Therapeutic alternatives research for the treatment of ocular inflammatory diseases

ESCOLA DE DOUTORAMENTO INTERNACIONAL DA USC Xurxo García Otero Tese de doutoramento Therapeutic alternatives research for the treatment of ocular inflammatory diseases Santiago de Compostela, 2022 Programa de Doutoramento en Investigación e Desenvolvemento de Medicamentos TESE DE DOUTORAMENTO THERAPEUTIC ALTERNATIVES RESEARCH FOR THE TREATMENT OF OCULAR INFLAMMATORY DISEASES Xurxo García Otero ESCOLA DE DOUTORAMENTO INTERNACIONAL DA UNIVERSIDADE DE SANTIAGO DE COMPOSTELA PROGRAMA DE DOUTORAMENTO EN INVESTIGACIÓN E DESENVOLVEMENTO DE MEDICAMENTOS SANTIAGO DE COMPOSTELA 2022 II III DECLARACIÓN DO A UTOR DA TESE Therapeutic alternatives research for the treatment of ocular inflammatory diseases D. Xurxo García Otero, Presento a miña tese seguindo o procedemento axeitado ao Regulamento e declaro que: 1) A miña tese abarca os resultados da elaboración do meu traballo. 2) De selo caso, na tese faise referencia ás colaboracións que tivo este traballo. 3) A tese é a versión definitiva presentada para á súa defensa e coincide coa versión enviada en formato electrónico. 4) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. En Santiago de Compostela, a 6 de Novembro de 2022 Asdo. Xurxo García Otero IV V AUTORIZACIÓN DOS DIRECTORES Therapeutic alternatives research for the treatment of ocular inflammatory diseases D. Francisco Javier Otero Espinar D. Anxo Fernández Ferreiro D. Pablo Aguiar Fernández D. José Blanco Méndez INFORMAN: Que a presente tese, correspóndese co traballo realizado por D. Xurxo García Otero, baixo a nosa dirección/titorización, e a utorizamos a súa presentación , considerando que reúne os r equisitos esixidos no R egulamento de Estudos de Doutoramento da USC, e que como directores desta non incorre nas causas de abstención establecidas na Lei 40/2015. De acordo co indicado no Regulamento de Estudos de Doutoramento, declara tamén que a presente tese de doutoramento é idónea para ser defendida en base á modalidade de Monográfica con reprodución de publicacións, nos que a participación do doutorando foi decisiva para a súa elaboración e as publicacións se axustan ao Plan de Investigación. En Santiago de Compostela, a 6 de Novembro de 2022 Asdo. Francisco J. Otero Espinar Asdo. Anxo Fernández Ferreiro Asdo. Pablo Aguiar Fernández Asdo. José Blanco Méndez (Titor) VI VII AGRADECIMIENTOS VIII XV SUMMARY 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. XVI 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 XVII 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. XVIII 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). XIX 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 XX 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. Zirconium89 (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 XXI 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. XXII 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. XXIII RESUMO A calidade de vida de millóns de persoas en todo o mundo vese comprometida por causa de diferentes afeccións e patoloxías oculares. Un dos retos máis complexos para os farmacéuticos é conseguir tratamentos eficaces e efectivos para o ollo, xa que a súa anatomía e fisioloxía presenta moitos mecanismos de protección, o que fai que sexa un órgano realmente complexo de tratar. Por esta razón, é importante que a biodispoñibilidade do fármaco sexa adecuada naqueles lugares de acción específicos das diferentes estruturas oculares onde sexa necesario un tratamento. Os avances alcanzados respecto ás tecnoloxías de administración de fármacos foron evolucionando co paso do tempo, aínda que quedan grandes desafíos no que se refire ó tratamento de enfermidades oculares. Estes sistemas de administración por vía ocular deben manter as concentracións terapéuticas do fármaco, reducir a frecuencia de dosificación e atravesar as diferentes barreiras oculares. O uso da instilación tópico-oftálmica para o tratamento de enfermidades que afectan ó segmento anterior do ollo é a vía de administración máis desexable. Estes sistemas presentan varias vantaxes xa que considéranse fáciles de manexar, teñen unha aceptación alta por parte do paciente e tamén son rendibles economicamente. Non obstante, tamén presenta certas limitacións en canto á baixa biodispoñibilidade ocular, isto é debido á elevada taxa de renovación lacrimal o que equivale a un aclaramento importante a través do drenaxe nasolacrimal, repercutindo na repetición constante de instilacións frecuentes. Polo tanto, é necesario que moitos dos esforzos enfóquense en abordar este problema. Esta solución pasa por desenvolver sistemas que permitan aumentar a permanencia na superficie ocular e que faciliten a entrada nos tecidos. Por outro lado, a administración directa mediante inxeccións intravítreas de fármacos é útil no tratamento de patoloxías oculares do segmento posterior do ollo. Esta vía presenta algunhas vantaxes en comparación ca administración sistémica eludindo as barreiras hemato-oculares, permitindo alcanzar niveis de fármaco máis elevados na cavidade ocular e limitando os efectos secundarios sistémicos. Sen embargo, tamén presenta algunha limitación xa que considérase XXIV unha ruta invasiva que normalmente require inxeccións repetidas, por este motivo, é necesario coñecer a farmacocinética intravítrea dos fármacos para poder desenvolver novos sistemas de liberación sostida, e así reducir o número de administracións. As desvantaxes da vía tópico-oftálmica e das inxeccións intravítreas vense reflexadas nos tratamentos de patoloxías inflamatorias oculares que afectan ó segmento anterior e posterior do ollo como poden ser a uveíte no seu amplo espectro ou a queratoconxuntivite, patoloxías nas que se centra esta tese de doutoramento. A uveíte é o termo empregado para un grupo moi heteroxéneo de enfermidades que causan inflamación ocular. Debido a que a súa orixe e lugar de afectación poden ser moi diversos, para o tratamento destas enfermidades é necesario empregar diferentes vías e estratexias segundo o lugar e o grado de afectación, polo que un amplo abanico de fármacos e rutas de administración son necesarias para unha cura eficiente da enfermidade. Ó longo do tempo, foise estudando o uso de diferentes familias de fármacos con acción terapéutica, así como a optimización e mellora das vías de administración para reducir os posibles efectos adversos sistémicos producidos polos fármacos. A familia de corticosteroides é unha das máis empregadas xa que produce efectos favorables rapidamente, sen embargo, tamén é frecuente atopar efectos adversos non desexados nalgunhas estruturas oculares e toxicidade sistémica en tratamentos de longa duración. Por esta razón, xurdiron novas familias de fármacos que demostraron un efecto terapéutico fronte a estas enfermidades, como son o caso dos inmunosupresores (tacrolimus) ou fármacos biolóxicos (adalimumab), ambos fármacos estudados nesta tese de doutoramento. O capítulo 1 contén unha revisión bibliográfica que pretende dar unha visión xeral dos principais aspectos que interveñen na farmacocinética dos fármacos oculares destinados a tratar diferentes enfermidades oculares. En primeiro lugar, analizáronse os diferentes factores que interveñen na administración ocular de fármacos, incluíndo as diferentes rutas de entrada no ollo. As barreras fisiolóxicas e as vías de transporte dos fármacos foron detalladamente descritas, e tamén discutíronse as vantaxes e inconvenientes das diferentes vías de administración ó ollo. As vías de administración convencionais, como a tópica ou a sistémica, normalmente presentan importantes limitacións, xa sexa pola baixa penetración ocular ou pola aparición de efectos secundarios ligados á posoloxía, entre outros. Polo tanto, son necesarios novos sistemas de administración de fármacos (DDS) que axuden a prolongar e adecuar os intervalos de administración nas patoloxías oculares. Sen embargo, o desenvolvemento destes novos sistemas é XXXI RESUMEN La calidad de vida de millones de personas en todo el mundo se ve comprometida por causa de diferentes afecciones y patologías oculares. Uno de los retos más complejos para los farmacéuticos es conseguir tratamientos eficaces y efectivos para el ojo, ya que su anatomía y fisiología presenta muchos mecanismos de protección, lo que hace que sea un órgano realmente complejo de tratar. Por esta razón, es importante que la biodisponibilidad del fármaco en aquellos lugares de acción específicos de las diferentes estructuras oculares donde sea necesario un tratamiento sea adecuada. Los avances conseguidos respecto a las tecnologías de administración de fármacos han ido evolucionando con el paso del tiempo, aunque todavía quedan grandes desafíos en lo que se refiere al tratamiento de enfermedades oculares. Estos sistemas de administración por vía ocular deben mantener las concentraciones terapéuticas del fármaco, reducir la frecuencia de dosificación y atravesar las diferentes barreras oculares. El uso de la instilación tópico-oftálmica para el tratamiento de enfermedades que afectan al segmento anterior del ojo es la vía de administración más deseable. Estos sistemas presentan varias ventajas ya que se consideran fáciles de manejar, tienen una aceptación alta por parte del paciente y también son rentables económicamente. No obstante, también presenta ciertas limitaciones en cuanto a la baja biodisponibilidad ocular, esto es debido a la elevada tasa de renovación lacrimal lo que equivale a un aclaramiento importante a través del drenaje nasolacrimal, repercutiendo en la repetición constante de instilaciones frecuentes. Por lo tanto, es necesario que muchos de los esfuerzos estén enfocados en abordar este problema. Esta solución pasa por desarrollar sistemas que permitan aumentar la permanencia en la superficie ocular y que faciliten la entrada en los tejidos. Por otro lado, la administración directa mediante inyecciones intravítreas de fármacos es útil en el tratamiento de patologías oculares del segmento posterior del ojo. Esta vía presenta algunas ventajas en comparación con la administración sistémica eludiendo las barreras hemato-oculares, permitiendo alcanzar niveles de fármaco más elevados en la cavidad ocular y limitando los efectos secundarios XXXII sistémicos. Sin embargo, también presenta alguna limitación ya que se considera una ruta invasiva que suele requerir inyecciones repetidas, por este motivo, es necesario conocer la farmacocinética intravítrea de los fármacos para poder desarrollar nuevos sistemas de liberación sostenida, y así reducir el número de administraciones. Las desventajas de la vía tópico-oftálmica y de las inyecciones intravítreas se ven reflejadas en los tratamientos de patologías inflamatorias oculares que afectan al segmento anterior y posterior del ojo como pueden ser la uveítis en su amplio espectro o la queratoconjuntivitis, patologías en las que se centra esta tesis doctoral. La uveítis es el término utilizado para un grupo muy heterogéneo de enfermedades que causan inflamación ocular. Debido a que su origen y lugar de afectación pueden ser muy diversos, para el tratamiento de estas enfermedades es necesario emplear diferentes vías y estrategias según el lugar y el grado de afectación, por lo que un amplio abanico de fármacos y rutas de administración son necesarias para una cura eficiente de la enfermedad. A lo largo del tiempo, se han ido estudiando el uso de diferentes familias de fármacos con acción terapéutica, así como la optimización y mejora de las vías de administración para reducir los posibles efectos adversos sistémicos causados por los fármacos. La familia de corticosteroides es una de las más empleadas ya que produce efectos favorables rápidamente, sin embargo, también es frecuente encontrar efectos adversos no deseados en algunas estructuras oculares y toxicidad sistémica en tratamientos de larga duración. Por esta razón, han surgido nuevas familias de fármacos que han demostrado un efecto terapéutico frente a estas enfermedades, como son el caso de los inmunosupresores (tacrolimus) o fármacos biológicos (adalimumab), ambos fármacos estudiados en esta tesis doctoral. El capítulo 1 contiene una revisión bibliográfica que pretende dar una visión general de los principales aspectos que intervienen en la farmacocinética de los fármacos oculares destinados a tratar diferentes enfermedades oculares. En primer lugar, se analizaron los diferentes factores que intervienen en la administración ocular de fármacos, abarcando las diferentes rutas de entrada en el ojo. Las barreras fisiológicas y las vías de transporte de los fármacos fueron detalladamente descritas, y también se discutieron las ventajas e inconvenientes de las diferentes vías de administración al ojo. Las vías de administración convencionales, como la tópica o la sistémica, suelen presentar importantes limitaciones, ya sea por la baja penetración ocular o por la aparición de efectos secundarios ligados a la posología, entre otros. Por lo tanto, son necesarios nuevos sistemas de administración de fármacos (DDS) que ayuden a prolongar y adecuar los intervalos de administración en las patologías XXXIII oculares. Sin embargo, el desarrollo de estos nuevos sistemas es especialmente complicado debido a varios aspectos que deben considerarse, como la farmacocinética, la inmunogenicidad, la biodegradación, la tolerabilidad y la toxicidad. En las últimas décadas se ha observado un aumento exponencial en el diseño y desarrollo de nuevos sistemas de administración de fármacos destinados al tratamiento de patologías oculares. Desgraciadamente, los conocimientos sobre la capacidad de estos sistemas para vehiculizar los fármacos en el ojo siguen siendo escasos. Asimismo, se han producido grandes avances en cuanto a la investigación y el desarrollo de nuevas vías de administración alternativas para alcanzar concentraciones de fármaco en las diferentes partes del ojo. Todas ellas tienen características farmacocinéticas específicas que las hacen útiles para el tratamiento de patologías oculares concretas. No obstante, estas vías han mostrado diversas ventajas y limitaciones, donde la elección de una u otra depende, no sólo de la propia patología, sino de la forma farmacéutica, del fármaco utilizado y de la adherencia del paciente al tratamiento. El capítulo 2 engloba el estudio de elaboración y eficacia de una formulación oftálmica de tacrolimus al 0,03% (p/v) a partir de la presentación comercial sistémica (Prograf®) e introducida en tres tipos de vehículos: Balanced Salt Solution (BSS), solución de alcohol polivinílico (PVA, Liquifilm®) y una solución de ácido hialurónico. Para ello, se realizaron ensayos in vitro (estudios de estabilidad) e in vivo (tiempo de permanencia en la córnea mediante Tomografía de Emisión de Positrones) de las tres posibles formulaciones. El estudio de estabilidad a diferentes condiciones de temperatura se llevó a cabo durante 90 días en congelación (-20 °C), refrigeración (2-8 °C) y a temperatura ambiente (20-25 °C). Los parámetros de osmolalidad, pH y concentración de fármaco fueron estudiados durante este período. La única formulación que se mantuvo estable durante 90 días en condiciones de refrigeración y congelación fue la del vehículo de PVA, en BSS solo aguantó en congelación y en ácido hialurónico no se preservó en ninguna de las condiciones. Las medidas de pH y de osmolalidad se mantuvieron constantes con un pH de 7,5 y unos valores de osmolalidad demasiado altos (por encima de 1000 mOsm/kg) en comparación con la osmolalidad fisiológica de la superficie ocular. En cuanto al estudio de la permanencia ocular mediante PET se hizo un seguimiento de 3 horas tras la instilación del colirio en el ojo, en el que se pudo comprobar que el colirio de tacrolimus con PVA era el que menor aclaramiento producía, aumentando el tiempo de contacto en la superficie ocular. A continuación, se seleccionó la mejor formulación (tacrolimus en PVA), y se evaluó su perfil toxicológico y su eficacia clínica en comparación con el colirio XXXIV comercial de ciclosporina (Restasis®). El tacrolimus con PVA mostró menor citotoxicidad que la ciclosporina siendo mejor tolerado por las células epiteliales corneales en los primeros tiempos. Por otra parte, se realizó un estudio piloto en 8 pacientes en el que se mostraron mejoras significativas en los pacientes, sin reacciones adversas apreciables. Sin embargo, 7 de los 8 pacientes mostraron un leve picor ocular después de la instilación de este colirio de tacrolimus, seguramente provocado por la alta osmolalidad de la formulación y la presencia de excipientes poco adaptados a la vía ocular presentes en Prograf®. Basándose en los estudios de estabilidad, permanencia ocular, seguridad y eficacia clínica, es posible concluir que el colirio de tacrolimus-PVA es un candidato adecuado para su aplicación clínica en las enfermedades inflamatorias oftalmológicas. Con la finalidad de resolver los problemas derivados de la presencia de excipientes poco adaptados en el Prograf® en el capítulo 3 se abordó el diseño y desarrollo de dos tipos de formulaciones oftálmicas que contienen tacrolimus para el tratamiento de la uveítis. El tacrolimus es un fármaco inmunosupresor que se caracteriza por tener una muy baja solubilidad en agua y una baja estabilidad en medio acuoso, por esta razón el objetivo principal fue conseguir la solubilización del fármaco combinando diferentes proporciones de un derivado de la β-ciclodextrina, la 2-hidroxipropil-β-ciclodextrina (HPβCD). De esta manera, se logró una interacción entre el fármaco y la ciclodextrina formando la complejación del tacrolimus en la cavidad hidrofóbica de la HPβCD, generando un aumento en la solubilidad del fármaco. Toda esta información se consiguió realizando estudios de solubilidad, RMN y modelaje molecular. Tras haber conseguido la solubilización del tacrolimus se procedió a comprobar si este complejo podía funcionar en otros solventes acuosos, como en el caso de una solución de BSS (Balanced Salt Solution), y también en Liquifilm®, una lágrima lubrificante que contiene alcohol polivinílico (PVA). Diferentes parámetros para la optimización de la formulación fueron llevados a cabo, consiguiendo fijar la composición de cada colirio y el tiempo óptimo de solubilización del tacrolimus. De estos estudios resultaron cuatro formulaciones definitivas: dos formulaciones con el 20% (p/v) de HPβCD y 0,01% (p/v) de tacrolimus utilizando como vehículo BSS en el primer caso y Liquifilm® en el segundo, y otras dos formulaciones con el 40% (p/v) de HPβCD y 0,02% (p/v) de tacrolimus, también en BSS y Liquifilm®. Una vez que las formulaciones estaban a punto se procedió a la caracterización fisicoquímica, determinando parámetros como el pH, osmolalidad, tensión superficial de la formulación y la fuerza necesaria para dispensar una gota. Estos estudios concluyeron que las formulaciones tienen un rango de pH óptimo para XXXV la fisiología del ojo y valores de osmolalidad de ≃320 mOsm/kg. De esta manera, se solucionó el problema de osmolalidad existente en las formulaciones clásicas preparadas en la farmacia hospitalaria que tenían valores por encima de ≃1200 mOsm/kg. Los estudios de irritación y toxicidad ocular fueron realizados a través del test de permeabilidad y opacidad en córnea bovina y también con el ensayo en la membrana corioalantoidea de huevos fertilizados. De esta manera se comprobó que ninguna de las formulaciones generó el mínimo indicio de toxicidad o irritación. Otra característica importante que había que demostrar es que el tacrolimus permaneciese estable en solución