Development of topical ophthalmic formulations for fungal keratitis treatment

Resumen

Today, according to the World Health Organization's World Vision Report, at least 2.2 billion people suffer from visual impairment or complete blindness, of which just over half of these cases (1 billion) could have preserved their vision if they had access to effective treatment. Vision impairment or complete blindness can result from a wide and diverse range of conditions ranging from age-related macular degeneration, cataracts, corneal opacity, diabetic retinopathy or glaucoma, among others. The causes of vision impairment vary from country to country and depend mainly on the availability of adequate and effective ophthalmic care. The eye is an organ with a complex anatomy and physiology that provides insulation and protection. The cornea represents the main biological barrier of the eye, preventing the entry of microorganisms and foreign substances (including drugs) into the eye. In addition, the high tear fluid turnover rate, high nasolacrimal drainage and blinking effectively remove any substance from the ocular surface. For these reasons, topical-ophthalmic administration has a significantly low bioavailability. However, this route of administration is the most desirable, as formulations for this route are self-administered (improving patient adherence) and cost-effective. Over the years, research in the development of new formulations has mainly focused on two strategies to improve the bioavailability of drugs through the topical-ophthalmic route: increasing the biopermanence of the drug in the precorneal area and increasing the permeability of the drug through the cornea, sclera and conjunctiva. These goals can be achieved by developing systems based primarily on polymers that increase the viscosity of the formulations and increase their adhesion to the mucin layer, keeping the formulations longer on the ocular surface. Bioavailability can also be improved by increasing transcorneal permeability through the use of penetration promoters such as cyclodextrins. Fungal keratitis is a disease of infectious origin caused by different types of fungal species. Arpergillus spp. Curvularia spp., Penicillium spp. and Candida spp. are the most common species. The severity of the disease lies mainly in the lack of effective drugs available, the difficulty in diagnosis and the delay in starting effective treatment. These factors lead to blindness or even complete loss of the eyeball. It is an uncommon disease in developed countries, although in recent years an increase has been observed due to different factors, such as poor use of contact lenses, long treatments with corticoids or local antibiotics, immunosuppressed patients, etc. At the same time, there is only one FDA-approved topical-ophthalmic formulation and its efficacy is limited to superficial infections caused by filamentous fungi. For this reason, hospital pharmacy services are forced to reformulate drugs intended for other routes (mainly intravenous (IV)) by means of resuspension or redispersion with ophthalmic buffers. The main problem is that in the vast majority of cases, their toxicity, bioavailability and stability are unknown. In addition, in order to achieve therapeutic concentrations, intense dosages (every hour) are established, which cause systemic side effects and the abandonment of treatment by the patient, generally leading to a worsening of the clinical picture and hospitalization, increasing health care costs. This doctoral thesis is divided into two sections. The first section consists of the first three chapters. These chapters focus on the design, development and characterization of several topical-ophthalmic formulations for the administration of three different antifungals: econazole, voriconazole and natamycin. These formulations are mainly based on the formation of inclusion complexes with cyclodextrins to increase the aqueous solubility of the drugs and improve their corneal permeability, and their vehicleization in in-situ gelling hydrogels and mucoadhesive hydrogels to increase the permanence of the formulations on the ocular surface. In the second section, we focus on the study of the ocular safety of cyclodextrin solutions, as well as the study of their potential as permanence promoters on the ocular surface. This section is the fourth and final chapter. In the introduction of this thesis, a critical bibliographic review is carried out in which all the possible pharmacological treatments for the treatment of fungal keratitis are compiled, including a large number of active ingredients belonging to different pharmacological groups, such as polyenes, azoles or pyrimidines, among others. It also includes a section detailing the therapeutic regimen used in hospitals when a case of fungal keratitis occurs. In addition, surgical treatments such as keratoplasty, amniotic membrane transplantation or debridement are reviewed. Chapter 1 describes the design and characterization of ophthalmic hydrogels incorporating econazole. The aim of this chapter was to develop two formulations suitable for topical ophthalmic administration of econazole for the treatment of fungal keratitis. Econazole is an azole antifungal with low water solubility. For this