Therapeutic alternatives research for the treatment of ocular inflammatory diseases
- Autores: Xurxo García Otero
- Directores de la Tesis: Francisco Javier Otero Espinar (dir. tes.), Pablo Aguiar Fernández (codir. tes.), Anxo Fernández Ferreiro (codir. tes.), José Blanco Méndez (tut. tes.)
- Lectura: En la Universidade de Santiago de Compostela ( España ) en 2022
- Idioma: inglés
- Tribunal Calificador de la Tesis: Alvaro Ruibal Morell (presid.), Salvador Pastor Idoate (secret.), María Begoña Delgado Charro (voc.)
- Programa de doctorado: Programa de Doctorado en Investigación y Desarrollo de Medicamentos por la Universidad de Santiago de Compostela
- Materias:
- Enlaces
- Tesis en acceso abierto en: MINERVA
- Resumen
The quality of life of millions of people around the world is compromised by various eye conditions and pathologies. One of the most complex challenges for pharmacists is to achieve efficient and effective treatments for the eye, since its anatomy and physiology presents many protective mechanisms, which makes it a really complex organ to treat. For this reason, it is important that the bioavailability of the drug in those specific sites of action of the different ocular structures where treatment is needed is adequate.
Advances in drug delivery technologies have evolved over time, but major challenges remain in the treatment of ocular diseases. These ocular delivery systems must maintain therapeutic drug concentrations, reduce dosing frequency and cross the various ocular barriers.
The use of topical-ophthalmic instillation for the treatment of diseases affecting the anterior segment of the eye is the most desirable route of administration. These systems present several advantages as they are considered easy to handle, have high patient acceptance and are also economically cost-effective. However, they also present certain limitations in terms of low ocular bioavailability, this is due to the high tear turnover rate which equates to significant clearance through the nasolacrimal drainage, impacting on the constant repetition of frequent instillations. Therefore, much effort needs to be focused on addressing this problem. This solution involves the development of systems that increase the permanence on the ocular surface and facilitate entry into the tissues.
On the other hand, direct administration by intravitreal injections of drugs is useful in the treatment of ocular pathologies of the posterior segment of the eye. This route has some advantages compared to systemic administration by circumventing blood-ocular barriers, allowing higher drug levels to be reached in the ocular cavity and limiting systemic side effects. However, it also has some limitations since it is considered an invasive route that usually requires repeated injections, for this reason, it is necessary to know the intravitreal pharmacokinetics of the drugs in order to develop new sustained release systems, and thus reduce the number of administrations.
The disadvantages of the topical-ophthalmic route and intravitreal injections are reflected in the treatment of ocular inflammatory pathologies affecting the anterior and posterior segment of the eye, such as uveitis in its broad spectrum or keratoconjunctivitis, pathologies on which this doctoral thesis focuses.
Uveitis is the term used for a very heterogeneous group of diseases that cause ocular inflammation. Because their origin and site of involvement can be very diverse, the treatment of these diseases requires different routes and strategies depending on the site and degree of involvement, so that a wide range of drugs and routes of administration are necessary for an efficient cure of the disease. Over time, the use of different families of drugs with therapeutic action have been studied, as well as the optimization and improvement of the routes of administration to reduce the possible systemic adverse effects caused by the drugs. The corticosteroid family is one of the most widely used because it produces favorable effects quickly. However, it is also common to find undesirable adverse effects in some ocular structures and systemic toxicity in long-term treatments. For this reason, new families of drugs have emerged that have demonstrated a therapeutic effect against these diseases, such as immunosuppressants (tacrolimus) or biologic drugs (adalimumab), both drugs studied in this doctoral thesis.
Chapter 1 contains a literature review that aims to give an overview of the main aspects involved in the pharmacokinetics of ocular drugs intended to treat different ocular diseases. First, the different factors involved in ocular drug delivery were analyzed, covering the different routes of entry into the eye. The physiological barriers and drug transport pathways were described in detail, and the advantages and disadvantages of the different routes of administration to the eye were also discussed.