acuosa durante un periodo razonable de tiempo. Por lo que se realizó un estudio de estabilidad en los dos colirios que más tacrolimus contenían, 0,02% (p/v) en BSS y Liquifilm®, con este ensayo se demostró que los colirios podían preservarse durante al menos tres meses en refrigeración (4 ± 2 °C). El tiempo de permanencia de un colirio sobre la superficie ocular es otro parámetro sumamente importante para aumentar la biodisponibilidad del fármaco en la diana terapéutica. Por lo tanto, se realizaron estudios de mucoadhesion corneal ex vivo y de tiempo de permanencia en la superficie corneal in vivo mediante imagen PET. Tras analizar los resultados se observó que la HPβCD no solo aumenta la solubilidad y estabilidad del tacrolimus en solución, sino que también influye en las propiedades de mucoadhesividad, ya que aumentando la proporción de ciclodextrina mejora el tiempo de permanencia. La formulación con el 40% (p/v) de HPβCD, 0,02% (p/v) de tacrolimus disuelta en Liquifilm® duplicó el tiempo de permanencia en comparación con la formulación clásica de tacrolimus. El capítulo 4 se basa en la demostración del efecto antiinflamatorio en un modelo de uveítis inducido por endotoxina en ratas de la mejor formulación obtenida en el capítulo anterior. Para ello se dividieron 32 ratas en 4 grupos de 8 ratas cada uno: (a) un grupo de ratas sanas no tratadas, (b) un grupo de ratas con uveítis no tratadas, (c) un grupo de ratas con uveítis tratadas con el tratamiento estándar de gotas oftálmicas de dexametasona (Maxidex®) y (d) un grupo de ratas con uveítis tratadas con el colirio de TAC-HPβCD propuesto. La uveítis fue inducida inoculando 1 mg/kg de Lipopolisacárido de Escherichia coli (LPS) diluido en 0,1 mL de BSS en la pata derecha del animal. Las ratas con tratamiento recibieron la instilación de los colirios 3h horas antes de la inducción de la enfermedad y cada 3h hasta cumplir las 24 h de estudio. Una vez terminado el período de estudio, las ratas fueron eutanasiadas con dióxido de carbono para proceder a la recogida de muestras, y así poder analizar cuantitativamente la presencia de leucocitos en el humor acuoso, evaluar la histología ocular y por XXXVI último realizar la cuantificación mediante PCR de la expresión de niveles de IL6, IL-8, MIP-1α y TNFα en el ojo. Lo primero que se comprobó fue que el modelo de uveítis se indujo de manera adecuada, que supone que tras la administración de LPS se produce una activación de los TLRs, aumentando los niveles de mediadores proinflamatorios, desarrollándose la inflamación de la úvea anterior, la coroides y la retina y produciéndose la ruptura de la barrera hematohumoral, con exudación de leucocitos al humor acuoso. Este efecto se pudo comprobar gracias a los niveles significativamente mayores de TNFα, IL-6, IL-8, MIP-1α y leucocitos, así como la evaluación histológica del grupo con uveítis sin tratar en comparación con el grupo sano. Los niveles significativamente más bajos de TNFα, IL-6, MIP-1α y leucocitos, así como la evaluación histológica del colirio de TAC-HPβCD en comparación con el grupo enfermo confirma la eficacia de esta formulación en el tratamiento de la uveítis. En cuanto a la comparación entre la eficacia de nuestra formulación y el tratamiento estándar con dexametasona, no se encontraron diferencias estadísticamente significativas entre los niveles de citoquinas proinflamatorias del grupo de TAC-HPβCD y del grupo de dexametasona, lo que demuestra que los colirios de TAC-HPβCD podrían ser una alternativa al tratamiento tópico con corticoides en el tratamiento de este modelo de uveítis y su uso clínico traslacional debería estudiarse en futuros estudios. Además, en lo que respecta a la expresión del ARNm del TNFα, su reducción es estadísticamente significativa sólo entre el grupo de TAC-HPβCD y el de uveítis, pero no entre el grupo de dexametasona y el de uveítis, lo cual es coherente con el mecanismo de acción del tacrolimus y podría ser especialmente beneficioso en uveítis relacionadas con el aumento de este factor, como la uveítis asociada a HLA B27, la uveítis sarcoide y la uveítis asociada a la enfermedad de Behçet, entre otras entidades de uveítis no infecciosas. Puesto que, tal como se ha demostrado, el aumento de la expresión del TNFα juega un importante papel en las enfermedades inflamatorias oculares, en el capítulo 5 se planteó el estudio del perfil farmacocinético ocular y del aclaramiento y distribución en tejidos de un anticuerpo monoclonal inyectado intravítreamente que actúa inhibiendo los receptores de TNFα llamado adalimumab. El adalimumab es un fármaco anti-TNFα aprobado para el tratamiento de la uveítis por inyección subcutánea. Esta vía de administración expone a los pacientes a efectos adversos sistémicos y dificulta la obtención de XXXVII concentraciones terapéuticas del fármaco en el lugar de acción debido a las barreras anatómicas y fisiológicas del ojo. El uso de técnicas de imagen molecular en combinación con el radiomarcaje de anticuerpos permite no sólo la observación de su patrón de distribución en el organismo, sino también su cuantificación en tiempo real. En este sentido, la Tomografía por Emisión de Positrones (PET) representa una prometedora herramienta de imagen para la evaluación no invasiva de la farmacocinética de los anticuerpos, permitiendo realizar estudios longitudinales en los que se sigue a cada animal a lo largo del tiempo. Esto representa claramente la ventaja de reducir el número de animales, según los marcos de las 3Rs. El Zirconio-89 (89Zr) es un radionúclido que desempeña un papel importante en las técnicas de imagen de Inmuno-Tomografía por Emisión de Positrones (Inmuno-PET). Su larga vida media (3,3 días) es favorable para evaluar la distribución in vivo de los anticuerpos monoclonales (mAb), siendo de gran potencial en la monitorización de terapias basadas en anticuerpos. La técnica Inmuno-PET combina la sensibilidad del PET con la especificidad del anticuerpo, permitiendo conocer su distribución corporal y su comportamiento farmacocinético. Para lograr el objetivo de este estudio el adalimumab se conjugó con un agente quelante llamado deferoxamina cuya función es la de atrapar los núcleos de 89Zr. Una vez terminada la conjugación el anticuerpo conjugado fue radiomarcado con 89Zr con una actividad específica máxima de 10 MBq/mg, consiguiendo un resultado de radiomarcaje óptimo, ya que la pureza radioquímica tras la ultrafiltración fue del 99,69%. En cuanto al diseño del estudio se emplearon dos grupos de ratas: un grupo control de ratas sanas (n=3; 6 ojos) y un grupo de ratas con uveítis inducidas de la misma forma que en el capítulo 4 (n=6, 12 ojos). Cada uno de los animales fue inyectado en el vítreo con 4 µL que contenían ≃1,74 MBq de adalimumab marcado con 89Zr (en los dos ojos). La adquisición de imágenes del microPET se realizaron inmediatamente después de la inyección y en diferentes puntos temporales a lo largo de un estudio de 10 días, en esos mismos tiempos se recolectaron muestras de sangre a través de la vena de la cola. El análisis cuantitativo fue realizado en las imágenes PET obtenidas, en ellas se dibujaron diferentes regiones de interés (ROIs) en los órganos de estudio (ojos, corazón, hígado, bazo y ganglios linfáticos cervicales) con el fin de cuantificar la actividad radioactiva emitida por el 89Zr-adalimumab y así generar sus curvas cinéticas. Modelos monoy bicompartimentales fueron empleados para ajustar los datos experimentales. La farmacocinética ocular del anticuerpo se ajustó a un modelo monocompartimental, mostrando una semivida de eliminación intraocular de 15,57 horas para las ratas sanas y de 33,64 horas para las ratas con uveítis, lo que XXXVIII implica que el 89Zr-adalimumab permaneció alrededor de dos veces más en las ratas con la enfermedad en comparación con las sanas. Estos resultados muestran que, pese a que es esperable que el aclaramiento ocular de un fármaco en un ojo con inflamación sea más rápido debido al aumento de la permeabilidad de las barreras oculares, en este caso no ocurre esto. La capacidad del adalimumab de unirse selectivamente al factor de necrosis tumoral es bien conocida, por lo que este resultado es coherente con la sobreproducción de TNFα de los macrófagos y otras citoquinas en el proceso de uveítis, que activa las células dendríticas, iniciando la cascada inflamatoria en la que las células Th1 y Th17 migran y se infiltran en la barrera hemorretiniana causando daños. Los resultados del análisis farmacocinético compartimental en sangre muestran un comportamiento diferente entre los animales sanos y los enfermos. La actividad frente al tiempo en los animales sanos se ajusta a un modelo bicompartimental que muestra una rápida captación del anticuerpo desde el ojo simultáneamente al proceso de distribución tisular. Sin embargo, en el caso de los animales con uveítis, la actividad frente al tiempo se ajusta mejor a un modelo de un compartimento. Esto puede deberse a que el proceso de transferencia del ojo a la sangre es mucho más lento actuando como paso limitante, por lo que no se aprecia el proceso de distribución a los órganos. Este estudio muestra por primera vez el análisis farmacocinético ocular y sanguíneo de adalimumab en un modelo de uveítis en ratas aportando una información muy valiosa para poder desarrollar nuevos sistemas de liberación controlada de adalimumab que permitan espaciar la administración del fármaco y así mejorar la calidad de vida de los pacientes. En conclusión, en esta tesis doctoral se han propuesto diferentes formulaciones de tacrolimus para la administración tópico-oftálmica con un gran potencial, respaldadas por amplios estudios preclínicos con el fin de lograr una base consistente como alternativa a otros tratamientos farmacológicos para las enfermedades inflamatorias oculares. Esto fue confirmado con amplios estudios in vitro, ex vivo e in vivo. Por otra parte, en lo que respecta al estudio de inyecciones intravítreas de adalimumab, esta tesis doctoral ha dilucidado la distribución y farmacocinética intravítrea del anticuerpo monoclonal que permitirá el desarrollo de nuevos sistemas de administración. XXXIX INDEX XL 1 OBJECTIVES AND ORGANIZATION 2 3 OBJECTIVES AND ORGANIZATION STATEMENT OF THE PROBLEM Inflammatory eye diseases require effective treatment, which is related to achieving an effective drug concentration at the target site for long enough for the inflammation to subside. To accomplish this goal, special drug delivery routes or drug delivery systems that reach different parts of the eye are required. This is complicated by the fact that the eye is a singular and complex organ with a unique anatomy and physiology that contributes to its high level of protection. Consequently, access of exogenous substances to its internal structures is severely restricted. Ocular surface diseases undergo extensive clearance, which leads to a decrease in the bioavailability of the drug in the inflamed area. For the corresponding diseases of the posterior segment of the eye, the drug has to cross numerous barriers or has to be administered by more invasive injections. These two common limitations are reflected in the difficulties in the treatment of uveitis involving different parts of the eye. Location and severity of the inflammation will influence the change from topical to systemic administration route. The current treatment for uveitis is based on the administration of a first line of corticosteroids, both topically in the eye and systemically. Although these drugs have a highly and immediate efficacy, they also cause problems with repeated use. Long-term administration of these agents can cause serious systemic and ocular adverse effects There are more and more cases of patients with refractory problems due to treatment with corticosteroids, patients who do not respond to treatment or for whom the use of corticosteroids is contraindicated. For this reason, there is a need to use effective alternative treatments that improve the patient’s life quality. The use of immunosuppressants such as cyclosporine A or tacrolimus has become the second line of therapy for uveitis. These drugs are more focused on systemic administration, causing undesirable effects. Therefore, the development of new formulations that are much more targeted to the site of action in order to minimize these contraindications is one of the most challenging tasks for pharmacists. On the other hand, uveitis located in the posterior ocular segment with a remarkable severity requires therapy tailored to their characteristics. Due to the 4 anatomical features of the eye, the access of drugs to the target site is hampered. Special route of administration, such as intravitreal injection, is needed to achieve adequate drug levels in the deeper ocular layers since in clinical practice this type of administration is not used, employing the systemic route. The emergence of biological agents, such as anti-TNFa has opened up new routes of treatment, which have already demonstrated their efficacy. The use of these biological molecules by systemic administration has become increasingly important in recent times. However, there is still no comprehensive scientific basis for therapeutic doses or effective doses of these drugs inside the eye. Knowledge of the ocular pharmacokinetics after intravitreal injection of these anti-TNFa drugs or their distribution is another important aspect that needs to be resolved. Once the behavior of these biological molecules is clearly understood, controlled release systems can be developed to make delivery more efficient and limit invasiveness by reducing the number of injections. OBJECTIVES With these considerations in mind, the general objective of this thesis is to improve the pharmacological treatment of uveitis for ocular administration. In order to achieve these purposes, specific objectives linked to this line of research were set, including: 1. To understand the main factors affecting ocular delivery for anterior and posterior eye segment treatment. 2. Characterization of classical tacrolimus eye drops reformulated from the intravenous drug in Hospital Pharmacy Departments. 2.1. Preclinical characterization of tacrolimus eye drops. 2.2. Clinical evaluation of tacrolimus eye drops. 3. Development of a new and safe tacrolimus ophthalmic formulation intended for the topical treatment of uveitis. 3.1. Design, optimization, and physicochemical characterization of a hydroxypropyl-β-cyclodextrin topical ocular formulation containing tacrolimus. 3.2. Characterize the stability of the developed formulation and translate its preparation to hospital pharmacy departments. 3.3. Evaluate the residence time on the ocular surface of the developed eye drop. 4. Evaluation of the pharmacological effect of the tacrolimus/hydroxypropyl-βcyclodextrin eye drop in an endotoxin-induced uveitis model. 5 5. Evaluation of the intravitreal route for the administration of an anti-TNFa antibody (adalimumab) in uveitis. 5.1. Development of a radiolabeling procedure for adalimumab with Zirconium-89 (89Zr). 5.2. Evaluation of the pharmacokinetics and distribution of intravitreally injected adalimumab in healthy rat eyes. 5.3. Evaluation of the pharmacokinetics and distribution of intravitreally injected adalimumab in uveitis rat eyes. ORGANIZATION Based on the formulated objectives, this doctoral thesis is presented as a compendium of four original research works and one review article. This doctoral thesis has been organized to comply with the regulation of the International Doctorate School at the University of Santiago de Compostela (Spain) regarding thesis structure, languages, and ethical and intellectual properties. The present doctoral thesis is divided in three main sections correspondence with a general review and the two drugs studied. The first section corresponds to a deep review of the ocular routes. It is included in the first chapter, Chapter 1. Chapter 1 aims to provide an overview of the main aspects involved in ocular drug pharmacokinetics intended to treat eye diseases. A discussion of the different factors that are involved in the ocular drug delivery is first made, encompassing the different routes towards the posterior segment of the eye. Physiological barriers and drug transport pathways are described and the advantages and drawbacks of different administration routes to the eye are also discussed. The second section focuses on the development, characterization and evaluation of the pharmacological effect of tacrolimus eye drops for the treatment of uveitis and other inflammatory diseases. It is divided in three chapters, from Chapter 2 to 4. Chapter 2 comprises the characterization of three types of ocular vehicles containing tacrolimus reformulated from intravenous pharmaceutical form, the first vehicle is a Balanced Salt Solution (BSS®), the second one is a PVA solution (Liquifilm®) and the last one made from hyaluronic acid polymer. Moreover, an extensive preclinical and clinical evaluation have been performed. 6 Chapter 3 was based on the design, preparation, and characterization of tacrolimus/hydroxypropyl-β-cyclodextrin eye drops as topical ophthalmic formulation for uveitis treatment. A comparison has been made between the best formulation resulting from the Chapter 2 and the new tacrolimus/hydroxypropyl-β-cyclodextrin eye drop in different aspects: pH and osmolality, surface tension, squeezing force, ex vivo corneal mucoadhesion, ocular toxicity, stability study and in vivo evaluation of the residence time on the ocular surface. Chapter 4 was aimed to investigate the pharmacological effect of tacrolimus/hydroxypropyl-β-cyclodextrin eye drops developed in Chapter 3 in an endotoxin-induced uveitis (EIU) rat model. In this work, ocular levels of several pro-inflammatory cytokines were studied, leukocytes were quantified in the aqueous humor and histological evaluation was performed in 4 different groups of rats, comparing them with each other. These groups are divided into: healthy rats, EIU untreated rats, EIU rats treated with dexamethasone and EIU rats treated with tacrolimus/hydroxypropyl-β-cyclodextrin eye drops. The third section of the present thesis focuses on the study of the next therapeutic line for the treatment of uveitis, the use of biologic drugs such as monoclonal antibodies. It is also intended to deepen the knowledge of the intraocular pathway to help the drug reach the site of action. It consists of the last chapter, Chapter 5. Chapter 5 is based on the PET methodology for the study of the ocular and blood pharmacokinetics following the intravitreal injection of the anti-TNFa antibody adalimumab, as well as to know the distribution of adalimumab in the whole rat body. This follow-up was carried out for 10 days. For that purpose, adalimumab was radiolabeled to the radiotracer Zirconium-89 (89Zr) in order to visualize it via PET. 7 INTRODUCTION 8 9 INTRODUCTION 1. UVEITIS 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 so diverse, a proper classification according to different parameters is necessary so that some criteria and terminology can be standardized. There are multiple causes of uveitis including infections, inflammatory diseases, trauma and idiopathic cases. The main symptoms of uveitis comprise: eye pain, redness, photophobia, lacrimation, visual disturbances (mainly blurred vision and blind spots), floating bodies in the field of vision and being the loss of vision the most serious one. Regarding the classification of uveitis, there are several groupings depending on the type of criterion followed. Among these classification systems are according to anatomy, clinical course, cause of inflammation and histopathology. In this case the anatomical and clinical course classification will be detailed as they are the most useful. 