Pharmaceutical Development of Innovative Formulations for Ocular Drug Delivery

In the context of my PhD, focused on the development of new sustained ocular drug delivery systems (SDDS), three different formulations were developed: extruded and molded inserts, freeze-dried inserts and three nanomicelles-based formulations (eye-drops, solid inserts, and in situ gelling systems). First, from a collaboration with the University of Milan (research group held by Prof. Andrea Gazzaniga, Department of Pharmacy), hot-melt extruded (HME) and injection molded (IM) long-lasting intravitreal ocular inserts containing fluocinolone acetonide (FA) for the treatment of the age-related macular degeneration (AMD) were developed. Different polymeric materials were tested to produce the inserts with the two techniques. The inserts based on a high-amylose maize starch resulted the most promising, since it showed biocompatible characteristics and low swelling capacity, essential features for an intravitreal insert. They were then characterized by a physicochemical (DSC, FTIR, morphological analysis) and technological (in vitro release performance) point of view. From in vitro release experiments, reproducing the intravitreal environment, the insert produced by hot-melt extrusion resulted the most promising, since it released the drug slower than the same molded inserts up to 56 days. In conclusion, HME and IM techniques appeared suitable for developing long acting ocular inserts to apply in the posterior segment of the eye. Secondly, round-shaped ocular inserts produced by freeze-drying technique were explored as a promising platform for the delivery of antimicrobial peptides to treat ocular keratitis. Lyophilization was chosen, since protein-like drug are thermolabile and instable in aqueous solutions. Different mucoadhesive polymers, i.e. hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose (CMC), and sodium hyaluronate (HA), were used alone or in combination to improve the formulation resident time and drug bioavailability. Bulking (mannitol) and cryoprotectant (trehalose, T) agents were added. A preliminary scale-up of the developed formulation was done to evaluate the efficacy and reproducibility of the freeze-drying cycle, which resulted suitable for the manufacturing of lyophilized matrices, since no statistically significant physicochemical alterations (in terms of pH, osmolality, viscosity, and weight variation) after freeze-drying cycle neither biological incompatibility were found. Furthermore, matrices containing only HPMC and a mixture of HPMC and HA seem to hydrate slower than the matrix containing a mixture of HPMC and CMC and maintaining their mucoadhesive property even after high dilutions. Moreover, DSC results have shown that matrices containing trehalose are more homogeneous than the others developed matrices. Medicated matrices, loaded with vancomycin as model drug, were screened based on drug recovery and in vitro drug release performance. The matrices containing trehalose, used as cryoprotectant agent, have shown the best features in terms of drug recovery and in vitro drug release performance, whereby they were subjected in vivo precorneal pharmacokinetic. The formulation HPMC/T2/HA (containing HPMC and HA) was selected to be loaded with the target peptide. Peptide-loaded matrix showed a good drug recovery, no any chemical degradation up to at least 6 months, and antimicrobial activity up to at least 15 months, a very promising result considering the chemical lability of proteinaceous material. This part of experiment was in collaboration with the Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, research group held by Prof. Antonella Lupetti and Prof. Emilia Ghelardi. In the light of the results, freeze-dried matrix could be a good platform for the delivery of antimicrobial peptides for the topical treatment of ocular keratitis. Finally, three different naomicelles-based DDS were developed to increase the Cyclosporine-A (CyA) water solubility and ocular bioavailability. First of all, different nanomillar formulations were developed by adding different concentrations of sodium hyaluronate (0.001, 0.01, and 0.1%) to different total amount (1.5, 1.0, 0.5 %) of a mixture of Vit E-TPGS and OPEE (2.25:1) surfactants. A clear water dispersion was obtained for all the developed formulations. A DoE study was performed, from which the formulation Nano1HAB-CyA (containing the 1%wt of surfactants and the 0.01% of HA) was selected as the most potential performing formulation, since it was able to entrap a high amount of CyA, allowing to reach the desired drug concentration of 0.1%, as the marketed emulsion Ikervis®. From a preliminary scale-up of the selected formulation, a good reproducibility of the manufacturing method was found. Moreover, Nano1HAB-CyA revealed a suitable size (about 12 nm) to have a good biocompatibility, thermal and chemical stability, and capacity to enhance drug transport in a hydrophilic environment. The studied formulation was not cytotoxic, and HA seemed to have a protective effect. In vitro experiments on Nano1HAB-CyA using reconstituted corneal tissues showed that the nanostructured system enhanced CyA transport through and the accumulation in the tissue preserving its integrity. Furthermore, ex-vivo transscleral permeation experiments showed that the drug released from Nano1HAB-CyA tent mostly to accumulate into the scleral tissue, acting as potential reservoir for the delivery of CyA to the back of the eye. Moreover, fluorescent nanomicelles