Conventional routes of administration, such as topical or systemic, often present important limitations, either because of low ocular penetration or because of the occurrence of side effects linked to the dosage, among others. Therefore, new drug delivery systems (DDS) are needed to help prolong and adapt administration intervals in ocular pathologies. However, the development of these new systems is particularly challenging due to several aspects that need to be considered, such as pharmacokinetics, immunogenicity, biodegradation, tolerability and toxicity. The last few decades have seen an exponential increase in the design and development of new drug delivery systems for the treatment of ocular pathologies. Unfortunately, knowledge about the ability of these systems to deliver drugs into the eye remains scarce.
There have also been great advances in the research and development of new alternative routes of administration to achieve drug concentrations in the different parts of the eye. All of them have specific pharmacokinetic characteristics that make them useful for the treatment of specific ocular pathologies. However, these routes have shown various advantages and limitations, where the choice of one or the other depends not only on the pathology itself, but also on the pharmaceutical form, the drug used and the patient's adherence to treatment.
Chapter 2 includes the study of the development and efficacy of an ophthalmic formulation of tacrolimus 0.03% (w/v) based on the systemic commercial presentation (Prograf®) and introduced in three types of vehicles: Balanced Salt Solution (BSS), polyvinyl alcohol solution (PVA, Liquifilm®) and a hyaluronic acid solution.
For this purpose, in vitro (stability studies) and in vivo (permanence time in the cornea by Positron Emission Tomography) tests of the three possible formulations were carried out. The stability study at different temperature conditions was carried out for 90 days in freezing (-20 °C), refrigeration (2-8 °C) and at room temperature (20-25 °C). Osmolality, pH and drug concentration parameters were studied during this period. The only formulation that remained stable for 90 days under refrigeration and freezing conditions was that of the PVA vehicle, in BSS it only withstood freezing and in hyaluronic acid it was not preserved under any of the conditions. The pH and osmolality measurements remained constant with a pH of 7.5 and osmolality values too high (above 1000 mOsm/kg) compared to the physiological osmolality of the ocular surface. Regarding the study of ocular permanence by PET, a 3-hour follow-up was carried out after instillation of the eye drops in the eye, in which it was possible to verify that tacrolimus eye drops with PVA produced the least clearance, increasing the contact time on the ocular surface.
The best formulation (tacrolimus in PVA) was then selected, and its toxicological profile and clinical efficacy was evaluated in comparison with commercial cyclosporine eye drops (Restasis®). Tacrolimus in PVA showed less cytotoxicity than cyclosporine and was better tolerated by corneal epithelial cells in the early stages. On the other hand, a pilot study was performed in 8 patients in which significant improvements were shown in the patients, with no appreciable adverse reactions. However, 7 of the 8 patients showed a slight ocular itching after instillation of this tacrolimus eye drop, probably caused by the high osmolality of the formulation and the presence of excipients poorly adapted to the ocular route present in Prograf®.
Based on the stability, ocular permanence, safety and clinical efficacy studies, it is possible to conclude that tacrolimus-PVA eye drops are a suitable candidate for clinical application in ophthalmic inflammatory diseases.
In order to solve the problems arising from the presence of poorly adapted excipients in Prograf® in Chapter 3, the design and development of two types of tacrolimus-containing ophthalmic formulations for the treatment of uveitis was addressed. Tacrolimus is an immunosuppressant drug characterized by very low solubility in water and low stability in aqueous media, for this reason the main objective was to achieve solubilization of the drug by combining different proportions of a derivative of β-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (HPβCD). In this way, an interaction was achieved between the drug and the cyclodextrin forming the complexation of tacrolimus in the hydrophobic cavity of HPβCD, generating an increase in the solubility of the drug. All this information was obtained by performing solubility, NMR and molecular modeling studies.
After having achieved the solubilization of tacrolimus, it was proceeded to test whether this complex could work in other aqueous solvents, as in the case of a BSS (Balanced Salt Solution) solution, and also in Liquifilm®, a lubricating tear containing polyvinyl alcohol (PVA). Different parameters for the optimization of the formulation were carried out, managing to fix the composition of each eye drop and the optimal tacrolimus solubilization time. These studies resulted in four definitive formulations: two formulations with 20% (w/v) HPβCD and 0.01% (w/v) tacrolimus using BSS as vehicle in the first case and Liquifilm® in the second, and two other formulations with 40% (w/v) HPβCD and 0.02% (w/v) tacrolimus, also in BSS and Liquifilm®.