reason, the main objective was to increase its solubility. Based on the results of the solubility study, ¿-cyclodextrin (¿CD) was chosen as the solubilizing agent. The formation of the inclusion complexes was studied by nuclear magnetic resonance (NMR) and molecular modelling studies, suggesting that the imidazole ring of econazole is deeply embedded in the cavity of ¿CD. The inclusion complexes formed between econazole and ¿CD were vehicled into two types of hydrogels using different polymers. An ion-sensitive hydrogel consisting of a mixture of gellan gum (GG) and kappa carrageenan (CK) (4:1) and a second mucoadhesive hydrogel consisting of 0.4 % (w/v) hyaluronic acid were prepared. Once the hydrogels were prepared, the in vitro release of econazole was studied, demonstrating the ability of the hydrogels to control its release. In addition, ex vivo transcorneal permeability was studied using bovine corneas, demonstrating that the econazole included in these systems is able to pass through the cornea to reach deeper tissues. Two different types of studies were performed to assess ocular irritation and toxicity, the corneal permeability and opacity test or BCOP and the in vitro ocular irritation test HET-CAM. In the BCOP test, a change in transparency was observed, however, corneal permeability was not affected. In the HET-CAM test, using chorioallantoic membrane (CAM) from fertilized eggs, no change in the blood vessels of the membrane was observed, demonstrating that these formulations are non-irritating. On the other hand, to determine mucoadhesion in vivo, the ability of the formulations to remain on the ocular surface was studied. For this purpose, the hydrogels labelled with a radiopharmaceutical (18F-FDG) were administered to the ocular surface of sprague dawley rats, establishing their clearance over time using experimental molecular imaging techniques based on the use of positron emission tomography (µPET). The study demonstrates that both hydrogels are able to remain on the ocular surface for around 70 minutes, well above the time that the econazole/¿-cyclodextrin inclusion complex solution remains without bioadhesive polymers. The antifungal capacity of several antifungals (econazole, voriconazole, fluconazole, amphotericin B and natamycin) used in the treatment of fungal keratitis was evaluated on different fungal species (Candida Albicans, Aspergillus Fumigatus and Paecilomyces) using the disc diffusion method. In this study, econazole was found to have large zones of inhibition far superior to those obtained with amphotericin B and natamycin. Based on all the results obtained, the formulated econazole eye drops have great potential for use in the treatment of fungal keratitis, although due to the modification of corneal transparency they cause, their use should be limited to use in cases where other effective amphotericins with similar activity (such as voriconazole) are not available or where treatment outweighs the risks associated with their use. Chapter 2, like chapter 1, focuses on the development, optimisation and characterisation of two types of mucoadhesive hydrogels with high ocular permanence for the topical-ophthalmic administration of voriconazole: an ion-sensitive hydrogel composed of a mixture of GG:CK and a hyaluronic acid hydrogel. Despite the great potential of this antifungal for the treatment of fungal infections in different organs and tissues, which makes it increasingly used by ophthalmologists, there is currently no FDA- or EMA-approved ophthalmic formulation of voriconazole. As a result, hospital pharmacy departments are forced to prepare ophthalmic formulations of voriconazole from commercial intravenous formulations by dilution in biocompatible ocular vehicles. This chapter compares the voriconazole mucoadhesive hydrogels we have developed with one of the formulations produced in hospital pharmacy departments from Vfend®, a commercial intravenous injectable of voriconazole. The main problem with this formulation is the limited residence time on the ocular surface, which requires intensive treatment leading to systemic side effects. Voriconazole is an azole antifungal with very limited water solubility. For the development of the hydrogels, solubility studies of voriconazole with cyclodextrins were carried out. The formation of inclusion complexes between voriconazole and the cyclodextrins 2-hydroxypropyl-ß-cyclodextrin (HPßCD), 2-hydroxypropyl-¿-cyclodextrin (HP¿CD) was studied by nuclear magnetic resonance (NMR) and molecular modelling. Based on these results, HPßCD (20% w/v) was chosen for the development of the formulations. Previous studies have shown that this cyclodextrin is able to produce a significant improvement in the amount of drug permeated through the cornea and has decreased the ocular toxicity of certain drugs. The hyaluronic acid gel was formulated at a concentration of 0.4% (w/v) and the ion-sensitive hydrogels were prepared at different ratios of GG and CK (1:1, 2:1, 4:1). The 1:1 mixture was chosen as the best formulation for further development as it was the one that showed the best performance in terms of admisnistration. In vitro release and ex vivo transcorneal permeation studies were carried out in bovine corneas to determine the ability of the