1.1 Anatomic classification As set forth by the International Uveitis Study Group (IUSG) and confirmed by Standardization of Uveitis Nomenclature (SUN) anatomical classification is based on the place where the inflammation predominates (1,2). It divides uveitis into anterior, intermediate, posterior and panuveitis. Anterior uveitis are those inflammations that affect the anterior chamber of the eye (iris and ciliary body involvement). Intermediate uveitis is primarily derived from inflammation of the vitreous cavity and pars plana. Pars planitis is reserved only if snowballs (vitreous aggregates in the form of snowballs) are found in the vitreous. In the case of posterior uveitis, the involvement is located in the retina and choroid. Finally, the term panuveitis is reserved only when the inflammation does not have a specific location and the anterior chamber, retina and/or choroid are involved. 10 Table 1. Uveitis classification according to anatomical location of inflammation (2). Uveitis type Initial site of inflammation Includes Anterior uveitis Anterior chamber Iritis Iridocyclitis Anterior cyclitis Intermediate uveitis Vitreous Pars planitis Posterior cyclitis Hyalitis Posterior uveitis Retina and choroid Choroiditis Chorioretinitis Retinochoroiditis Retinitis Neuroretinitis Panuveitis Anterior chamber, vitreous, retina and choroid - 1.2 Clinical course classification Due to the inconsistency of the terminology used in the literature, a nomenclature was established to define uveitis according to the clinical course. The onset of the uveitis, the duration of an attack and the course of this disease were agreed upon by the SUN. Table 2. Uveitis classification according to the clinical course (2). Category Descriptor Specification Onset Sudden - Insidious Duration Limited ≤ 3 months duration Persistent > 3 months duration Course Acute Episode characterized by sudden onset and limited duration Recurrent Repeated episodes separated by periods of inactivity without treatment ≥ 3 months in duration Chronic Persistent uveitis with relapse in < 3 months after discontinuing treatment 2. ETIOLOGY AND PATHOGENESIS OF UVEITIS Inflammation can be defined as a biological reaction of the immune system that can be triggered by any etiological agent (pathogens, damaged cells and toxic compounds), whose main function is to resolve, destroy or wall off the pathogenic organism and repair the damage (3). 17 therapeutic spectrum. Some studies show their efficacy and usefulness in patients refractory to conventional therapy. These biological agents selectively suppress immune response by targeting specific molecules in effector mechanisms of autoimmunity and inflammation (50). Currently, a large therapeutic arsenal is available to combat uveitis. The use of different drug families with therapeutic action has been studied, as well as the optimization and improvement of the administration routes in order to reduce the possible systemic adverse effects caused by the drugs used. Moreover, within the different delivery routes to the eye, a great deal of effort has also been devoted by pharmaceutical researchers to developing new systems and vehicles to improve bioavailability and ocular toxicity. One of the major concerns regarding the treatment of uveitis is systemic side effects, which is precisely why local administration of corticosteroids, immunosuppressive agents and biological agents represent a broad area of work to be developed (52). With this in mind, ocular administration routes of these drugs include topical application, subconjunctival and sub-tenon injections or intravitreal route (injection or implant) (53–55). A summary of uveitis treatments and their site of action within the inflammatory process can be seen in Figure 4. Figure 4. Diagram of the drugs included in the therapeutic arsenal for uveitis and their site of action within the inflammatory process. Created with BioRender.com. 18 5.1 Corticosteroids Corticosteroid treatment is the gold standard in clinical practice because of its rapid pharmacological effect, low cost and lack of major complications (56–58). The fact that they are very potent drugs can also cause quite a few side effects, so they should be used until the benefits outweigh the risks. Depending on the degree and site of inflammation, topical corticosteroid treatment is indicated for anterior uveitis and systemic corticosteroids for posterior uveitis or panuveitis (48). An initial high dose of oral corticosteroids should not be prolonged for more than 1 month, at which time the dose should be gradually reduced. The major problem with these drugs is the ocular and systemic effects of long-term use. Many uveitis specialists have raised concerns about the safety of corticosteroids, and indeed "corticosteroid-free" has been one of the main goals for future developments (57). Despite this, the use of corticosteroids remains irreplaceable to this day, which is why the development of new corticosteroids that reduce adverse effects and maintain local anti-inflammatory activity remains a challenge (49,59). 5.2 Mydriatics and cycloplegics These drugs are administered topically and are used in the treatment of acute inflammation of anterior uveitis, as they relieve pain secondary to ciliary muscle spasm and help prevent synechiae. The most commonly used drugs are: cyclopentolate, tropicamide, phenylephrine, homatropine or atropine. 5.3 Immunosuppressive agents The therapeutic second line for uveitis is immunosuppressants. The indication for these drugs is focused on chronic uveitis that compromises or could compromise the patient's vision. The use of immunosuppressants in uveitis is indicated in corticosteroid-refractory eye disease or after systemic side effects’ appearance. These drugs are sometimes given in combination with oral corticosteroids in the acute phase of severe ocular inflammation. In this way, a larger number of patients can be controlled with low doses of corticosteroids and minimal side effects (60). In patients who are refractory to corticosteroid therapy, immunosuppressive drugs can play a greater role in controlling inflammation and can sometimes even serve as rescue therapy (61,62). Due to the success of immunosuppressive agents in the control of other autoimmune inflammatory disorders or in transplant rejection, they have been 19 introduced into clinical practice to treat uveitis (63). A large body of evidence demonstrating the advantages of immunosuppressive drugs in the treatment of uveitis has been collected, although the indications for choosing one immunosuppressant over another are still uncertain (64,65). Most of the evidence are observational case series and clinical trials comparing different drugs and different types of uveitis are needed (65). The choice of immunosuppressive agents made by uveitis specialists depends on complex factors such as individual familiarity with the drug, safety/tolerability, efficacy, patient compliance and cost. This broad pharmacological group of immunosuppressive or immunomodulatory therapies can be divided into T-lymphocyte inhibitors, antimetabolites and alkylating agents. 5.3.1 T-lymphocyte inhibitors Cyclosporin A was the first T-cell inhibitor used for the systemic treatment of uveitis. Multiple studies showed the satisfactory therapeutic efficacy of this drug, making it the only immunosuppressant for this disease at that time (66). Thus, it was found to be similar to oral corticosteroids in terms of efficacy (67). However, some adverse reactions, such as renal toxicity and hypertension, required its dose reduction or discontinuation in a large proportion of patients. These results suggest that cyclosporin A is not suitable for long-term control of inflammation at high doses as a single agent (66). Close monitoring of patients on treatment with this drug is required to avoid irreversible damage (60). Tacrolimus is a macrolide with a great immunosuppressive activity (10-100 times more potent than cyclosporine A) (68) having demonstrated in clinical studies more effectiveness at lower concentrations and a better safety profile than cyclosporine A (60,69,70). Tacrolimus acts by inhibiting T-cell activation by blocking calcineurin, calcineurin inhibition suppresses dephosphorylation of the nuclear factor of activated T cells and its transfer into the nucleus, which results in the suppressed formation of T-cell lymphokine (IL-2) production. This inhibition is going to lead in the release inhibition of inflammatory cytokines and decreased stimulation of other inflammatory cells (71). The tacrolimus mechanism of action is shared with cyclosporine A (72). Systemic treatment with tacrolimus showed high efficacy in patients with refractory uveitis with excellent tolerability. Adverse effects are similar to, but always less than, those of cyclosporin A. Given their good results, attempts were 20 made to adapt the systemic formulations to the topical ocular route, with good results in terms of treatment efficacy. However, due to its poor water solubility and relatively high molecular weight, it has great difficulty penetrating the corneal epithelium and accumulates in the corneal stroma (73). In addition, as it is not designed as an ophthalmic drug, patients suffer discomfort because of its high osmolality, decreasing their success rate by discontinuing treatment. The potential role of topical tacrolimus in treating patients with uveitis it should be determined yet. Further studies are needed to detect the optimum concentration and formula of topical tacrolimus (74). 5.3.2 Antimetabolites Within the antimetabolites therapy, there are three drugs with therapeutic indications in uveitis: methotrexate, azathioprine and mycophenolate mofetil. Each of them has a different application depending on which part of the eye is affected. Methotrexate was the first antimetabolite to be added to the therapeutic arsenal against uveitis. The effectiveness of this drug is widely reported in the literature (75), including specific uveitis such as Behçet's disease (76), sarcoidosis-related panuveitis (77) or juvenile idiopathic arthritis-associated uveitis (51). Several studies indicate that their effectiveness is moderate, but they are well tolerated by patients and rarely present adverse effects. Because of this safety profile, it is one of the drugs of choice for uveitis specialists. However, it is worth noting that most patients require 6 months to have a full effect. Remarkably, methotrexate has a higher remission success in patients with anterior uveitis and scleritis (65,75). In the case of azathioprine, it has more recently been used for the treatment of uveitis. Its effectiveness has been demonstrated in corticosteroid-resistant uveitis, juvenile idiopathic arthritis associated iridocyclitis and intermediate uveitis but not in Behçet's disease (78,79). Although the overall efficacy of azathioprine is very close to that of methotrexate, its toxicity is a major limitation even though it may be reversible, as is the limitation due to interaction with other treatments (80). Mycophenolate mofetil is considered a relatively new immunosuppressant and therefore experience in clinical practice is more limited. However, its acceptance has been growing due to the publications generated (64,81), showing effectiveness in the different areas of ocular inflammation. Some studies show that remission of inflammation is achieved faster with mycophenolate mofetil than with methotrexate and azathioprine (82). 21 5.3.3 Alkylating agents This pharmacological group has a high percentage of disease remission after treatment. Therefore, it can be said that alkylating agents are highly effective against ocular inflammation (65,78,83). Although the success of chlorambucil (84–86) and cyclophosphamide (87,88) treatment in inflammatory eye pathology is a fact, numerous side effects are associated with the use of these agents. The risk is higher with these drugs than with alternative agents, and therefore close monitoring is very necessary in these cases (89). With the high risk of side effects and the emergence of new immunosuppressive drugs, alkylating agents have become the last therapeutic option. Their use is only reserved in severe or recalcitrant cases of ocular inflammation (89). 5.4 Biological agents Biological therapy has recently become increasingly important due to the discovery of new pathogenic mechanisms related to the role of pro-inflammatory cytokines and other factors causing ocular inflammation (90). TNFa is a cytokine that takes on great importance in regulating the functions of cells involved in the inflammatory process (91). TNFa, which is secreted by monocytes and macrophages, lymphocytes and mast cells, appears to play a key role in ocular inflammatory diseases (90). Infliximab was the first anti-TNFa to be used to treat uveitis, showing good results in short-term disease remission, the effect is temporary and has a high risk of treatment failure. This is due to the development of self-antibodies against the murine component of the molecule (92,93), causing a major limitation. Adalimumab is a humanized anti-TNFa monoclonal antibody used in the treatment of autoimmune diseases, directly blocking TNFa. Because of this, it was thought that it could also be helpful in the uveitis treatment. Adalimumab therapy efficacy has been widely demonstrated in the scientific literature. Compared to infliximab, adalimumab has shown better long-term efficacy and more adequate safety/toxicity profiles, this may be due to the fact that in this case there is no murine component in the molecule. Some side effects can occur with anti-TNFa, limiting their use for severe ocular inflammation and/or uveitis refractory to one or more immunosuppressants. In terms of side effects, both antibodies behave similarly, the only difference between infliximab and adalimumab is the local reactions after subcutaneous injection of adalimumab. For this reason, improvement in the routes of administration of this anti-TNFa antibody to achieve a localized effect is necessary. In the case of etanercept, there 22 are no conclusive data clearly showing its efficacy in the treatment of inflammatory eye diseases (94). Another biological therapy is interferons (IFNs), these molecules are low molecular weight glycoproteins that possess antiviral, antiproliferative, proor anti-apoptotic and immunomodulatory properties (95). IFNα is a natural cytokine that is secreted in response to viral infections, having an immunomodulatory effect and is thought to be a key mediator of "systemic" autoimmune diseases (96). Other biological agents act at other levels of the inflammatory response, overproduction of pro-inflammatory cytokines such as IL-1 and IL-6 are involved in the immune response. Two biologic drugs act on this mechanism by counteracting this effect, anakinra as anti-IL-1 (97) and tocilizumab for IL-6 (98), both molecules have been used in the treatment of uveitis. 6. IN VIVO ANIMAL MODELS During the last years, a large number of animal models have been developed to provide a valuable insight into the different pathways involved in uveitis pathogenesis. In each animal model the part of the eye undergoing inflammation is different, thus simulating different types of human uveitis as well. The cause of the inflammation can be caused by various retinal antigens, but other nonretinal and non-ocular antigens, such as bacterial endotoxin and cytokines, can also be used. Heterogeneity in the inflammatory response in these animal models is very pronounced, as even animals of the same species have variable responses to the same antigen. For this reason, the combination of uveitis animal models is very common. Table 5 shows some of the most representative models for the study of uveitis. Table 5. Relationship between different uveitis animal models, the antigen/endotoxin with which it is induced and the type of uveitis it generates. Uveitis model Antigen/Endotoxin Types of uveitis EAU S-antigen IRBP Rhodopsin Recoverin Phosducin Posterior uveitis EMIU Melanin protein Anterior uveitis Posterior uveitis Diffuse uveitis EAE/AU Myelin binding protein Anterior uveitis EIU E.coli lipopolysaccharide endotoxin (LPS) Anterior uveitis Diffuse uveitis 23 6.1 Experimental Autoimmune Uveitis model (EAU) The first developed experimental model of uveitis was Experimental Autoimmune Uveitis (EAU), this disease model is a human endogenous uveitis that can be induced in susceptible animals by immunization with retinal antigens. This disease induction resembles human uveitis in the sense that both are mediated by T-cells (Th1) targeting the neural retina and adjacent tissues (99). The EAU clinical and pathological characteristics depend on the species and the antigen used for induction, but T-cell mediated inflammatory response is always present, mainly CD4+ cells. EAU has been induced in a variety of species including rats, mice, rabbits and monkeys (100). No animal model in itself represents the complex spectrum of human uveitis. Although, there is no single animal model that covers the entire field of human uveitis, several models are often used to approach it. Among the above models, the rat model has the advantage of being very well characterized immunologically and immuno-genetically (101). Within the diversity of EAU models, the most common is the Classical EAU in which the mouse is immunized by the subcutaneous injection of bovine S-antigen with the Complete Freund’s adjuvant (CFA) as vehicle (102). On this basis, over the years, variations have been included in the model that have enriched the broad pathogenicity of the disease. Other antigens used to generate this inflammation are IRBP (Interphotoreceptor retinoid-binding protein) (103), rhodopsin (104), recoverin (105) and phosducin (99). 6.2 Experimental Melanin Protein-Induced Uveitis model (EMIU) In addition to the use of retinal proteins to induce uveitis in animals, the use of non-retinal proteins such as melanin or tyrosinase-related proteins is also worth mentioning. Depending on which antigen is used for induction, uveitis of different duration and form will be produced. In the 1990s the induced EMIU model was developed by Broekhuyse et al. with non-soluble melanin proteins extracted from pigmented bovine eyes (106). Following subcutaneous injection of melanin protein in combination with Hunter's adjuvant in rats, after 10-14 days, ocular inflammation is evident in the iris, ciliary body and choroid. Fundamentally, this model can be suited to the study of anterior uveitis (iridocyclitis), where melanin is found, as well as its spontaneous recurrence. This relapsing characteristic of the model makes it useful for the evaluation of therapies initiated during established disease and aimed at preventing recurrence. 24 6.3 Experimental Autoimmune Encephalomyelitis-Associated Anterior Uveitis model (EAE/AU) In this animal model, the protein used to generate the immunization of rodents is myelin basic protein, following subcutaneous injection with CFA (107,108). This induction usually causes dilation of the iris vessels, anterior uveitis for 30 days, and is therefore ideal for the study of anterior uveitis and the inflammatory relapses associated with this disease. 