Once the formulations were ready, physicochemical characterization was performed, determining parameters such as pH, osmolality, surface tension of the formulation and the force required to dispense a drop. These studies concluded that the formulations have an optimal pH range for the physiology of the eye and osmolality values of ≃320 mOsm/kg. This solved the osmolality problem existing in classical formulations prepared in the hospital pharmacy that had values above ≃1200 mOsm/kg.
Ocular irritation and toxicity studies were performed through the permeability and opacity test on bovine cornea and also with the test on the chorioallantoic membrane of fertilized eggs. In this way it was proved that none of the formulations generated the slightest indication of toxicity or irritation. Another important characteristic that had to be demonstrated was that tacrolimus remained stable in aqueous solution for a reasonable period of time. Therefore, a stability study was performed on the two eye drops containing the most tacrolimus, 0.02% (w/v) in BSS and Liquifilm®, which showed that the eye drops could be preserved for at least three months under refrigeration (4 ± 2 °C).
The residence time of an eye drop on the ocular surface is another extremely important parameter to increase the bioavailability of the drug in the therapeutic target. Therefore, ex vivo corneal mucoadhesion and in vivo corneal surface permanence time studies were performed using PET imaging. After analyzing the results, it was observed that HPβCD not only increases the solubility and stability of tacrolimus in solution, but also influences the mucoadhesion properties, since increasing the proportion of cyclodextrin improves the permanence time. The formulation with 40% (w/v) HPβCD, 0.02% (w/v) tacrolimus dissolved in Liquifilm® doubled the dwell time compared to the classical tacrolimus formulation.
Chapter 4 is based on the demonstration of the anti-inflammatory effect in an endotoxin-induced uveitis model in rats of the best formulation obtained in the previous chapter. For this purpose, 32 rats were divided into 4 groups of 8 rats each: (a) a group of untreated healthy rats, (b) a group of rats with untreated uveitis, (c) a group of rats with uveitis treated with the standard treatment of dexamethasone eye drops (Maxidex®) and (d) a group of rats with uveitis treated with the proposed TAC-HPβCD eye drops.
Uveitis was induced by inoculating 1 mg/kg of Escherichia coli lipopolysaccharide (LPS) diluted in 0.1 mL of BSS into the animal's right paw. The treated rats received the instillation of the eye drops 3h hours before the induction of the disease and every 3h until 24 h of study. Once the study period was over, the rats were euthanized with carbon dioxide to proceed to sample collection, in order to quantitatively analyze the presence of leukocytes in the aqueous humor, evaluate the ocular histology and finally perform PCR quantification of the expression of IL-6, IL-8, MIP-1α and TNFα levels in the eye.
The first thing that was proven was that the uveitis model was induced in an appropriate manner, which implies that after LPS administration there is an activation of TLRs, increasing levels of proinflammatory mediators, developing inflammation of the anterior uvea, choroid and retina and resulting in the breakdown of the blood-tumoral barrier, with exudation of leukocytes into the aqueous humor. This effect could be verified by significantly higher levels of TNFα, IL-6, IL-8, MIP-1α and leukocytes, as well as histological evaluation of the untreated uveitis group compared to the healthy group.
The significantly lower levels of TNFα, IL-6, MIP-1α and leukocytes, as well as the histological evaluation of the TAC-HPβCD eye drops compared to the diseased group confirms the efficacy of this formulation in the treatment of uveitis. Regarding the comparison between the efficacy of our formulation and standard treatment with dexamethasone, no statistically significant differences were found between the levels of proinflammatory cytokines in the TAC-HPβCD group and the dexamethasone group, demonstrating that TAC-HPCD eye drops could be an alternative to topical corticosteroid therapy in the treatment of this model of uveitis and their translational clinical use should be studied in future studies.
Moreover, regarding TNFα mRNA expression, its reduction is statistically significant only between the TAC-HPβCD and uveitis groups, but not between the dexamethasone and uveitis groups, which is consistent with the mechanism of action of tacrolimus and could be especially beneficial in uveitis related to the increase of this factor, such as HLA B27-associated uveitis, sarcoid uveitis, and uveitis associated with Behçet's disease, among other non-infectious uveitis entities.
Adalimumab is an anti-TNFα drug approved for the treatment of uveitis by subcutaneous injection. This route of administration exposes patients to systemic adverse effects and makes it difficult to obtain therapeutic concentrations of the drug at the site of action due to the anatomic and physiologic barriers of the eye.