were developed and used in the in vitro permeation studies on cornea and sclera, revealing an enhancing ability of sodium hyaluronate to improve the nanomicelles interaction with the targeted tissues thanks to its well-known mucoadhesive properties. Finally, in vivo pharmacokinetics studies demonstrated that the combined system (nanomicelles + HA) prolonged the permanence of CyA in the precorneal area, without producing any evident irritation phenomena on the rabbit eye, suggesting a good tolerability of the developed formulation. Secondly, to optimize the previous nanomicellar formulation (Nano1-CyA), HA was removed and Nano1-CyA formulation loaded into a polymeric insert. This project was realized in collaboration with Kingston University London, Department of Pharmacy, research group held by Prof. Raid Alany. The ocular devices were prepared by mixing the nanomicellar and polymeric dispersions together and then the film was produced by solvent casting. Then, the film was cut to obtain final round-shaped inserts (diameter 7 mm). Developed inserts were assessed for drug-polymer interactions (DSC and FTIR), physical characteristics (weight, thickness, drug content, surface pH), and mechanical properties (tensile strength and folding endurance). Inserts showed a CyA content corresponding to a daily dosage of Ikervis® Europe marketed emulsion (0.1%CyA). Moreover, contact angle measurements, morphological analysis (SEM), and in vitro dissolution tests were performed on all the developed inserts. From these results, the best formulation in terms of physicochemical characteristics, drug dissolution velocity and morphological analysis contained a mixture of mucoadhesive polymers, i.e. poly(vinyl alcohol) (PVA), carboxymethylcellulose (CMC), xanthan gum (XG), and sodium alginate (ALG) with Nano1-CyA dispersion (F3). Besides, it presented a smooth surface, without debris and no incompatibilities among the components. Then, the Nano-CyA ability to reorganize itself after insert rehydration in water was confirmed by DLS. Moreover, after UV-sterilization using UVC ray, F3 insert showed improved mechanical properties in term of flexibility thus potentially more comfortable for long wearing, since the insert was thought for a daily administration. Furthermore, the homogeneity of nanomicelles into F3 insert was evaluated by CLSM analysis on the insert containing Coumarin-6 (C6), a fluorescence probe, inside the nanomicelles. The images showed homogeneous fluorescence at C6 excitation wavelength (512 nm), suggesting a successful incorporation and distribution of the laden nanomicelles. Finally, in vivo pharmacokinetic of the selected insert was performed on New Zealand rabbits, and data have shown a medium retention time of the formulation 7-fold higher than the nanomicelles dispersion, prolonging the formulation resident time in the precorneal area, as aimed. Furthermore, the insert was well tolerated by the animal, since no irritation phenomena was observed. The combination of nanomicelles with solid insert device seems to be a promising platform to obtain not toxic and biocompatible formulations able to prolong CyA resident time in the precorneal area of the eye for DES treatment. Finally, the same nanomicellar formulation Nano1-CyA was introduced into in situ gelling system to take advantage of nanomicelles ability to incorporate insoluble drugs and of in situ gelling system ability to improve the residence time. Different sol-gel dispersions, using gellan gum as polymer, were developed to obtain a viscoelastic gel triggered by ions concentration changes when in contact with the tear fluid. Three different calcium ion concentrations were added to a dispersion of 0.1%w/w gellan gum maintaining a constant concentration of the monovalent cations (Na+ and K+) to obtain the formulations NanoG1, NanoG2 and NanoG3, having Ca2+ increasing concentration. Nano1-CyA formulation was directly obtained by mixing the surfactants with the gellan gum dispersion, leaving the gel to stabilize for 12 hours before any use. The formulations were characterized measuring pH, osmolality, size distribution and clarity, to assess the presence of the nanomicelles into the gel system, analyze their size in view of ocular administration, and their stability. The technological parameters of the formulations resulted suitable for ocular administration and the nanomicelles diameter was comparable to the original formulation Nano1–CyA. The rheological behavior of the polymeric dispersions in presence or absence (reference) of nanomicelles were measured at 32°C immediately after preparation to verify any possible surfactant influence and to evaluate the increase in viscosity after dilution of the polymeric dispersions with Artificial Tear Fluid (ATF, ratio 3:0.7). All the tested formulation presented a pseudoplastic behavior. On the basis of the obtained data, NanoG2 formulation, was selected to load CyA (NanoG2-CyA), since was able to pass from sol to gel when in contact with ATF. The dynamic light scattering analysis and the amount of drug encapsulated were determined on three different batches of NanoG2-CyA to verify the reproducibility of the preparation method. These preliminary results suggest the possible use of nanomicelles loaded into in situ gelling system for ocular administration, taking in account the instillation of a liquid formulation, easy to apply, that became a gel in contact with the tear fluid extending the drug resident time in the precorneal area, thus improving CyA ocular bioavailability