6.4 Endotoxin Induced Uveitis model (EIU) The most widely used model of autoinflammatory uveitis driven largely by innate immune mechanisms is endotoxin-induced uveitis (EIU), it was also developed in rats, and involves the systemic injection of Escherichia coli lipopolysaccharide endotoxin (LPS) to induce an acute anterior uveitis (30,109,110). This model is generally considered to be an inflammation of the anterior uvea, with some changes in the posterior segment (vitreous and retina), producing a severe immune response in animals. Thus, stimulation of the production of inflammatory cytokines such as tumor necrosis factor-a (TNFa) and other cytokines and inflammatory mediators are triggered. 7. IN VIVO IMAGING IN PRECLINICAL RESEARCH Modern imaging techniques provide anatomical, functional and molecular information about what is happening in tissues through the physical phenomena that penetrate living tissue, such as magnetic resonance imaging (MRI), computed tomography (CT), single-photon emission computed tomography (SPECT) and positron emission tomography (PET), among others. In recent years, an adaptation of these imaging techniques has been carried out to be used in small animal models, by increasing the spatial resolution and the sensitivity of the scanners (111). These models are mainly rodents (mice and rats) that are too small to be managed accurately. Thus, to overcome these limitations, dedicated and miniaturized versions of clinical scanners are currently available for small animal imaging in preclinical research (112). In a very few years, dedicated preclinical imaging scanners have reached enormous translationality by achieving high resolution and extreme sensitivity. These techniques are currently considered as an important tool in the field of preclinical and clinical research to accelerate the drug development process (113). The most promising tool of this technology focuses on non-invasive visualization of anatomical structures and physiological activity in both animal model (preclinical research) and patients (clinical practice) using exactly the same technology, which greatly maximizes its translationality. (111). It features 25 advantages over conventional study methods because it is possible to take measurements from the subject at different times without causing harm and in real-time (114). Furthermore, the reduction of the number of animals used for each study in accordance with the 3Rs principle (replacement, reduction and refinement) is an important advantage that also helps to reduce costs with the power of longitudinal studies (115,116). The combination of preclinical imaging and classical practices can achieve a considerable reduction in the expense and time spent on the development of new molecules and biologics. These molecular imaging techniques can be differentiated into structural and functional, in the former case an animal anatomical image is obtained, while in the latter case a physiological assessment at the molecular and cellular level can be carried out. 7.1 Structural imaging techniques 7.1.1 Micro-Computed Tomography (µCT) Computed tomography is an application of X-ray imaging that provides detailed images of organs, blood vessels, bones and soft tissue. µCT consists of a source of X-rays and a detector placed opposite each other that rotates around the subject in the center of the CT scanner. The X-rays will pass through the object to a greater or lesser extent depending on the density of the tissues, thus allowing its differentiation. The CT scan generate three-dimensional anatomical image, from which sectional or spatial images can be reconstructed (111). The use of some contrast agent is very common, as it allows the observation of soft tissue that would otherwise be difficult to differentiate. The main application of µCT in eye-related studies is to provide an anatomical framework with high image quality for combination with functional studies, particularly in PET and SPECT imaging, as was done in several works (114,117). 7.1.2 Micro-Magnetic Resonance Imaging (µMRI) Magnetic resonance imaging (MRI) is based on the interaction of nuclear spin with an external magnetic field. According to the properties of hydrogen nuclei (protons) contained in water or magnetically nuclei (Gadolinium, Gd), spatial maps are produced. This technique is especially useful for imaging soft tissues depicting anatomic details with high resolution in three dimensions (3D) (111). This imaging technique has been used to study the structure of the eye (118,119), in studies of ophthalmic drug delivery (120,121) and some pathologies (122,123), as it allows real-time determination of the drug distribution in the eye without 26 damaging the tissue. Some preclinical studies have been developed to determine permanence on the ocular surface with µMRI (124). 7.2 Molecular imaging techniques Molecular imaging technology is the visualization, characterization, and measurement of biological processes at the molecular and cellular levels in humans and other living systems (125,126). Molecular imaging technology can provide valuable information on the in vivo pharmacokinetics and distribution of drug biomarkers, to understand the permanence of a drug at its site of action, to confirm its biological activity, to show the mechanisms of a disease, or to validate the efficacy of drug treatment. Maximizing the collection of in vivo biological information during the preclinical phases of drug development can be valuable during the selection/optimization of promising drug candidates (111). In vivo imaging techniques such as PET and SPECT also allow pharmacokinetic and pharmacodynamic studies in animal models without the need to sacrifice animals at every study time. “Motion frozen” PET and SPECT imaging in pharmacokinetic and pharmacodynamic studies is feasible because of the option of dynamic and static imaging. As PET and SPECT provide quantitative information, it is possible to quantify the amount of drug in the organs of interest and to monitor it over time (111). 7.2.1. Micro-Single Photon Emission Computed Tomography imaging (µSPECT) Single-photon emission computed tomography is a cross-sectional nuclear imaging technique that uses gamma rays resulting from radionuclide decay, providing a three-dimensional spatial distribution. Some common radionuclides used in clinical practice are 99mTc, 67Ga, 111In or 123,125,131I (127), which require to be injected into the subject. The single-photon emissions are ejected from the source multi-directionally with equal probability. Because this occurs, an aperture composed of highly attenuating material is required to identify the source path of each emission before constructing an image with a gamma camera (111). The extended half-life of these isotopes translates to longer scan times and/or scanning periods without the need for secondary isotope administration. This means that smaller quantities of isotope are needed. 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PET/CT imaging of I-124-radiolabeled bevacizumab and ranibizumab after intravitreal injection in a rabbit model. Invest Ophthalmol Vis Sci. 29 de julio de 2011;52(8):5899-903. 142. Verel I, Visser GWM, Boellaard R, Walsum MS van, Snow GB, Dongen GAMS van. 89Zr Immuno-PET: Comprehensive Procedures for the Production of 89Zr-Labeled Monoclonal Antibodies. J Nucl Med. 1 de agosto de 2003;44(8):1271-81. 39 CHAPTER 1 DRUG DELIVERY TO THE POSTERIOR SEGMENT OF THE EYE: BIOPHARMACEUTIC AND PHARMACOKINETIC CONSIDERATIONS 40 41 CHAPTER 1 DRUG DELIVERY TO THE POSTERIOR SEGMENT OF THE EYE: BIOPHARMACEUTIC AND PHARMACOKINETIC CONSIDERATIONS ABSTRACT The treatment of the posterior-segment ocular diseases, such as age-related eye diseases (AMD) or diabetic retinopathy (DR), present a challenge for ophthalmologists due to the complex anatomy and physiology of the eye. This specialized organ is composed of various static and dynamic barriers that restrict drug delivery into the target site of action. Despite numerous efforts, effective intraocular drug delivery remains unresolved and, therefore, it is highly desirable to improve the current treatments of diseases affecting the posterior cavity. This review article gives an overview of pharmacokinetic and biopharmaceutics aspects for the most commonly used ocular administration routes (intravitreal, topical, systemic, and periocular), including information of the absorption, distribution, and elimination, as well as the benefits and limitations of each one. This article also encompasses different conventional and novel drug delivery systems designed and developed to improve drug pharmacokinetics intended for the posterior ocular segment treatment. Keywords: ocular pharmacokinetics; ocular drug delivery systems; ocular routes of drug administration; intravitreal administration; topical administration 42 1. INTRODUCTION The posterior segment of the eye comprises the back two-thirds of the eye, including the vitreous humor, the retina, the choroid and the optic nerve. Posterior Segment Eye Diseases (PSEDs) are then defined as the disorders that affect these tissues with the common main outcome of varying degrees of visual impartment and blindness. The most prevalent diseases are glaucoma, agerelated macular degeneration (AMD) and diabetic retinopathy (DR) (see Figure 1). Nowadays, millions of people are suffering from retinal and choroid diseases and the number is increasing every year, as the incidence significantly increases with age. Both disorders are characterized by their severity and difficulty of treating. Despite numerous efforts, effective intraocular drug delivery remains unresolved and therefore, it is highly desirable to improve the current treatments of diseases affecting the vitreous cavity. Figure 1. Prevalence of the main ocular pathologies. Data from the world report on vision published by the World Health Organization (WHO), 2019 [1]. In clinical practice, the standard procedure in treating these disorders is the intravitreal administration of injected drugs, although topical and systemic administration have also been addressed with limited results. Thus, other approaches have been developed for the treatment of posterior segment diseases such as periocular, suprachoroidal, and subretinal administration (Figure 2). All these routes of drug administration consist of the drug/system injection in the surroundings of the target site. Periocular administration includes subconjunctival, sub-Tenon’s, peribulbar, retro bulbar, and posterior juxtascleral injection. However, these injections might not result in therapeutic drug levels in the target site due to the necessity of crossing several barriers to reach the P r e v a le n c e Y e a r N u m b e r p e o p le (m illio n s ) 2015 2020 2025 2030 0 100 200 300 A M D G la u c o m a O th e r o c u la r p a th o lo g ie s 49 (although uncommon) are eye infections or vitreous hemorrhage. Depending on the disease being treated, a follow-up visit to the specialist should be scheduled for the administration of repeated intravitreal injections, always needed in chronic disorders such as DP or AMD [21]. Despite the associated complications to the injection procedure and discomfort after administration, repeated intravitreal injections are the only alternative to administer drugs as anti-VEGF, steroids, and antibiotics to treat pathologies affecting the posterior segment of the eye. 2.1.3. Pharmacokinetics Nowadays, the pharmacokinetics and pharmacodynamics of pharmacological agents after intravitreal administration remain relatively unexplored and poorly understood. The vitreous-humor clearance mechanisms of intraocularadministered drugs limit the duration of their effect and injection’s repeated administration. Nevertheless, there remains a real need to develop novel drug delivery systems intended to sustain release and more effective therapy. Pharmacokinetics studies of intravitreal injection have demonstrated the drawbacks of an invasive administration route and the difficulty of acquiring direct samples. Most of the in vivo studies have been performed in rabbits as an experimental animal model [22]. Vitreous humor samples collection may require animal sacrifice, even though novel techniques such as micro-dialysis can be applied for continuous sample collections without animal detriment [23]. According to the 3Rs regulatory frameworks, the alternative is to extract aqueous humor or blood samples and then make a correlation with the drug concentration in the vitreous humor. The drug concentration at any certain time after injection depends on the drug distribution volume, the initial injected dose and the elimination rate. Therefore, the two factors that predominantly affect the pharmacokinetics of injected drugs into the vitreous are their vitreous humor distribution and their clearance. Drug Distribution in the Vitreous Humor Drug distribution in the posterior segment of the eye is a crucial step in ocular pathologies treatment in order to obtain a pharmacological effect at the right target site. The factors that affect the drug distribution in the vitreous humor are predominantly its diffusion through the vitreous, the effect of the convective flow and the possible drug interactions with the vitreous humor elements. Moreover, other ocular barriers may limit the drug passage depending on the target site within the vitreous cavity [24]. 50 Another aspect that should be taken into consideration is the effect of the injection itself, as the needle creates a channel through the vitreous humor. After the needle is being removed, it leaves behind a low resistance pathway for the passage of the drugs due to a change of the integrity of the vitreous humor [25]. The impact of both diffusion and convection flow in the drug pharmacokinetics within the vitreous humor has been studied based on different experimental data and mathematical models [7,26–29]. 1. Diffusion Once the drug is injected into the vitreous humor, it will diffuse through the vitreous humor until reaching its target tissue. The diffusion speed of this process depends on the drug physiochemical properties and the vitreous retention effect. The mesh size of the bovine vitreous humor was calculated to be approximately 500 nm [30]. Also, the different structural components presented in the vitreous constitute a barrier to the diffusion of the drugs depending of the characteristics on the drug. Drug diffusion through the vitreous humor is created as drug concentration gradients from the injection site, which provides the moving force for the drugparticles’ movement until a concentration balance is reached, where not more diffusion occurs. Regarding the drug physicochemical properties, the molecular weight and the drug net’s charge are the parameters that most influence in the diffusion speed through the vitreous humor. Generally, low molecular-weight drugs (e.g., fluorescein, glycerol, mannitol) exhibit high diffusion coefficients as they do not have the diffusion restriction through the vitreous meshwork [31,32]. In fact, the diffusivity in an aqueous solution could be a specific representation of this parameter in the vitreous humor. Diffusion of high molecular-weight molecules (e.g., FITC dextran) can be limited by the vitreous structure [33]. On the other hand, the drug net’s charge could affect its diffusion since the vitreous humor is a negatively charged polymer network. Anionic and neutral molecules will not show any type of restriction in the vitreous humor considering their charge. However, cationic drugs could electrostatically interact with the negative charges of the vitreous humor [30,34]. This will lead to a decrease in the drug diffusion due to its retention in the vitreous humor. 2. Convection Convection is the process of moving a fraction of volume of the aqueous humor produced by the ciliary processes throughout the vitreous humor towards the 51 retina [7,26,28,29]. This process takes place due to the differences in pressure and temperature between the anterior chamber and the retinal surface [7]. The significance of the vitreous outflow effect on drug distribution depends on drug diffusivity in the vitreous [28]. It has been seen that drugs with high diffusivity values within the vitreous are not affected by the convective flow due to the high net movement through the vitreous humor, while low molecular weight drugs do not suffer any type of restriction through the vitreous. On the other hand, the relevance of the convective flow in the movement of drugs with low diffusivity values is quite unclear, even though it is generally accepted that the convective flow has a major effect when the diffusivity is lower and the flow is increased. Park et al. have calculated the drug diffusivity values that could be affected by the convective flow, using a mathematical model. The convection does not affect the drug distribution with high diffusivity values within the vitreous (1 × 10−5 cm2/s) but it starts to become relevant in the case of drugs with low diffusivity values (1 × 10−5 cm2/s), particularly if the flow is increased [26,28]. Moreover, Xu et al. predicted that convection could be responsible for the 30% of the drug movement through the vitreous humor [27]. In the light of these results, it can be concluded that the convection is not a major contributing factor in the drug distribution through the vitreous humor [3]. Since the intravitreal convective flow depends on pressure, it seems obvious that an increase in the intraocular pressure could change flow values in the vitreous humor. As an example, in some pathologies such as glaucoma and retinal detachment, where higher intraocular pressure values are noticed, an increase in the convection has been observed [26,29]. 3. Drug Interaction with Vitreous Humor Hyaluronic acid, as one of the main vitreous components (65–400 mg/mL concentration range), may establish charge interactions with intraocularly administered drugs that are positively charged [35]. This binding could importantly decrease the cationic-drug diffusion through the vitreous humor (e.g., poly-l-lysine) [3]. However, up to now, the effect of this binding on drug pharmacokinetics remains to be elucidated. 4. Drug Interaction with Proteins Protein concentration in the vitreous humor was estimated to be 4.7 ± 1.2 µg/µL in human samples [36], being the albumin the most abundant one (60–70% of the total protein) [37]. Between 1000 and 2500 different proteins have been identified 52 in the vitreous cavity [36,38]. Although their concentration is very low in comparison with plasma concentration, the interaction between drugs and vitreous proteins can occur in the same way with plasma proteins. This interaction will lead to a free drug decrease to exert a pharmacological effect. Furthermore, the drug binding to a protein could slow down its diffusion through the vitreous humor increasing the residence time in the vitreous. In some vitreoretinal pathologies, such as diabetic vitreoretinopathy, an increment in protein concentration in the vitreous humor is observed, as well as new proteins are expressed and linked to inflammation and immunity processes [36]. However, there is no available data regarding the impact of the increased protein concentration in the drug interaction with the proteins in these chronic diseases. 