quantifying the radioactivity concentration in the eye. The biopermanence of the formulations was determined by measuring the radioactivity concentration at different times after topical application. Additionally, cellular and ex vivo safety assays were performed to assess the safety of the performed procedures. Results Differences were observed in the ocular pharmacokinetics of the two formulations. After 1.5 hours of contact, 90% of the hydrogel remained in the ocular surface, while only 69% of the control solution remained. Furthermore, it was observed that flickering had a very important role in the approach of the trial. The application of 18F-FDG in the eye was neither irritating nor cytotoxic for human corneal epithelial cells. Conclusions The use of small-animal PET and 18F radiotracers in ocular pharmacokinetics of ophthalmic formulations is feasible and could be a safe method for future ocular pharmacokinetic studies in humans.

In vivo eye surface residence determination by high-resolution scintigraphy of a novel ion-sensitive hydrogel based on gellan gum and kappa-carrageenan

&NA; In last years, sensitive hydrogels have become a breakthrough in ophthalmic pharmaceutical technology aimed at developing new strategies to increase the residence time of active substances. In a previous paper, we qualitatively demonstrated the capacity of a new ion sensitive hydrogel to increase the residence time. Nevertheless, the clearance of the gel from the ocular surface was not quantifiable with the used methodology. The aim of the present work was to use a well‐established approach based on scintigraphy to quantitatively estimate the residence time of the previously proposed hydrogel. The rat corneal residence time of a topic ophthalmic formulation containing gellan gum and kappa carragenan (0.82% w/v) labeled with 99mTc‐DTPA radiotracer was evaluated and compared with the residence of an aqueous solution. Ophthalmic safety studies such as eye irritation or passage through the cornea were also carried out. After 1.5 h of contact, 77% of the hydrogel remained in the ocular surface, presenting kinetics of disappearance one‐phase decay and a half time of 262 min. We conclude that the novel ophthalmic hydrogel developed with kappa carrageenan and gellan gum remains for long periods of time on the corneal surface, presenting a drop that fits an exponential decay. Graphical abstract Figure. No caption available.

Análisis de la toxicidad ocular de los colirios de voriconazol y fluconazol con HET-CAM

Purpose: The objective of the study is to provide toxicological information through the HET-CAM test of Fluconazole and Voriconazole eye drops prepared commonly in Pharmacy Servi - ces for the treatment of fungal keratitis. Method: Experimental Study. The ocular toxicity of topical voriconazole 10 mg/ml and fluconazole 2 mg/ml were evalua ted by the hen‘s egg test (HET) on the chorioallantoic membrane (CAM). The effects on blood vessels were based on its behavior during 300 seconds and processes that may occur at each time, then we calculated the irritation index (is, irritation score). Results and conclusions: Both eye drops, voriconazol and fluconazole have been proven to be safe, since the IS that we obtained was zero for both samples and did not present significant signs of irritation. Therefore, these eyedrops are conside red suitable for ocular use from a toxicological point of view.

Cysteamine polysaccharide hydrogels: Study of extended ocular delivery and biopermanence time by PET imaging

Cystinosis is a rare autosomal recessive disorder in which cystine crystals accumulate within the lysosomes of various organs, including the cornea. Ocular treatment is based on the administration of cysteamine eye drops, requiring its instillation several times per day. We have introduced the cysteamine in two types of previously developed ocular hydrogels (ion sensitive hydrogel with the polymers gellan gum and kappa-carrageenan and another one composed of hyaluronic acid), aiming at increasing the ocular retention in order to extend the dosing interval. The biopermanence studies (direct measurements and PET/CT) show that these formulations present a high retention time on the ocular surface of rats. From the in vitro release study we determined that both hydrogels can control the release of cysteamine over time, showing a zero order kinetics during four hours. At the same time, these hydrogels could act as corneal absorption promoters, as they allow a higher permeation of cysteamine through bovine cornea compared to a solution. HET-CAM test and cytotoxicity assays show no irritation on the ocular surface. These results demonstrate that the developed formulations present a high potential as vehicles for the topical ocular administration of cysteamine.