hydrogels to release voriconazole in a controlled manner and to improve the transcorneal permeation of voriconazole. The osmolality and pH values obtained for all formulations developed except Vfend® were found to be within the appropriate range for ophthalmic administration. The ophthalmic solution prepared using Vfend® shows osmolality values much higher than those recommended for ocular formulations, due to the presence of the cyclodextrin sulfobutileter-ß-cyclodextrin (SBEßCD) used to solubilise the voriconazole in the injectable. This is an anionic cyclodextrin that has several sulphobutyl radicals in the form of a sodium salt in its structure and therefore contributes a high concentration of sodium ions to the medium. Ocular irritation and toxicity tests performed by BCOP and HET-CAM showed that all formulations evaluated are safe for ophthalmic use. To determine the permanence time on the ocular surface, ex vivo corneal mucoadhesion studies and in vivo corneal surface permanence time studies were performed using PET imaging. These tests confirmed the mucoadhesive properties of the newly prepared voriconazole hydrogels and their superior ocular permanence compared to Vfend® solutions. Based on all these results, it is possible to conclude that the developed voriconazole hydrogels are excellent candidates for clinical application in cases of fungal keratitis. The severity of fungal keratitis is aggravated by the emerging resistance of fungal species to current treatments. Combination therapy of several antifungals is often more effective than monotherapy, so chapter 3 of this doctoral thesis addresses the design and development of several eye drops containing two molecules with antifungal activity: natamycin and voriconazole. Natamycin is the molecule of first choice in the early treatment of fungal keratitis caused by filamentous fungi. The main problem is that there is only one FDA-approved ophthalmic formulation, Natacyn®, which is based on a conventional natamycin suspension with low transcorneal penetration and is therefore limited to superficial infections. In this chapter, we study improving the water solubility of natamycin through the use of cyclodextrins. As with voriconazole, the cyclodextrin of choice after solubility studies was HPßCD. Due to the type of solubility diagram obtained (AL) (the negative deviation shows the formation of aggregates of the inclusion complexes) the solutions of natamycin, voriconazole and HPßCD were analysed by transmission electron microscopy (TEM). The images demonstrate the presence of nanometre-sized spherical aggregates that may enhance the ability to permeate through the cornea. The inclusion complex formed between natamycin and HPßCD was studied by NMR. Using this technique, competition studies on complex formation with HPßCD between natamycin and voriconazole were also performed. As could be observed, natamycin competes with voriconazole for the same binding site to HPßCD, which leads to the need to adjust and optimise the concentration of the cyclodextrin. In our case, the use of HPßCD concentrations of 40% (w/v) allowed the preparation of formulations at the desired concentrations (natamycin 7 mg/mL (w/v) and voriconazole 10 mg/mL). Once the inclusion complexes formed had been studied, two hydrogels were prepared. One hydrogel was made with hyaluronic acid (0.4% (w/v)) and the other with a commercial hydrogel (Liquifilm®) based on polyvinyl alcohol (PVA) (1.4% w/v) frequently used in the preparation of formulations in hospital pharmacy services. The hydrogels were characterized in terms of pH, osmolality, viscosity and transparency. These studies showed that the values obtained are within the accepted range for ophthalmic topical formulations. In vitro release studies showed characteristic Fickian-type diffusion process profiles for all new formulations. In addition, ex vivo corneal permeability studies demonstrated a significant improvement in the transcorneal permeation of natamycin compared to the commercial formulation Natacyn®. The developed formulations did not show any indication of irritation according to the results of the HET-CAM and BCOP tests and can therefore be considered safe formulations. The in vitro antifungal effectiveness of the eye drops was evaluated by the disc diffusion method, showing that the developed formulations show activity against the tested fungal species (Candida albicans ATCC 90231), Candida albicans ATCC 90028, Paelomyces lilacinus ATCC 90028, Aspergillus fumigatus, Paelomyces lilacinus, and Fusarium solanii). Ex vivo and in vivo mucoadhesion studies demonstrated that the mucoadhesive capacity shown by the new formulations is mainly due to the incorporated cyclodextrin, as no significant differences were observed when HA or PVA were incorporated at the concentrations studied. During the search for published information on the ophthalmic application of cyclodextrins carried out during the research conducted in the previous 3 chapters, it was found that there is very little information on their toxicity and safety at the ophthalmic level and on their mucoadhesive capacity on the ocular surface. Therefore, in chapter 4, the results of safety and ocular permanence studies carried out on cyclodextrin solutions at high