5. Liquefaction One factor influencing both drug diffusion and convection processes in the vitreous humor is its own liquefaction, that is, the degeneration process of the vitreous humor associated with aging. The vitreous humor is observed in both liquid and jelly forms at the same time in the vitreous cavity [39]. Nevertheless, the proportion between these two forms gets modified with age, being observed as an increase in the liquid proportion and a decrease in the gel one [7], due to the disruption of the fibers mesh composing the vitreous humor. Vitreous liquefaction could cause an increase in the drug diffusivity, particularly in those that showed a limited diffusion, since the fibers mesh presents less movement restriction of molecules in its interior. This expanded diffusion can lead to an elimination increase, although the liquefaction itself does not directly affect the drug elimination from the posterior segment of the eye. The vitreous humor higher liquefaction, the more closely the diffusivity of the molecules in it approximates to their diffusion in water [3]. On the other hand, liquefaction and the vitreous homogeneity loss through aging are also associated with an increase in the convection flow [40]. Such data entails that the treatment of different-age-group patients with the same dosing scheme might be inappropriate, leading to overdose or insufficient dosage situations. Even though, so far it is not clear whether the vitreous liquefaction can affect the intravitreal pharmacokinetics or not [41,42]. It is also worthwhile to mention that the injection position during the administration and the injected volume may have an influence on the distribution of molecules in the vitreous humor. Different injection sites affect the drug distribution and permeability through the retina [43], whereas the injected 53 volume could affect the drug elimination in different degree, depending on the injection position [43]. On the other hand, special patient situations, such as vitrectomy (procedure where the vitreous humor is removed), also determine the drug behavior into the vitreous. The motivations to perform a vitrectomy are varied, where the most common ones are: 1) Removal of infection, 2) obtention of a better access to the retina, 3) removal of scar tissue, and 4) correction of retinal detachment. After the vitrectomy procedure, the drug elimination is greatly increased, regardless of the elimination pathway. In different animal studies, the half-life reduction of intravitreal drugs has been detected after a vitrectomy was performed, including: Amikacin [44], amphotericin B [45,46], cefazolin [47], ceftazidime [48], ciprofloxacin [49], and vancomycin [50], as well as some biologics, such as bevacizumab [51,52], ranibizumab [51,53], and aflibercept [53]. It also must be taken into account that these studies were performed in animal models and that its extrapolation to humans lacks of enough knowledge. However, it seems to be a general decrease in the half-life in humans and might affect the efficacy of the intravitreal drug, although this aspect is still under investigation [54]. Moreover, a higher risk of retinal toxicity is expected in vitrectomized eyes. The anti-infective is supposed to be placed in contact to the retinal surface on a high concentration, instead of being distributed all over the vitreous humor, consequently causing the retinal toxicity [55]. This is extremely important in drugs as amikacin since it has proven to produce retinal toxicity [56]. Surgical vitrectomy is normally followed by the filling of the vitreous chamber with silicone oil, which acts as a long-term buffer in the management of vitreous detachment. Injected silicone oil can also behave as a slow-release reservoir for some drugs [7], although it must be taken into consideration that most antiinflammatory drugs are not soluble in the silicone oils. Several studies confirmed the drug injection into silicone oil-filled eyes led to its migration to the aqueous interface, resulting in an increase in the local concentration and the ulterior precipitation. This may cause retinal toxicity [57,58], supposedly caused by a decrease on the preretinal-space, which can also affect the drug distribution and half-life. Therefore, the drug pharmacokinetics in silicone oil-filled eyes is still not well defined, although some authors recommend a substantial reduction of the drug dose (1/4–1/10 of the standard dose) to prevent these phenomena [59]. 54 Drug Distribution to Surrounding Tissues Depending on the type of disease, the target site could be located in the vitreous humor itself (e.g., infections), the retina (e.g., AMD, diabetic macular degeneration), or the choroid (e.g., serpiginous choroiditis). Therefore, the drug distribution to the surrounding tissues could be considered as part of the elimination process if the site of action is in the vitreous humor or as part of the drug distribution in order to achieve its target site. Nevertheless, studies performed in rabbit eyes have shown that the distribution volume is close to the anatomical volume of the vitreous chamber which implies that the surrounding tissues do not contribute too much to the distribution process [3]. The vitreous body is limited by the anterior blood–aqueous barrier (BAB) and posterior BRB. The inner BRB allows for the permeation of small molecules, where molecules with a size higher than 2 nm are prevented from their diffusion. Likewise, the RPE is a tight cellular layer between the photoreceptors and the choroid, which its permeability depends on the molecule’s size and lipophilicity [3]. Once the drug reaches to the choroid, the drug diffusion is quite rapid, because of the higher permeability of the choroid and, subsequently, the drug is quickly removed to the blood circulation [3]. There is evidence of BRB influx-and-efflux carriers that ensure the retina is constantly supplied of nutrients and ions [7,8,60]. The evidence of efflux transporters at the BRB has been recently investigated, as the studies performed in animal models might not correlate with the results that could be obtained in humans. MDR1, BCRP, some MRP, and OATP are some of the main carrier families that have been detected in the BRB [61]. It should be noted that some drugs can be substrates of the BRB active transporters, but their contribution to the drug pharmacokinetics is still unclear. Firstly, it is needed to be addressed that the presence of active transport at the BRB could be an advantage if the drug target is in the choroid, as it will help the drug to reach the target site or even ensure that some drugs, that normally are not able to cross the BRB, can achieve the choroid. Conversely, this fact could be a disadvantage if the target site is prior to the retina or in the retina itself, the active transport will act as an elimination pathway. Overall, the active transport contribution on the drug movement through the BRB is quite low, the effect being also reduced over the time. As the drug concentration at the vitreous humor is usually very high after administration, the transporters are prone to be saturated [61]. The drug elimination from the vitreous humor involves the drug possible metabolism in the ocular tissues and the removal from the ocular compartments to the systemic blood flow. 55 Drug metabolism in the vitreous humor has not been deeply investigated. Mainly, studies have aimed at the enzyme identification in the vitreous humor, but not at analyzing its impact of drug pharmacokinetics [45]. For example, the presence of enzymes such as esterases or peptidases in rabbits´ vitreous humor should be mentioned here [62]. The drug in situ metabolism have been exploited for the development of prodrugs, such as ganciclovir esters (prodrugs with no pharmacological activity) which are biotransformed into ganciclovir (drug with pharmacological activity) once injected into the vitreous humor [62]. Metabolic enzymes have been detected in other ocular tissues posterior to the elimination of the drug from the vitreous humor, such as retina, ciliary body, and iris [63]. There are two major routes of drug elimination from the vitreous: Anterior and posterior clearance (Figure 4). Figure 4. Schematic representation of the anterior and posterior clearance from the vitreous humor. 1. Anterior Route After intravitreal injection, the drugs will be eliminated following the anterior route from the vitreous by a diffusion process across the lens and the ciliary body, to enter afterwards into the posterior chamber. From there, drugs are removed through the aqueous humor turnover to the anterior chamber, where they are subsequently removed along with aqueous humor by the trabecular and uveoscleral outflow [8]. The rapid turnover of the aqueous humor into the anterior chamber is the main force for the anterior clearance [7]. This elimination route is accessible for all type of drugs as they can freely move across the hyaloid membrane, avoiding the lens. However, drugs that are typically removed from this pathway are hydrophilic and large molecules that are not able to cross the retina [7]. The elimination of high molecular-weight 56 compounds by the anterior route has been widely studied [40,42,64] (see Table 1). In fact, there is an inverse relationship between the molecular weight and the elimination rate from the vitreous. Experimental data have determined that drugs which are removed from the anterior pathway exhibit higher half-lives than the ones that are removed from the posterior route. The relationship between vitreous half-life and aqueous humor/vitreous humor ratio is not broadly clarified. However, it is known that the presence of the drug is higher in the aqueous humor, i.e., it is removed by the anterior route, as the half-life is higher [65]. Table 1. Vitreous half-life times for intravitreally administered drugs with different pharmacokinetic characteristics. Pharmacologic Group Drug Characteristics Half-Life Time (h) Ref. Corticosteroids Dexamethasone Low molecular weight Water insoluble 3.48 [66] Antibiotics Ceftizoxime Low molecular weight Water soluble 5.70 [67] Somatostatin analogues Octreotide acetate High molecular weight Water soluble 16.00 [68] Antiviral ISIS 2922 High molecular weight Water soluble 62.00 [69] 2. Posterior Route In the posterior route, administered drugs permeate through the retina and subsequently are cleared by the choroidal blood flow. Drugs that are removed by the posterior route exhibit short half-lives due to the large surface area available for permeation and the presence of active transport mechanisms [65]. In posterior elimination processes, a relationship between the drug physicochemical properties and their half-lives within the vitreous humor has been identified. Durairaj et al. established that the drug molecular weight, its lipophilia, and the dose/solubility ratio at pH 7.4 are the major parameters that affect the drug half-life in the vitreous [70]. Therefore, the posterior route is the main elimination pathway for small and lipophilic molecules since they can easily cross the retina. The diffusion process could take place via the paracellular and/or transcellular route. As can be seen in Table 2, some important differences are shown between the parameters affecting the anterior and posterior elimination route of drugs from the vitreous humor. 57 Table 2. Comparison of the anterior and posterior route of drug elimination from the vitreous humor [65,70]. Features Anterior Route Posterior Route Tissue involved BAB BRB Elimination pathway Aqueous humor outflow Choroidal flow Molecule characteristics Hydrophilic High molecular weight Lipophilic Small molecular weight 2.1.4. Drug Delivery Systems Several reviews about the development of drug delivery systems have been published previously [71–74]. For this reason, in this article is not going to be treated in depth. Biodegradable implants [75], non-biodegradable implants [76], biodegradable microspheres [71], nanoparticles [77], dendrimers [78], and hydrogels [79] have been used for intravitreal drug administration. Moreover, some sophisticated systems have been developed for the treatment of chronic and refractory ocular diseases, such as a microelectromechanical system (MicroPump) [80] and a port delivery system (PDS) [81]. In addition, to the current research on new systems of intravitreal release [82], there are already commercialized formulations such as LucentisÒ, OzurdexÒ, EyleaÒ, AvastynÒ, among others (see Table 3). Table 3. Summary of the main key pharmacokinetic parameters for different intravitreally administered drugs. Pharmacologic Group Subgroup/Drug Half-Life Time (h) MRT (h) Cmax (µg/mL) Ref. Nonsteroidal antiinflammatory drugs Ketorolac 4.3 6.16 175 [83] Diclofenac 2.05 2.95 65 [83] Antibiotics Penicillines 10–20 5–25 1000–5000 [84–86] Cephalosporines 5–15 5–30 1000–2250 [48,85,87] Tetracyclines 10–20 NA 125–400 [86,88] Fluoroquinolones 3.5–5.5 0.25–5 100–500 [49,89,90] Monobactams 7.5 NA 1000 [91] Carbapenems 2.5–10 NA 50–100 [92,93] Macrolides 40–60 NA 100–200 [85,94,95] Antibodies Bevacizumab 4.32 5.92 400 [96] Ranibizumab 2.88 4.03 162 [97] 58 2.2. Topical Administration Ophthalmic topical administration by eye drops is commonly used for the treatment of anterior-segment diseases [98,99]. Most of the topically applied drugs are intended for the treatment of diseases that affect different layers of the cornea, the conjunctiva, iris, or the ciliary body [5]. However, topical administration for the treatment of posterior ocular diseases is considered an ineffective pharmacological strategy since therapeutic drug concentrations are not reached in the posterior segment of the eye due to low drug penetration. 2.2.1. Ocular Barriers for the Entry of Drugs: Precorneal Factors After topical eye-drops administration, the first tissue barrier that drug molecules must overcome to access the target is the tear drainage of the excess volume through the nasolacrimal duct. In normal conditions, this drainage occurs at 1.45 µL·min−1 and it results in a drug loss into systemic circulation, especially related to hydrophilic molecules [100]. In fact, the loss of eye drop solution occurs until the tear volume returns to a normal range (7–9 µL). Likewise, the thin precorneal tear film secreted by different glands and the Globet cells, which is about 8 µm thickness and with a 7 µL volume, also acts as a barrier in terms of drug absorption. It is composed of three layers: mucin, an aqueous and a lipid layer. The rate of drug elimination from the tear fluid is in the range of 0.5–1.0 µL·min−1 [101,102]. As a result of these facts and the systemic absorption through the conjunctiva, the ocular drug absorption is limited to less than 5% (<5%) when this delivery method is used [102]. 2.2.2. Corneal and Anterior Compartment Barriers Cornea The cornea is the transparent portion surrounding the sixth anterior part of the eyeball with a 0.5 mm thickness and a 12 mm diameter. Tear film and aqueous humor provide nourishment and oxygen as it lacks blood vessels. The cornea consists of a collagen structure organized in six layers: Epithelium, Bowman’s membrane, stroma, Dua´s layer, Descemet’s membrane, and endothelium (Figure 5). The stratified, squamous and non-keratinized epithelium is the most critical barrier to penetration with a 10−7 –10−5 cm−1 drug permeability rate because of the fact that tight junctions impair the permeation of low lipophilic molecules [12,100,103]. 65 intravenous, or intramuscular). Once the drug reaches the bloodstream, the absorption will take place through conjunctival, episcleral and/or choroidal vessels to get the vitreous cavity, although most of the drugs do not pass the main blood–ocular barriers [6]. 2.3.1. Advantages and Limitations Systemic drug administration shows a series of advantages and disadvantages compared to other administration routes to treat posterior segment diseases. Firstly, systemic administration is very effective in the case of concomitant ocular and systemic diseases as they can both be treated with only one treatment. Although ocular effects slowly appear with the systemic administration compared to other routes, the duration of the effect is more prolonged [127]. The oral administration to treat ocular diseases presents some positive features: Non-invasive, no need to use strict sterile conditions, high patient compliance, and adherence to treatment and width availability of pharmaceutical forms that provide great drug stability [5,103,128]. Oral administration is usually combined with topical ocular administration [5]. However, the systemic route presents certain drawbacks [5,103]. The presence of physiological barriers (BAB and BRB mainly) prevents the drug passage to the eye, leading to a drug bioavailability of less than 2% [5]. This low bioavailability forces the administration of high doses of the drug to obtain therapeutic concentrations into the vitreous which may lead to systemic toxicity and severe side effects. In addition, a lag time occurs between the drug administration and onset of pharmacological effect. An important prerequisite for a drug to be administered by oral route (for ocular applications) is to have a high drug oral bioavailability [5]. Even though, the limited access to the posterior segment of the eye implies the administration of high doses of drug or an increased administration frequency to obtain a significant therapeutic response [103,116,129]. However, these procedures can exacerbate drug toxicity as a consequence of the drug nonspecific absorption to other organs [116,130,131]. Trained personnel are also required in case of intravenous or intramuscular administration. Likewise, parenteral administration is also a systemic administration route used as an alternative pathway in posterior ocular pathologies. Ocular drugs can be administered by intravenous injection, although its use is less frequent than oral administration route [132]. 66 2.3.2. Pharmacokinetics Absorption to Ocular Tissues Systemically administered drugs can easily reach the choroid due to the high vascularization of this tissue, as more than 85% of the ocular blood flow takes place in this layer, with a value of 43 mL/h [133], and choriocapillaris fenestrations [5,130]. Drug transport from blood circulation to the ocular cavity is strictly regulated by two anatomical and physiological barriers: The BAB and the BRB [8,134]. These barriers limit drug bioavailability by restricting its intercellular permeation to the anterior and posterior segment of the eye. This barrier effect is mainly due to the presence of highly complex tight junctions among epithelial and endothelial cells, which are located on the interface between blood flow and intraocular tissue. Moreover, it was observed that the drug passage improves during inflammatory conditions [135]. This is due to an increase in vascular permeability that leads to greater extravasation of components from the bloodstream to extravascular tissue [136]. Drug entry into posterior ocular tissues is mainly governed by the BRB. Thus, the relationship between drug permeability and physicochemical factors has been demonstrated, concluding that drug permeability increases with decreasing molecular weight and/or protein binding but improves with increasing lipophilicity [127]. As a result, small and lipophilic compounds can easily cross eye barriers (RPE, non-pigmented internal ciliary epithelium and retinal blood vessels), while hydrophilic and large compounds penetration is restricted [133]. However, hydrophobicity and high molecular weight tend to increase drug’s half-life time in the posterior segment of the eye [137]. Therefore, RPE presents a selective permeability to highly hydrophobic drugs, whereas the penetration of hydrophilic and/or large substances being much more limited. The type (influx or output flow) and/or location (vitreous or blood side) of the ocular transporters also condition the drug absorption and its concentration in the intraocular cavity [138]. Recently, many drug efflux pumps were identified in the ocular barriers. Among transporters with the greatest influence on the drug´s arrival to the posterior segment of the eye are the efflux transporters, these being a part of the ATPbinding cassette (ABC) protein family located in the RPE [139]. Specifically, two efflux pumps related to the development of chemoresistance were described, these being: P-glycoprotein (ABCB1) and the multidrug-resistance associated protein (MRP1) (ABCC1). 