The use of molecular imaging techniques in combination with radiolabeling of antibodies allows not only the observation of their distribution pattern in the body, but also their quantification in real time. In this sense, Positron Emission Tomography (PET) represents a promising imaging tool for the non-invasive evaluation of antibody pharmacokinetics, allowing longitudinal studies in which each animal is followed over time. This clearly represents the advantage of reducing the number of animals, according to the 3Rs frameworks. Zirconium-89 (89Zr) is a radionuclide that plays an important role in Immuno-Positron Emission Tomography (Immuno-PET) imaging techniques. Its long half-life (3.3 days) is favorable for assessing the in vivo distribution of monoclonal antibodies (mAb), being of great potential in monitoring antibody-based therapies. The Immuno-PET technique combines the sensitivity of PET with the specificity of the antibody, allowing to know its body distribution and pharmacokinetic behavior.
Since, as has been shown, increased TNFα expression plays an important role in ocular inflammatory diseases, in Chapter 5 it was set out to study the ocular pharmacokinetic profile and the clearance and distribution in tissues of an intravitreally injected monoclonal antibody that acts by inhibiting TNFα receptors called adalimumab.
To achieve the objective of this study, adalimumab was conjugated with a chelating agent called deferoxamine whose function is to trap the 89Zr nucleus. Once the conjugation was completed, the conjugated antibody was radiolabeled with 89Zr with a maximum specific activity of 10 MBq/mg, achieving an optimal radiolabeling result, since the radiochemical purity after ultrafiltration was 99.69%. Regarding the study design, two groups of rats were used: a control group of healthy rats (n=3; 6 eyes) and a group of rats with uveitis induced in the same way as in Chapter 4 (n=6, 12 eyes). Each of the animals was injected into the vitreous with 4 μL containing ≃1.74 MBq of 89Zr-labeled adalimumab (in both eyes). MicroPET image acquisition was performed immediately after injection and at different time points throughout a 10-day study, at the same times blood samples were collected through the tail vein.
Quantitative analysis was performed on the PET images obtained, in them different regions of interest (ROIs) were drawn in the study organs (eyes, heart, liver, spleen and cervical lymph nodes) in order to quantify the radioactive activity emitted by 89Zr-adalimumab and thus generate their kinetic curves. One and two-compartment models were used to fit the experimental data.
The ocular pharmacokinetics of the antibody was adjusted to a one-compartment model, showing an intraocular elimination half-life of 15.57 hours for healthy rats and 33.64 hours for rats with uveitis, implying that 89Zr-adalimumab remained about twice as long in rats with the disease compared to healthy rats. These results show that, despite the fact that ocular clearance of a drug in an eye with inflammation would be expected to be faster due to the increased permeability of the ocular barriers, this is not the case here. The ability of adalimumab to selectively bind tumor necrosis factor is well known, so this result is consistent with the overproduction of TNFα from macrophages and other cytokines in the process of uveitis, which activates dendritic cells, initiating the inflammatory cascade in which Th1 and Th17 cells migrate and infiltrate the blood-retinal barrier causing damage.
The results of compartmental pharmacokinetic analysis in blood show different behavior between healthy and diseased animals. The activity versus time in healthy animals conforms to a two-compartment model showing a rapid uptake of the antibody from the eye simultaneously with the tissue distribution process. However, in the case of animals with uveitis, the activity versus time conforms better to a one-compartment model. This may be because the transfer process from the eye to the blood is much slower acting as a limiting step, so that the distribution process to the organs is not appreciated.
This study shows for the first time the ocular and blood pharmacokinetic analysis of adalimumab in a rat model of uveitis, providing valuable information for the development of new controlled release systems of adalimumab that allow spacing the administration of the drug and thus improve the quality of life of patients.
In conclusion, in this doctoral thesis different tacrolimus formulations for topical-ophthalmic administration have been proposed with great potential, supported by extensive preclinical studies in order to achieve a consistent basis as an alternative to other pharmacological treatments for ocular inflammatory diseases. This was confirmed with extensive in vitro, ex vivo and in vivo studies. Moreover, with regard to the study of intravitreal injections of adalimumab, this doctoral thesis has elucidated the intravitreal distribution and pharmacokinetics of the monoclonal antibody that will allow the development of new delivery systems.