concentrations are presented. For this purpose, different varieties of cyclodextrins of interest in the development of ophthalmic drug delivery systems have been included in the study. The effect of ¿CD, ß-cyclodextrin (ßCD), ¿-cyclodextrin (¿CD), 2-hydroxypropyl-¿-cyclodextrin (HP¿CD), HPßCD, HP¿CD, SBEßCD, and partially methylated ß-cyclodextrin (RMßCD) solutions on the choriolantoid membrane of fertilized eggs was studied using the HET-CAM assay and on bovine corneas using the BCOP method. According to the results obtained in these tests, all cyclodextrins are safe, with the exception of ¿CD and RMßCD. The 20% (w/v) solutions of RMßCD caused a significant change in corneal transparency but showed no changes in the chorioallantoic membrane vessels. The changes in transparency are due to the low pH value obtained when dissolving this partially methylated variety (2.447±0.025). At acidic pH, alteration of proteins and other components of the mucosa and epithelium can occur, causing these alterations. The negative effect on corneal transparency was resolved by adjusting the RMßCD solution to pH 7.4. The ¿CD solutions (in unnaturalized and neutralized solutions at pH 7.4), did not cause hemorrhage, lysis or coagulation on the chorioallantoic membrane vessels of the eggs, but the formation of a thin white precipitate was observed. In addition, a significant change in corneal transparency was also observed during the BCOP test. This phenomenon is probably due to the interaction described for ¿CD and different phospholipids and lipid components of the membrane, which form insoluble inclusion complexes, which can precipitate on the membranes. The permeability of fluorescein across corneas studied through BCOP was affected by most of the cyclodextrin solutions studied. Increased fluorescein permeability is normally interpreted as a sign of toxicity; however, it is known that cyclodextrins can interact with cell membrane components to enhance permeability across different epithelia and barriers without showing toxic effects, including transcorneal permeability. The mucoadhesive capacity of cyclodextrins was studied by in vitro, ex vivo and in vivo assays. These tests demonstrated the ability of the cyclodextrins to interact and bind to the ocular mucosa. The ex vivo studies showed that at the concentrations studied there are no significant differences between the different cyclodextrins, and they exhibit similar levels of mucoadhesion to those observed for some biodhesive polymers (such as HA and PVA) and for their blends with cyclodextrins. In vitro studies performed on mucin blends and cyclodextrin solutions showed the establishment of interactions between cyclodextrins and mucin in solution, probably through the establishment of weak hydrogen bonding or Van der Waals interactions. In vivo studies demonstrated the excellent ability of the cyclodextrin solutions to remain on the ocular surface. All the permanence values (with the exception of the RMßCD solutions) decreased when the solutions were adjusted to pH 7.4. This may be due to an increased difficulty in establishing hydrogen bonds between the cyclodextrins and mucin caused by the ionization of sialic acid. In the case of RMßCD, in vivo ocular permanence was increased by neutralizing the dilution. pH values as acidic as those observed for this unneutralized solution exceed the buffering capacity of the eye, producing irritation and damage to the epithelium, which promotes tearing and blinking, increasing clearance at its surface. ¿CD was the cyclodextrin that showed the highest MRT value, probably due to the formation of insoluble aggregates formed with the phospholipids of the epithelial cells. HP¿CD, HPßCD and HP¿CD showed the highest t1/2 values, suggesting that it is the hydroxylated cyclodextrins that remain on the ocular surface the longest. Therefore, taking into account the results obtained on the safety and ocular mucoadhesion of the different cyclodextrins, as well as their capacity to promote the transcorneal penetration of other molecules, make cyclodextrins excellent candidates for the development of ophthalmic formulations of drugs that are poorly soluble in water or with low corneal permeability. In conclusion, this doctoral thesis has addressed the rational design of different topical-ophthalmic formulations for the treatment of ocular fungal diseases. Using three antifungals with different activity spectra, different formulations for topical-ophthalmic administration have been developed. In addition, a complete preclinical characterization of these formulations has been carried out. These formulations have great potential for the treatment of fungal keratitis caused by different species, which can fill the current therapeutic gap. All the formulations developed have shown adequate levels of safety, in vitro activity and excellent mucoadhesive behaviour, with high residence times on the ocular surface. Finally, safety and bioadhesion studies carried out with cyclodextrin solutions show that even at relatively high concentrations they are suitable excipients for the preparation of ophthalmic formulations. However, they should be supplemented by further in vivo studies to complete the knowledge on the effect of cyclodextrins on the ocular surface.