67 P-glycoprotein is an efflux protein located in the iris, cornea and ciliary muscle, as well as in conjunctival epithelium, non-pigmented ciliary epithelium and RPE. It is a 170 KDa membrane protein that is expressed on the apical surface of the aforementioned cells [139]. It actively promotes drug molecules’ exit from retinal endothelial cells, reducing drugs accumulation within them [139]. For its part, MRP1 is a 190 KDa efflux protein encoded by the MRP1 gene on chromosome 16p13.1 and bounded to the choroidal side of the retinal barrier. It is an atypical ABC transporter with two cytosolic nucleotide-binding domains (NBD) and seventeen membrane-spanning domains (MSD) [140]. It functions as a multispecific organic anion transporter, mediating an ATP-dependent transport of drugs and other xenobiotics [141]. In any case, molecular mechanisms of these drug transporters are not completely known. Distribution to Ocular Tissues In general, systemically administered drugs reach the target tissues from the blood. Drug plasma transport is protein-binding dependent, giving rise to 1) free drug fraction, which is active and susceptible of excretion/metabolism and 2) protein-bound fraction that acts as a drug inactive reservoir. Only the free drug fraction can cross biological membranes and, consequently, reach the ocular tissues. Specifically, drug distribution through blood–ocular barriers is a key factor in the achievement of an effective ocular treatment with systemically administered drugs. Before mentioned ocular barriers regulate drug transfer between blood circulation and the eye in both directions. Drugs whose transport is predominantly carried out by passive diffusion, distribution and clearance are independent of the drug transport direction, although mediated permeation by transporters could lead to a drug preferred transport direction (inward or outward) [3,128]. In any case, it must be taken into account that, although the eye barriers structure and main permeability trends have been known for some time, the distribution process from the blood circulation to the posterior segment of the eye is not yet fully elucidated. However, pharmacokinetic simulation models have been created for the drug concentrations prediction in the vitreous humor, depending on the free drug concentration and the blood flow between the general circulation and the posterior segment of the eye [133]. Once drugs reach the vitreous humor, their distribution and elimination follow the same pattern as the followed after an intravitreal administration. The 68 parameters that affect the drug pharmacokinetics within vitreous humor have been discussed in the section of intravitreal administration. 1. Proteins and Biological Binding The free drug may accumulate more than expected in any posterior ocular layers if it binds to cellular components or if it acts as a substrate for a significant active transport process. Specifically, drugs binding to ocular tissues’ proteins and pigments, mainly melanin, significantly affects their transport to the posterior segment of the eye [6,130,142,143]. Melanin is a polyanionic biological pigment located in the uvea and the RPE in the ocular tissues as melanosomes or pigment granules, which are melanoprotein complexes where melanin is bound to a protein matrix [142]. Menon et al. determined the existence of 6–8 mg melanin at the ocular level [6,144]. Drug binding to melanin and proteins in ocular tissues is an important factor in drugs administration by systemic route since they can modify the drug bioavailability in the target site and, consequently, reduce its pharmacological activity [5,145]. Specifically, ocular melanin has a significant influence on drug pharmacokinetics and permeation through the retina [5,146] due to its capacity to bind (mainly reversible binding) free radicals and chemicals, especially basic (pKa values above 7) and lipophilic drugs by electrostatic, charge transfer, and van der Waals forces [5,147]. Main pharmacological consequences of the drug-melanin complexes are drug accumulation and retinal toxicity, besides the fact that larger doses are needed to obtain a therapeutic response due to the bound-to-melanin drug inactivation (e.g., betaxolol, metoprolol, and phosphodiesther oligonucleotides) [142]. Nevertheless, this drug depot may act as a reservoir over a long time, prolonging drug effects [145,146]. Drug Elimination Regarding drug elimination process by this route, it must be taken into account that the administered drug not only faces passive barriers but also active barriers, such as clearance through the choroidal blood flow, which is extremely high, and the number and size of the choriocapillaris fenestrations that make up this system [6,148], by presenting a 70–80 nm pore size and a number between 30–50 fenestrations/µm2 [149,150]. In general, drug elimination from the posterior segment of the eye can be carried out by two different ways, anterior and/or posterior pathways [103], following the same pattern described for the intravitreal administration route. 69 2.3.3. Drug Delivery Systems The design of ophthalmic drugs systemic administration forms is aimed at achieving therapeutic concentrations in the posterior segment of the eye without causing undesired side effects. Drug targeted administration to these tissues from the systemic circulation has only been studied in preclinical animal models. Studies were carried out based on qualitative investigations performance and assessed by microscopy, immunohistochemistry, and/or angiography techniques [3]. Some of the novel systemic targeting systems studied for drug transport through the choroid to the posterior segment of the eye include [137]: 20 nm gold nanoparticles [151], polyethylene glycol (PEG) conjugated immunoliposomes [103], PLGA nanoparticles or Visudyne®. Currently, none of them is commercialized except for Visudyne®, an intravenous administration formulation used in photodynamic therapy for age-related wet macular degradation treatment [151]. Even so, several strategies related to the design of advanced delivery systems are currently under study, such as: 1) Structurally modified drugs that can effectively avoid MRP1 efflux transporter to improve ocular penetration, 2) hydrophilic drug administration through advanced delivery systems directed by transporter/receiver (superficially marked systems with an ocular-receptor specific substrate and/or vehicles attached to substrates that show a high affinity for ocular tissues) [139], and 3) drug affinity improvement to BRB transporters, such as amino acid, oligopeptides or cation and anion transporters, in order to increase drug transport to the posterior segment of the eye [152]. 2.3.4. Systemic Drugs for Posterior Segment Eye Diseases Drug systemic administration is not the preferable route in the treatment of posterior ocular segment pathologies, although it is still useful in many cases, being an administration pathway carried out by different routes (e.g., oral, intravenous, intramuscular). Thus, some drugs are administered orally (see Table 4). 70 Table 4. Summary of orally administered drugs for the treatment of posterior segment ocular diseases. Pharmacologic Group Drug Pathology Administration Route Ref. Analgesics Paracetamol Ocular trauma treatment-associated pain Oral [153] NSAIDs (Flurbiprofen, Ketorolac, Diclofenac, Bromfenac and Nepafenac) Ocular trauma treatment-associated pain Oral [153] Antibiotics Doxycycline Neovascularization Oral [110,154] Tetracycline Ocular rosacea Oral [111,155] Erythromycin Orbital cellulitis Oral [111] Minomycline Ocular rosacea Oral [110] Corticosteroids Dexamethasone Giant cell arteritis Oral [153] Immunosuppressants Cyclosporine Idiopathy or related-toBehçet’s-disease uveitis Oral [156] Carbonic anhydrase inhibitors Acetazolamide (Diamox sequel®) Glaucoma Oral [113, 157] Etoxolamide Glaucoma Oral [114,158] Apart from these, orally administration formulations with antineoplastic and antiviral agents have also been studied [103]. In relation to the latter one, it must be considered that the most frequent ocular posterior segment pathologies with viral etiology are associated with immunosuppression states (e.g., AIDS or transplants). These include: Cytomegalovirus retinitis, whose treatment is based on the antivirals intravenous administration (valganciclovir, ganciclovir, foscarnet, and/or cidofovir) as well as acute retinal necrosis and progressive external retinal necrosis, whose etiology is broad (varicella-zoster, herpes simplex, cytomegalovirus, or Epstein-Bar virus) and whose treatment is based on the acyclovir (very effective), ganciclovir or foscarnet administration [159]. Similarly, parenteral administration formulations have also been developed for the same purpose, including different intramuscular and intravenous preparations. These encompass: Parenteral antibodies for the uveitis treatment [3] and hydroxocobalamin intramuscular injections (B12 vitamin, Alpha Redisol) for the treatment of B12 vitamin deficiency states, as well as certain antibiotics (e.g., penicillin, gentamicin, ceftazidime, or amikacin) used in the subsequent ocular infection’s treatment (uveitis, scleritis, and/or pseudoscleritis) (see Table 5). Likewise, the antibiotic combinations administration by continuous perfusion for the serious-eye-diseases treatment, such as endophthalmitis, is quite frequent 71 [127,160]. As an example, ceftazidime is the best studied cephalosporine due to its activity spectrum against Gram-negative bacilli (including Pseudomonas aeruginosa). It is more frequently used as a first election treatment in this pathology, in monotherapy or combined with other antibiotics, since it allows reaching ocular concentrations in the order of 35.4 mg/l after intravenous administration of a 100 mg/kg antibiotic dose [161]. Table 5. Summary of systemically administered drugs for the treatment of posterior segment ocular diseases. Pharmacologic Group Drug Pathology Target Route Ref. Antibodies Secukinumab Tocilizumab Adalimumab Uveitis Inflammatory cytokines Intravenous [162,163] Ustekinumab Subcutaneous Abatacept T-cell activation Rituximab B-cell targeting Subcutaneous Vitamins B12 Vitamin B12 Deficiency Optic Neuropathy Folate receptor Intramuscular [164] Antibiotics Penicillin Gentamicin Ceftazidime Amikacin Uveitis Scleritis Pseudoscleritis Endophtalmitis Bacteria Intravenous [165–167] In addition to these, different intravenous drug delivery strategies have been tested for drug arrival to the posterior segment of the eye [168]. Specifically, photodynamic therapy with verteporfin has been practiced, being a choroidal neovascularization effective treatment by stopping the neovascular membrane growth. Apart from this, ocular posterior segment diagnostic intravenous techniques have also been developed, being the most prominent the fluorescein digital angiography, an exploratory technique for the retinal vasculature visualization by means of the sodium fluorescein injection (vegetable origin orange dye). In order to point out, eyes can be also exposed to systemic drugs (as a kind of side effects) and xenobiotics not intended for ophthalmic treatment, such as bisphosphonates (whose action mechanism is based on the bone-resorption inhibition, being used in the osteoporosis prevention and treatment), which can cause ocular inflammatory reactions (uveitis, neuritis, iritis, scleritis, or pseudoscleritis) [169]. Reformulation studies have also been carried out on these drugs in order to reduce their passage from the blood to the eye, making them more selective in order to decrease associated ocular side effects [169]. 72 2.4. Periocular Administration The periocular route has been considered as a promising drug administration route for the posterior ocular segment diseases treatment due to the high concentrations obtained with this kind of formulations’ inoculation. This route allows the drug deposition on the scleral external surface and includes the following administration routes: subconjunctival, subTenon´s, retrobulbar, peribulbar and posterior juxtascleral. These pathways differ from one another in the location and/or injection direction in the proximity of the sclera. 2.4.1. Advantages and Limitations Periocular injections are associated with greater adherence to treatment by patients compared to intravitreal injections [139] since it is considered a less invasive administration route and capable of providing a relatively great drug bioavailability in the posterior ocular segment [150,170]. The sclera provides a relatively large area for the drug absorption (approximately 17 cm2) [171] compared to other ocular surfaces, like the cornea. Moreover, periocular administration takes advantage of the high scleral permeability. These two factors contribute to the potential effectiveness of the periocular administration compared to other ocular routes. On the other hand, the main drawback is that the drug needs to diffuse from the site injection to the target site, with the possibility of drug losses. 2.4.2. Pharmacokinetics In general, although this type of administration avoids the corneal-conjunctive barrier, the drug must cross several barriers to reach the target sites in the choroid, the RPE, or the neural retina [65,150]. To do this, it must overcome several dynamic, static, and metabolic barriers that limit drug access to the posterior segment of the eye. Two different types of barriers should be mentioned: 1) Physical barriers, which include sclera, choroid (its high blood flow can remove a significant fraction of drug before it can reach neural retina), and RPE, and 2) physiological barriers, as occurs with conjunctival, episcleral, and choroidal lymphatic flow [150], whose drug elimination ability is relatively fast [3,65,103]. In any case, episcleral blood and lymphatic flows are seen as the main limiting factors in drug periocular distribution, while anatomical barriers and choroidal blood flow are less important [116,150,170]. 73 As a result, drug is removed into the systemic circulation, decreasing ocular bioavailability thereof. However, molecules that escape from the conjunctival vasculature can penetrate through the sclera and choroid to reach the neural retina and photoreceptor cells. In addition, permeability through sclera is lipophilicity-independent (e.g., inulin, methazolamide, pilocarpine, hydrocortisone) [172], unlike corneal and conjunctival layers, being dependent on molecular radio [4,42,137,148]. Drug reflux after periocular administration is the initial loss factor and contributes to its low bioavailability in the posterior segment of the eye [116,173]. It was demonstrated that the use of an adequate injection technique, volume and/or formulation type can improve drug bioavailability and ocular penetration [116,173–175]. Absorption Drugs administered by periocular injections can reach its target site in the posterior segment of the eye through three different routes: Transscleral or direct penetration route, systemic circulation route, and anterior route [6,176]. In the anterior route, the drug diffuses directly through the sclera and the ciliary body, or indirectly through the lacrimal fluid and the cornea because of the conjunctival circulation reflux. In the systemic circulation route, the drug goes to systemic circulation through conjunctival, episcleral and/or choroidal vessels, where it is diluted, and then it returns to the eye by the blood flow. In the direct penetration route, drug reaches the vitreous humor through the underlying tissues; it represents the most important absorption route in terms of drug penetration and distribution to the posterior chamber of the eye. In any case, it should be taken into consideration that the scleral permeability depends on the molecular radio, scleral hydration, and intraocular pressure [176,177] instead of molecular lipophilia [17]. Although the latter improves permeability through the RPE, it also increases drug loss from the choroidal and subconjunctival space to the bloodstream [3]. Regardless of the absorption route, only a small portion of the drug reaches the posterior segment of the eye [116,178] mainly due to the drug loss through periocular space, BRB, choroidal circulation, efflux transporters, and drug binding to ocular tissue proteins [116]. Elimination Once the drug reached its target site, small molecules are rapidly removed from the administration site, presumably through conjunctival and episcleral blood and lymphatic flow [103,139,150], whereas larger molecules have much slower 74 elimination kinetics, around 10 times lower [150], so that their residence time is much greater. 2.4.3. Subconjunctival Route Subconjunctival injection has been used to administer drugs in the anterior segment of the eye, achieving higher concentrations in the anterior chamber compared to the topical administration. However, this route has also been investigated as an alternative pathway to intravitreal injection for the drug administration of retinal diseases treatment [6] due to the fact that it is considered a less invasive technique, comparing it with the intravitreal administration route [116,179]. It also minimizes the side effects, mainly endophthalmitis, cataracts, and retinal damage [116]. The drug is injected under the conjunctival membrane that covers the sclera, avoiding the conjunctival epithelial barrier. In this way, direct access to the transscleral route is achieved [116], increasing its bioavailability in aqueous humor in comparison with the topical route, which presents the corneal barrier as an impediment. Advantages and Limitations Subconjunctival administration shows two effective types of sustained drug delivery to the retina, both the topical and intravitreal administration. Moreover, it is an alternative route in order to allow easy and better accessibility and reduce the side effects caused by the intravitreal injection procedure (e.g., intraocular pressure and cataracts) [3]. The injection volume with the same drug concentration could be higher in this route in comparison with the intravitreal injection [3], enabling a wide dose range. In addition, the enzyme absence in the injection area is an important advantage due to the low enzymatic drug degradation. The availability of pharmacokinetic and pharmacodynamic data about this route is limited. However, the drug delivery to the back of the eye through this route is better compared with topical and systemic administration routes [150]. This pathway also has some limitations regarding the concentration that can cross to the retina. The elimination via systemic circulation and to the tear cause a reduced bioavailability. Nevertheless, the permeability is modified by the age, according to the patient get older, the permeation through the sclera is less prevented so the amount of drug that can reach the retina is higher. 81 Pharmacokinetics Suprachoroidal administration leads to greater vitreous bioavailability for small lipophilic (1.5%) and hydrophilic (0.19%) molecules, while macromolecules (4.2%) show a 6–23 times improvement in terms of ocular bioavailability compared to the drug administration by subconjunctival route [150]. Compared with small molecules, macromolecules have a much longer half-life time in ocular tissues where steady state is slowly reached. Drug levels in the vitreous humor are generally reached 15 h for small lipophilic drugs, 70 h later for small hydrophilic drugs and 500 h later for larger molecules. Therefore, it was seen steady-state drug levels are quickly reached for small molecules, becoming 100– 1000 times slower for macromolecules [150]. Several studies showed a significant fraction of the drug administered by the suprachoroidal route was removed through conjunctival and regional lymphatic nodes, including those of high molecular weight [6,175]. In addition, it should be taken into account that choroidal blood flow also removes a huge part of the inoculated drug (96–99%) in the case of low molecular weight compounds, whereas high molecular weight drugs will be removed from the tissue almost equally through the choriocapillaris (54%) and the subconjunctival space (41%) [150]. Drug Delivery Systems Currently, there are no pharmaceutical forms commercialized to be administered by suprachoroidal injections. Nevertheless, this type of administration was widely studied by using fluorescein and fluorescently tagged dextrans (40 and 250 kDa), bevacizumab and polymeric particles (20 nm to 10 µm in diameter). Sulforhodamine B microneedle injection was also studied as well as nanoparticle and microparticle suspensions into the suprachoroidal space [215,222]. TA formulations were also researched as a way of DMA alternative treatment, as well as ranibizumab and bevacizumab injections, although recent studies showed these large biologic proteins are quickly removed from the suprachoroidal space [220]. 2.4.9. Subretinal Route Drug subretinal route has emerged as an alternative administration route to intravitreal administration due to its side effects and lower adherence to treatment by patients derived from the latter [223,224]. Thus, subretinal administration involves drug inoculation into the subretinal space [224], an ocular space located between RPE cells and photoreceptors [224,225]. 82 Advantages and Limitations Subretinal injection is an especially useful route for the posterior ocular pathologies treatment by providing a direct route with a very precise location through a minimally invasive injection. A typical volume of around 150 µl is injected, leading to a transient detachment between these two layers [2]. A lower drug dose is needed to accurately reach subretinal-space cells. Compared to intravitreal administration, subretinal injection has a direct effect on subretinal space cells. Unlike vitreous cavity, subretinal space is an isolated closed anatomical area, which also has a greater immunological defense mechanism providing a safer route of administration in case of entry of bacteria [178,224]. Basically, three subretinal injection techniques have been studied, these being: 1) a transcorneal route through the pupil, surrounding the lens, and passing the vitreous humor and the retina [226], 2) a transscleral route through pars plana or limbus, crossing the vitreous humor and the opposite side of the retina into the subretinal space [227], and 3) a transscleral route through the choroid and Bruch’s membrane without penetrating the retina [224]. All routes were equally effective regardless of the chosen one. The administration procedure is performed under direct visualization by using a surgical microscope and where blister formation should be observed as a sign of the procedure success. Drug Delivery Systems Subretinal administration has been considered as one of the best strategies for gene therapy using viral vectors, a carried-out treatment effectively achieved for pigmentary retinitis (PR) and Leber’s congenital amaurosis (LCA) [225,227]. Gene expression is however limited to the injection site, suggesting that the primary barrier for efficient therapy following subretinal injection is the retina itself [2]. In addition, it was reported macrophages subretinal injection leads to pathological fibrosis, which could be used for the advanced AMD evaluation [224]. Subretinal delivery can also be used for stem cell transplantation in ocular degenerative diseases, which was reported in vivo studies and aimed at clinical applications [224]. 3. CONCLUSIONS The eye is one of the most inaccessible organs in terms of obtaining therapeutic drug concentrations, especially in the treatment of posterior segment ocular pathologies. Conventional administration pathways, such as topical or systemic 83 routes, usually show important limitations, either by low ocular penetration or by the appearance of side effects linked to the posology, among others. New drug delivery systems (DDS) are needed to prolong the administration intervals for posterior segment ocular pathologies, even though the development of novel DDS is particularly complicated due to several aspects must be considered, such as pharmacokinetics, immunogenicity, biodegradation, tolerability and toxicity, among others. Apart from these, different technological requirements must be taken into account, including reproducible manufacturing, clinical performance and sterility. In the last few decades, an exponential increase in the design and development of novel DDS intended for the treatment of posterior segment ocular pathologies was observed. Biodegradable and non-biodegradable implants, microparticles, nanoparticles, microneedles, and microelectromechanical systems are the most innovative ones. Unfortunately, knowledge about drug targeting to the posterior segment of the eye is still sparse and, thus, there is not many DDS in clinical trials. 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PLoS ONE 2015, 10, e0136523. 101 CHAPTER 2 PRECLINICAL CHARACTERIZATION AND CLINICAL EVALUATION OF TACROLIMUS EYE DROPS 102 103 CHAPTER 2 PRECLINICAL CHARACTERIZATION AND CLINICAL EVALUATION OF TACROLIMUS EYE DROPS ABSTRACT Severe allergic ocular diseases as atopic keratoconjunctivitis can induce corneal damage due to inflammatory substances released from giant papillae. Tacrolimus eye drops are one of the current therapeutic alternatives for its treatment. This work is aimed at developing and characterizing a 0.03% tacrolimus ophthalmic formulation, which was introduced in three types of vehicles (BSS, PVA and Hyaluronic Acid). For this, we have performed in vitro (stability studies) and in vivo assays (corneal permanence time measured directly and by Positron Emission Tomography) of three potential formulations. Next, the best formulation was selected, and its toxicological profile and clinical effectiveness have been evaluated. The ocular permanence studies (direct measurements and PET/CT) showed that the formulations with PVA and Hyaluronic Acid present more retention time on the ocular surface of rats than PBS. From the stability study, we have determined that tacrolimus with PVA in cold storage is the best option. Tacrolimus with PVA has shown lower cytotoxicity than cyclosporine at early times. On the other hand, the pilot study performed has shown significant improvements in patients, with no noticeable adverse reactions. Based on stability, ocular permanence, safety and clinical effectiveness studies, we concluded that tacrolimus-PVA eye drops are a suitable candidate for its clinical application in inflammatory ophthalmology diseases. Keywords: corneal residence time; cytotoxicity; effectiveness; eye drops; stability; tacrolimus 104 1. INTRODUCTION Severe allergic ocular diseases as atopic keratoconjunctivitis (AKC) or vernal keratoconjunctivitis (VKC) can induce corneal damage due to inflammatory substances released from giant papillae [1,2]. Moreover, the inflammation has been shown to be a key factor in the pathogenesis of other pathologies as dry eye, a pathology that affects thousands of people around the world [3], or other complex ocular diseases as uveitis [4]. The pathogenesis involving these diseases is probably multifactorial; however, inflammation plays a major role in which Th2-driven mechanism with the involvement of mast cells, eosinophils, and lymphocytes has been suggested [5]. Maintaining an appropriate eye health is essential to down-regulate the ocular immune response, preserve the integrity of the ocular architecture and decrease the associated symptomatology [6]. The current treatment of AKC is based on the use of topical antiallergic agents and corticosteroids, but the chronic use of topical corticosteroids may increase intraocular pressure and susceptibility to opportunistic infections [7]. The second line of treatment is based on topical immunosuppressive drugs as cyclosporine and tacrolimus. Cyclosporine and tacrolimus are not structurally related; however, their mechanisms of action are similar. These drugs bind to high affinity to immunophilin proteins that are present in most cells and the drug-receptor complex inhibits calcineurin. Cyclosporine binds to cyclophilins while tacrolimus binds to FK506 binding protein (FKBP). The formed complex of tacrolimus-FKBP-12, calcium, calmodulin and calcineurin inhibit the phosphatase activity of calcineurin. This lead to an inhibition of the translocation of some transcription factors (NF-AT), which ultimately decrease the transcription for genes which encode IL-2, IL-3, IL4, IL-5, GM-CSF and TNFa, all of which cause the reduction of T-cell activation [8]. Its mechanism of action has promoted its use in pathologies such as corneal graft rejection [9,10], inflammatory conjunctival and corneal diseases [11,12], uveitis [13,14] or graft-versus-host disease [15]. The use of ophthalmic cyclosporine has been described for several decades, yet only a few products have been successful in reaching the pharmaceutical marketplace. On the other hand, when it is necessary to use higher concentrations of cyclosporine, the Hospital Pharmacy Department (HPD) is responsible for its formulation as a sterile pharmaceutical compound [16,17]. Unlike cyclosporine, tacrolimus eye drops are not marketed and all its use rests in the elaboration in HPD. Nowadays, most of the topical ophthalmic solutions manufactured in these departments are based in vehicles with short retention time in corneal surface and consequently the need of frequent to obtain a sustainable benefit [18,19]. Several types of tacrolimus formulations as ointments [20,21], emulsions [22], liposomes [23], dextran conjugates [24] or cyclodextrins complexation [25] have 105 been described by other authors; however, if these are not synthesized to be marketed, their elaboration in HPD is complicated. The use of new vehicles can be a feasible alternative that could help to overcome the high ocular clearance of conventional eye drops [26]. The increase of patient’s compliance and the establishment of appropriate dosing schedules are key factors for improving the treatment of many ophthalmic pathologies [27]. It is also important to determine safety as well as the stability of the new formulations in order to be administered to patients. Nowadays, there are few studies on tacrolimus eye drops characterization, probably due to the short time as a therapeutic option. In this regard, the aim of this work is to develop and characterize a 0.03% tacrolimus ophthalmic formulation. For this, we have performed in vitro (stability studies) and in vivo (corneal permanence time measured directly and by Positron Emission Tomography) assays of three potential formulations. Next, the best formulation has been selected. Its toxicological profile has been tested in vitro and its clinical effectiveness has been evaluated in patients. 2. MATERIALS AND METHODS 2.1. Preparation of formulations 50 mL of each three ophthalmic formulations with 0.03% (300 µg/mL) tacrolimus were prepared. Three milliliters of 5 mg/mL tacrolimus (Prograf® ampoules) were added into 47 mL of each vehicle. The first formulation (TBS) used BSS® (Balanced Salt Solution, Alcon®) as vehicle. The second formulation (TLI) used Liquifilm® (Allergan®; Composition: 1.4% Polyvinyl alcohol, Sodium chloride, Sodium phosphate dibasic, Sodium phosphate monobasic, Benzalkonium chloride, Edetate disodium and Purified water). The third formulation (THA) used hyaluronic acid (Acofarma®, Spain. Molecular weight 1.5 x106 - 2.0x106 Da) dissolved in BSS® to obtain a mucoadhesive hyaluronic acid hydrogel at 0.4%. 2.2. Stability study Three batches of each formulation (TBS, TLI, THA) were prepared for each storage condition. The formulations were stored in three different temperatures: room temperature (20 ± 2°C), refrigerated (2 to 8 °C) and frozen (–15 to –20 °C), in all cases protected from light. A sample of 100 µL from each formulation and from each preservation condition was withdrawn with a micropipette immediately after preparation and at days 3, 7, 15, 30, 60 and 90. Osmolality, pH, microbiological control growth and concentration of drug were measured. All samples were also visually inspected for any macroscopic changes (e.g., colour, 106 turbidity, precipitation). t90 was calculated in order to characterize the stability properties. t90 is the time that the concentration of tacrolimus in the formulations is maintained above the 90% of the initial concentration. 2.2.1. Quantification of tacrolimus amount The determination of tacrolimus was performed using an Agilent 1260 series HPLC system (Agilent Technologies, USA) equipped with Diode Array Detector HS, a solvent delivery quaternary pump system, maximum pressure of 400 bar and an autosampler with thermostat. The software model OpenLAB CDS 3D UV (PDA) was used for the data processing. The analysis was performed in an isocratic method. The column used was a ZORBAX Eclipse Plus C18 (4,6x50 mm 5 µm) and at a temperature of 60°C. The mobile phase was water-acetonitrile (35:65 v/v) using a flow rate of 1.5 mL.min-1. A wavelength of 210 nm was employed for the quantification of tacrolimus. The volume of the injected sample was 10 µL and the retention time was 3.3 minutes. Each sample of each formulation (100 µL) was blended using a Vortex Mixer RSLAB-6PRO with 900 µL of water (HPLC grade) and then incorporated to the HPLC to determine the drug concentration. Frozen and refrigerated samples were kept at room temperature for 10 minutes before quantification. Each sample was assayed in triplicate. The analytical method was validated according to International Conference on Harmonization guideline recommendations [28]. 2.2.2. Osmolality, pH and Microbiological control growth The pH measurements were performed with a HI5221 HANNA® pHmeter and the osmolality was measured with a MicroOsmometer Fiske Model 210. In order to study the microbiological stability, 1 mL of each formulation were added in blood agar plates, sabouraud agar and liquid thioglycate medium. These samples were grown at 37 °C for a period of 48h, 15 days and 10 days respectively. At the end of each incubation period, microbiological growth was observed and determined. 2.2.3. Statistical analysis The margins set as indicated in Pharmaceutical Codex [29] have been established, with the expiration of the formulation being established when the concentration of active ingredient has been reduced by 10% with respect to the initial concentration. The percentage of drug unaltered vs time was fitted to a first order kinetics using GraphPad Prism® v.5.0b and the degradation constant k and the t90 was calculated. On the other hand, monitoring has been performed in order 209 Figure 7. Activity (SUV) in the different organs (heart, liver, spleen and cervical lymph nodes) versus time after intravitreal injection of 89Zr-adalimumab in both rat groups (Healthy and Uveitis). Filled dots (measured by PET) and error bars represent the mean ± SD of the observed values, whereas the solid lines represent the predicted values obtained by a first-order absorption one-compartment model. 4. DISCUSSION To our knowledge, this is the first work addressing intravitreal pharmacokinetics of adalimumab. In this sense, regarding the ocular permanence of adalimumab, although the group of healthy animals shows a slight two-compartment behavior, data obtained from PET analysis fits well with a one-compartment model for both Healthy and Uveitis groups. The one-compartment model providing significant differences in the k elimination constant from the eye, and subsequently in the time adalimumab remains in the eye (half-life of 15.57 ± 2.64 hours for Healthy group and 33.64 ± 6.69 hours for Uveitis group). These results seem to be in contradiction with the behavior observed in a previous work comparing the pharmacokinetics of two radiopharmaceuticals, 18F-NaF and 18FFDG [23]. In this earlier work, an increase in 18F-FDG clearance caused by inflammation was observed, while 18F-NaF remained unchanged. The 0 24 48 72 96 120 144 168 192 216 240 0.1 1 10 Time (hours) SUV (g/mL) Liver Healthy Uveitis 0 24 48 72 96 120 144 168 192 216 240 0.1 1 10 Time (hours) SUV (g/mL) Cervical lymph nodes Healthy Uveitis 0 24 48 72 96 120 144 168 192 216 240 0.1 1 10 Time (hours) SUV (g/mL) Heart Healthy Uveitis 0 24 48 72 96 120 144 168 192 216 240 0.1 1 10 Time (hours) SUV (g/mL) Spleen Healthy Uveitis 210 explanation for the possible causes of the observed increase in 18F-FDG clearance in uveitis was the increased permeability of blood-retinal barrier and the GLUT transporter. However, the ability of the adalimumab to selectively bind to the tumor necrosis factor is well known. Thus, this result is consistent with the overproduction of TNFa from macrophages and other cytokines in the uveitis process, which activates dendritic cells, starting the inflammatory cascade in which Th1 cells and Th17 cells migrate and infiltrate to the blood-retinal barrier causing damage [37]. This overproduction of TNFa was shown in a previous work of our group where rats with uveitis presented a higher presence of TNFa mRNA in the eye tissues than healthy rats [29]. These measures of TNFa levels have also been carried out by other authors in the preclinical and clinical setting [8,14]. Due to this biochemical mechanism, adalimumab is specifically blocking the binding of human TNFa to the receptors in the Uveitis group. Since adalimumab binds to TNFa receptors located in the uvea, its ocular clearance will be diminished. The results of the pharmacokinetic compartmental analysis in blood show a different behavior between healthy and diseased animals. The activity vs time in healthy animals fits to a two-compartment model showing a rapid uptake of the antibody from the eye simultaneous to the tissue distribution process. However, in the case of animals with uveitis, the activity vs time fits better to a onecompartment 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. In order to compare the rate and magnitude of adalimumab transfer from the eye to the blood, a non-compartmental analysis was used. Regarding to this, our results provided similar blood terminal half-life between the Healthy (339.00 ± 96.90 hours) and Uveitis groups (295.93 ± 46.92 hours), as well as MRT (172.97 ± 18.67 hours and 175.18 ± 17.54 hours, respectively), but significant differences in the tmax between Healthy group and Uveitis group (12 ± 0.0 and 84 ± 13.15, respectively), probably due to the fact that, as can be observed in Figure 6, up to 72h the adalimumab amount in the blood of healthy rats is higher than that of uveitis rats. This could be caused by the binding between antibody and TNFa when the eyes are inflamed. Although ocular inflammation usually increases the permeability of ocular barriers and clearance of intravitreal drugs [23], our opposite results are due to the interaction between adalimumab and the TNFa [38] present in the ocular cavity, which achieves increased values in uveitis as demonstrated in previous studies [29]. After 72 h, blood values are equalized until the end of the study. 211 Intravitreal pharmacokinetic studies in patients are performed from the aqueous humor due to the impossibility of obtaining vitreous samples [39]. This procedure has also been carried out in the preclinical setting, specifically on animals sacrificed at different time points in order to obtain vitreous samples. In this sense, PET imaging has the advantage of avoiding invasive sampling and obtaining concentrations from the same animal over time, with the consequent decrease in the number of animals per study group [23]. Additionally, PET methodology avoids the intrusiveness of the pharmacokinetic studies, which is very important taking into account the invasiveness of the intravitreal injection itself. PET imaging requires the use of radioactive derivatives which are stable over time. In this study, 89Zr was selected because it allows to visualize the distribution of antibodies for up to 10 days due to its long half-life. In order to obtain radiolabeled adalimumab, deferoxamine was previously bound to this drug to act as a chelator of 89Zr. As studied in previous works of our group [25], 89Zr-labelled deferoxamine without monoclonal antibody has a completely different kinetic behavior, the rate of elimination from both the vitreous cavity and blood is much faster (24h) and that vitreous elimination is independent of the deferoxamine chelator, only the molecule has an influence. The main limitation of our study regarding intravitreal pharmacokinetics is that all ocular cavities are measured together, including aqueous and vitreous humor, as well as surrounding tissues. However, this is partially counteracted by the fact that drug concentration declines with the same decay rate in the vitreous humor, aqueous humor and retina, as several authors state [40,41]. As well as other species, rats represent a good model for intravitreal pharmacokinetic studies [42]. However, comparison with humans should be cautiously made due to the anatomical and physiological ocular differences. With this regard, rats have a larger len and a simpler inner limiting membrane, which could be a poorer barrier to diffusion [43,44], and around 50 µL of vitreous humor [45], which is well below that volume in the human eye (4 mL). In addition, it is necessary to take into account that the stability of the metal complex formed between 89Zr and the bifunctional chelate used (TFP-N-sucDf) was studied by Perk et al. who reported a small loss of 89Zr from conjugate over a 7-day period (4.7 ± 0.5%) [46], however, our study lasted 10 days so this loss could be slightly higher. Regarding adalimumab pharmacokinetics outside the ocular cavity, interaction between the anti-TNFa that is removed from the eye and the major inflammatory agents is going to occur. The immune system is closely related to the lymphatic system, so an accumulation in the different lymph nodes close to the site of administration is expected, as can be confirmed by our results. In this sense, the highlighted area in the inferior part of the mandible (Figure 5-sagittal plane) corresponds to the cervical lymph nodes, as described by Suami et al. [47]. The elimination process of adalimumab in the body cannot be clearly appreciated by 212 observing the pharmacokinetic results of the main drug elimination organs. This is due to the fact that anti-TNFa has a large molecular weight (≃150 KDa), so filtration by the kidneys and elimination through urine is not possible, except under pathological conditions [48,49]. Thus, the main route of elimination occurs through intracellular catabolism by lysosomal degradation, transforming the monoclonal antibody into peptides and amino acids that can be re-used in the body for the de novo synthesis of proteins [50]. 5. 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Pharmacokinetics and Safety of Elotuzumab Combined With Lenalidomide and Dexamethasone in Patients With Multiple Myeloma and Various Levels of Renal Impairment: Results of a Phase Ib Study. Clin. Lymphoma Myeloma Leuk. 2016, 16, 129–138, doi:10.1016/j.clml.2015.12.007. 49. Ryman, J.T.; Meibohm, B. Pharmacokinetics of Monoclonal Antibodies. CPT Pharmacomet. Syst. Pharmacol. 2017, 6, 576–588, doi:10.1002/psp4.12224. 50. Waldmann, T.A.; Strober, W. Metabolism of Immunoglobulins. Prog. Allergy 1969, 13, 1–110, doi:10.1159/000385919. 217 CONCLUSIONS 218 225 CONCLUSIÓNS Nesta tese de doutoramento realizouse unha investigación das alternativas terapéuticas para o tratamento das enfermidades inflamatorias oculares, o que nos permitiu extraer as seguintes conclusións: 1º - Realizouse unha revisión detallada das diferentes vías oculares e dos principais aspectos da farmacocinética ocular. Os diferentes factores que interveñen na administración ocular de fármacos, incluídas as barreiras fisiolóxicas e as vías de transporte dos mesmos, así como as vantaxes e desvantaxes das vías de administración oftálmicas foron tamén descritas. As vías de administración convencionais, como a tópica ou a sistémica, normalmente presentan importantes limitacións, xa sexa pola baixa penetración ocular ou pola aparición de efectos secundarios ligados á posoloxía, entre outros. Necesítanse novos sistemas de administración de fármacos para prolongar os intervalos de administración en patoloxías oculares, aínda que o desenvolvemento destes novos sistemas é especialmente complicado debido a varios aspectos que deben considerarse, como a farmacocinética, a inmunoxenicidade, a biodegradación, a tolerabilidade e a toxicidade. Nas últimas décadas observouse un aumento exponencial no deseño e desenvolvemento de novos sistemas de administración de fármacos destinados ó tratamento de patoloxías oculares. Desafortunadamente, os coñecementos sobre a vehiculización dos fármacos cara ó ollo seguen sendo escasos. Así mesmo, producíronse grandes avances en canto á investigación e o desenvolvemento de novas vías de administración alternativas para as diferentes partes do ollo. Todas elas teñen características farmacocinéticas específicas que as fan útiles para o tratamento de patoloxías oculares concretas. Sen embargo, estas vías mostraron diversas vantaxes e limitacións, onde a elección dunha ou outra depende, non só da propia patoloxía, senón da forma farmacéutica, do fármaco empregado e da adherencia do paciente ó tratamento. 2º - Co fin de aportar información sobre as características do colirio clásico de tacrolimus preparado nos Servizos de Farmacia do Hospital, realizouse un estudo comparativo noutros dous vehículos, unha solución de alcohol polivinílico e unha solución de ácido hialurónico. Foi observada unha maior permanencia na superficie ocular e unha maior estabilidade a diferentes 226 condicións de temperatura do colirio de tacrolimus en alcohol polivinílico. Así mesmo, os resultados obtidos en termos de citotoxicidade demostraron que os colirios de tacrolimus son mellor tolerados que o de ciclosporina. Aínda que os resultados do estudo en pacientes indicaron unha boa tolerancia, notificáronse algúns casos de irritación leve trala administración. Isto débese probablemente á elevada osmolalidade das formulacións e á presenza dalgúns excipientes, como o etanol e aceite de ricino hidroxenado e polioxietilenado, aportados pola presentación intravenosa do fármaco ca que se preparan as formulacións. 3º - Foi levado a cabo o deseño e desenvolvemento de diferentes formulacións tópico-oftálmicas que conteñen tacrolimus e ciclodextrina como alternativa ós colirios clásicos de tacrolimus reformulados a partir do fármaco intravenoso nos departamentos de farmacia dos hospitais. A interacción tacrolimus/HPβCD en solución confirmouse mediante o estudo de solubilidade, a RMN e os estudos de modelado molecular, estudando tamén a influencia do vehículo. O uso do 40% (p/v) de HPβCD permitiu preparar solucións de colirio cunha concentración de tacrolimus do 0,02% (p/v) que podería estar no rango terapéutico para o tratamento da uveíte. As formulacións desenvolvidas a base de HPβCD mostraron valores de pH, osmolalidade, tensión superficial e seguridade no rango óptimo para a administración tópico-oftálmica, mellorando as propiedades dos colirios estudados no capítulo 2. Os estudos de estabilidade non mostraron cambios nos colirios conservados en condicións de refrixeración durante polo menos 3 meses, o que facilita a programación da preparación na farmacia hospitalaria e mellora a comodidade e adherencia do paciente ó tratamento. Ademais, os estudos de mucoadhesión ex vivo e de permanencia ocular in vivo mostraron boas propiedades mucoadhesivas e un menor aclaramento ocular para a mencionada formulación de tacrolimus/HPβCD en Liquifilm®, case duplicando o tempo de permanencia na superficie ocular en comparación co colirio de tacrolimus clásico. O sinxelo proceso de elaboración dos colirios de TAC-HPβCD permite preparar esta formulación nos departamentos de farmacia hospitalaria, mellorando en termos de seguridade os actuais colirios de tacrolimus. 4º - A eficacia da formulación de tacrolimus desenvolvida no capítulo 3 avaliouse nun modelo de uveíte inducida por endotoxina en ratas. O colirio de TACHPβCD mostrou un efecto beneficioso neste modelo, polo que podería considerarse unha alternativa para o tratamento da uveíte en caso de resistencia ou intolerancia ós corticosteroides. Esta formulación demostrou reducir a inflamación ocular, a expresión de IL-6, TNFα, MIP-1α e a infiltración de leucocitos no humor acuoso. 227 5º - Por primeira vez, realizouse a avaliación do perfil farmacocinético do adalimumab inxectado por vía intravítrea nun modelo de rata, demostrando que a uveíte produce unha redución do aclaramento ocular do adalimumab. Isto débese á unión específica entre o anticorpo e os receptores do TNFα que se encontran en maior cantidade debido ó proceso inflamatorio producido pola enfermidade. A información farmacocinética obtida neste traballo será de gran utilidade para desenvolver novos sistemas de administración de adalimumab que permitan unha liberación máis controlada e prolongada do anticorpo monoclonal. Como conclusión xeral, nesta tese de doutoramento foron propostas diferentes formulacións de tacrolimus para a administración tópico-oftálmica, suxeitas por amplos estudos preclínicos co fin de lograr unha base consistente como alternativa a outros tratamentos farmacolóxicos para as enfermidades inflamatorias oculares. Isto foi confirmado con amplos estudos in vitro, ex vivo e in vivo. Por outra parte, no que respecta ó estudo de inxeccións intravítreas de adalimumab, esta tese de doutoramento dilucidou a distribución e farmacocinética intravítrea do anticorpo monoclonal que será de gran utilidade para o desenvolvemento de novos sistemas de administración. 228 229 APPENDIX 230 231 APPENDIX CONFLICT OF INTEREST The doctoral student declares that there is no conflict of interest regarding the doctoral thesis. ANIMAL EXPERIMENTS In the present thesis, male adult Sprague-Dawley rats with a 200-300 g average weight were used, supplied by the animal facility of the University of Santiago de Compostela (Santiago de Compostela, Spain). During the experiments, the animals were kept in individual cages with free access to food and water in a room under controlled temperature (22 ± 1°C) and humidity (60 ± 5%) and with day–night cycles regulated by artificial light (12/12 hours). The animals were treated according to the guidelines for the care and use of laboratory animals. Animal experiments complied with the ARRIVE guidelines (Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol. 2010 Jun 29;8(6):e1000412.). Experiments were performed at the Preclinical Imaging Service of the Experimental Biomedicine Center (CEBEGA) (REGA number: ES150780292901). Experiments were approved by the Consellería de Medio Rural da Xunta de Galicia (15012/2021/001 and 15010/2019/006) and were authorized by the Animal Research Committee of the Health Research Institute of Santiago de Compostela (IDIS). They followed the Spanish and European Union (EU) regulations for animal experimentation (86/609/CEE, 2003/ 65/CE, 2010/63/EU, and RD53/2013). CLINICAL STUDY In the present thesis, the prospective observational clinical study was performed in 16 eyes of 8 patients with severe ocular disease. This was performed at the outpatient clinic of the Department of Ophthalmology (University Clinical Hospital of Santiago de Compostela). The study is adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board/Ethics Committee of the Ethical Committee of Clinical Research of Galicia and the Consellería de Sanidade da Xunta de Galicia (2019/204). The committee complies with current legal requirements applicable to Research Ethics Committee, following the Standards of Good Clinical Practice (CPMP/ICH/135/95). 232 FUNDING AND ACKNOWLEDGEMENTS This thesis has been partially funded by Instituto de Salud Carlos III (ISCIII) through the projects PI20/00719, PI17/00940, RETICS Oftared, RD16/0008/0003 and RD12/0034/0017 and co-funded by the European Union, Axencia Galega Innovación (Grupos de Potencial Crecimiento IN607B2020/11, Grupo de Referencia Competitiva ED431C2021/01 and ED431C2021/26 and Proyectos de Excelencia IN607D 2021/001) and Spanish Ministry of Science, Innovation and Universities (RTI2018-099597-B-100). The doctoral student acknowledges the financial support of the IDIS (Health Research Institute of Santiago de Compostela). ARTICLES DERIVED FROM THIS THESIS In compliance with the rules of doctoral studies at Universidade de Santiago de Compostela in Regulamento dos estudos de doutoramento na USC, DOG de 16 de Setembro de 2020, it was provided some information regarding the articles on which this work is based and the journals that published those articles. In particular it was given the names of the authors, the title of the journals, the year each article was published, the publisher, the DOI-type link, the Journal Impact Factor and the quartile from the Journal Citation Reports, some relevant information regarding copyright and use of the articles and the author contribution. Þ Chapter 1 TITLE: Drug Delivery to the Posterior Segment of the Eye: Biopharmaceutic and Pharmacokinetic Considerations AUTHORS: Varela-Fernández, R.; Díaz-Tomé, V.; Luaces-Rodríguez, A.; CondePenedo, A.; García-Otero, X.; Luzardo-Álvarez, A.; Fernández-Ferreiro, A.; OteroEspinar, F.J. JOURNAL: Pharmaceutics YEAR: 2020 PUBLISHER: MDPI LINK: https://doi.org/10.3390/pharmaceutics12030269 JOURNAL IMPACT FACTOR: 6.525 (Q1) INFORMATION REGARDING COPYRIGHT AND USE: Information about permissions can be found on the website: https://www.mdpi.com/authors/rights where the following statement is placed: “For all articles published in MDPI journals, copyright is retained by the authors. Articles are licensed under an open access Creative Commons CC BY 4.0 license, 233 meaning that anyone may download and read the paper for free. The article may be reused and quoted provided that the original published version is cited.” AUTHOR CONTRIBUTION: The contributions of the Ph.D. candidate were essential in all the included articles. The candidate contributed to the information research and analysis, and the writing of the manuscripts. Þ Chapter 2 TITLE: Preclinical characterization and clinical evaluation of tacrolimus eye drops AUTHORS: Luaces-Rodríguez A.; Touriño-Peralba R.; Alonso-Rodríguez I.; GarcíaOtero X.; González-Barcia M.; Rodríguez-Ares MT.; Martínez-Pérez L; Aguiar P.; Gómez-Lado N.; Silva-Rodríguez J.; Herranz M.; Ruibal-Morell Á.; Lamas MJ.; OteroEspinar FJ.; Fernández-Ferreiro A. JOURNAL: European Journal of Pharmaceutical Science. YEAR: 2018 PUBLISHER: Elsevier LINK: https://doi.org/10.1016/j.ejps.2018.04.038 JOURNAL IMPACT FACTOR: 5.112 (Q2) INFORMATION REGARDING COPYRIGHT AND USE: Please note that, as the author of this Elsevier article, you retain the right to include it in a thesis or dissertation, provided it is not published commercially. Permission is not required, but please ensure that you reference the journal as the original source. AUTHOR CONTRIBUTION: The contributions of the Ph.D. candidate were essential in all the included articles. The candidate contributed to the design of the research and proofs, to the analysis of the results and to the writing of the manuscripts. Þ Chapter 3 TITLE: Development and Characterization of a Tacrolimus/Hydroxypropyl-βCyclodextrin Eye Drop AUTHORS: García-Otero, X.; Díaz-Tomé, V.; Varela-Fernández, R.; Martín-Pastor, M.; González-Barcia, M.; Blanco-Méndez, J.; Mondelo-García, C.; Bermudez, M.A.; Gonzalez, F.; Aguiar, P.; Fernández-Ferreiro, A.; Otero-Espinar, F.J. JOURNAL: Pharmaceutics YEAR: 2021 PUBLISHER: MDPI LINK: https://doi.org/10.3390/pharmaceutics13020149 JOURNAL IMPACT FACTOR: 6.525 (Q1) INFORMATION REGARDING COPYRIGHT AND USE: Information about permissions can be found on the website: https://www.mdpi.com/authors/rights where the following statement is placed: 234 “For all articles published in MDPI journals, copyright is retained by the authors. Articles are licensed under an open access Creative Commons CC BY 4.0 license, meaning that anyone may download and read the paper for free. The article may be reused and quoted provided that the original published version is cited.” AUTHOR CONTRIBUTION: The contributions of the Ph.D. candidate were essential in all the included articles. The candidate contributed to the design of the research and proofs, to the analysis of the results and to the writing of the manuscripts. Þ Chapter 4 TITLE: Anti-Inflammatory Effect of Tacrolimus/Hydroxypropyl-β-Cyclodextrin Eye Drops in an Endotoxin-Induced Uveitis Model AUTHORS: García-Otero, X.; Mondelo-García, C.; González, F.; Perez-Fernandez, R.; Avila, L.; Antúnez-López, J.R.; González-Barcia, M.; Adan, A.; Aguiar, P.; OteroEspinar, F.J.; Bermúdez, M.A.; Fernández-Ferreiro, A. JOURNAL: Pharmaceutics YEAR: 2021 PUBLISHER: MDPI LINK: https://doi.org/10.3390/pharmaceutics13101737 JOURNAL IMPACT FACTOR: 6.525 (Q1) INFORMATION REGARDING COPYRIGHT AND USE: Information about permissions can be found on the website: https://www.mdpi.com/authors/rights where the following statement is placed: “For all articles published in MDPI journals, copyright is retained by the authors. Articles are licensed under an open access Creative Commons CC BY 4.0 license, meaning that anyone may download and read the paper for free. The article may be reused and quoted provided that the original published version is cited.” AUTHOR CONTRIBUTION: The contributions of the Ph.D. candidate were essential in all the included articles. The candidate contributed to the design of the research and proofs, to the analysis of the results and to the writing of the manuscripts. Þ Chapter 5 TITLE: PET Study of Intravitreal Adalimumab Pharmacokinetics in a Uveitis Rat Model AUTHORS: García-Otero, X.; Mondelo-García, C.; Bandín-Vilar, E.; Gómez-Lado, N.; Silva-Rodríguez, J.; Rey-Bretal, D.; Victoria OteroEspinar, M.; Adan, A.; González-Barcia, M.; Aguiar, P.; Otero-Espinar, F.J.; Fernández-Ferreiro, A. JOURNAL: International Journal of Pharmaceutics YEAR: 2022