Matrix Metalloproteinases and Glaucoma Treatment.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade extracellular matrix (ECM) components such as collagen and have important roles in multiple biological processes, including development and tissue remodeling, both in health and disease. The activity of MMPs is influenced by the expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). In the eye, MMP-mediated ECM turnover in the juxtacanalicular region of the trabecular meshwork (TM) reduces outflow resistance in the conventional outflow pathway and helps maintain intraocular pressure (IOP) homeostasis. An imbalance in the MMP/TIMP ratio may be involved in the elevated IOP often associated with glaucoma. The prostaglandin analog/prostamide (PGA) class of topical ocular hypotensive medications used in glaucoma treatment reduces IOP by increasing outflow through both conventional and unconventional (uveoscleral) outflow pathways. Evidence from in vivo and in vitro studies using animal models and anterior segment explant and cell cultures indicates that the mechanism of IOP lowering by PGAs involves increased MMP expression in the TM and ciliary body, leading to tissue remodeling that enhances conventional and unconventional outflow. PGA effects on MMP expression are dependent on the identity and concentration of the PGA. An intracameral sustained-release PGA implant (Bimatoprost SR) in development for glaucoma treatment can reduce IOP for many months after expected intraocular drug bioavailability. We hypothesize that the higher concentrations of bimatoprost achieved in ocular outflow tissues with the implant produce greater MMP upregulation and more extensive, sustained MMP-mediated target tissue remodeling, providing an extended duration of effect

Structural and functional investigation of the trabecular outflow pathway

Primary open-angle glaucoma (POAG) is a leading cause of blindness in the world. A primary risk factor for POAG is elevated intraocular pressure (IOP), caused by increased aqueous humor outflow resistance. Currently, lowering the IOP is the only effective way of treating glaucoma; however, the cause of increased outflow resistance remains unclear. This thesis will present a series of studies which investigated structures of the trabecular outflow pathway, including Schlemm’s canal endothelium, juxtacanalicular tissue, and trabecular beams, and their roles in regulating aqueous outflow resistance. The studies were conducted in both human and animal models using ex vivo ocular perfusion as well as in vitro microfluidic systems. In the first study, we investigated the effects of Y27632, a derivative of Rho-kinase inhibitor that is being developed as next generation glaucoma drug with unclear IOP lowering mechanism, on aqueous humor outflow dynamics and associated morphological changes in normal human eyes and laser-induced ocular hypertensive monkey eyes. In the second study, we developed and validated a novel three-dimensional microfluidic system using lymphatic microvascular endothelial cells. The microfluidic system can be used to study Schlemm’s canal endothelial cell dynamics and aqueous humor transport mechanism in the future. In the last study, we characterized the morphological structure, distribution, and thickness of the endothelial glycocalyx in the aqueous humor outflow pathway of human and bovine eyes. Together these studies will help define new directions for therapy that will help control IOP and preserve vision throughout a normal life span

Numerical solution of flow resistance in outflow pathway and intravitreal drug delivery in vitrectomised eyes

In this study, numerical computations of the ocular fluid dynamics in a human eye are presented with a perspective of understanding the mechanisms of increased flow resistance. In the present study, the TM is represented as a multilayered-graded porous structure with specific pore size and void fraction. The flow patterns and pressure distribution in anterior chamber are analyzed to delineate key flow mechanism; the shear stresses on the lens, iris and IW of SC are also examined to locate the maximum values. Inside the human eye, the largest pressure drop occurs across JCT and IW of SC. The highest pressure in SC is at the midpoint between two collector channels (CC). The pressure falls near CC which implies that the IW of SC will experience more pressure difference towards CC, and the canal may show a greater tendency to collapse close to the CC exits. The maximum velocity is found in the vicinity of IW pores. It is also seen that AH velocity funneling out of the IW pores is higher in the region underlying the collector. Analysis is also carried out for glaucomatous condition where the IOP is increased to a high value of 8000 Pa. The later part of thesis is dedicated to the drug delivery to the posterior segment of the eye. The main objective of this study is to characterize the spatio-temporal evolution of drug distribution following intravitreal injection into a vitreous substitute such as silicone oil and in the case of vitreous liquefaction caused due to aging. Both direct injection of drugs and injection of time released drugs are studied. The results show that the concentration distribution is highly dependent on the vitreous substitute, diffusion coefficient of the drug and the permeability of the retinal surface. For drugs with high diffusion coefficients, convection plays a small role whereas for the drugs with low diffusion coefficients and low viscosity vitreous fluids, convection is seen to play a more important role and can lead to high drug concentrations on the retina which can be potentially toxic. Time-released drug injection is shown to avoid conditions of retinal toxicity

Biophysical properties of aqueous humour in ocular pathologies

El limbo corneoescleral contiene estructuras diseñadas para evacuar el humor acuso desde la cámara anterior del ojo hacia la circulación general. Estos elementos se encuentran entre el espolón escleral y la línea de Schwalbe que finalmente se continua con a membrana de Descemet del endotelio corneal. Su composición consta de tejido corneal, escleral, iridiano y del cuerpo ciliar; constituyendo elementos estructurales claves en el drenaje del humor acuoso como son: el canal de Schlemm, los canales colectores y la malla trabecular, y en ella sus tres regiones: uveal, corneoescleral y yuxtacanalicular. El canal Schlemm es un canal venoso circular de 36 µm de circunferencia y 350-500 µm de diámetro en su luz interna. La superficie de la luz está tapizada por células endoteliales que están unidas entre sí por uniones zonula occludens. En la pared interna del canal, a lo largo de su trayecto sinuoso, encontramos vesículas citoplasmáticas o vacuolas de grandes dimensiones de origen pinocítico que pueden llegar a medir 14 µm con aperturas puntuales hacia la luz del canal de 0.3 ¿ 2.0 µm postulandose que pueda ser un drenaje directo desde los espacios trabeculares hacia el canal de Schlemm. Existe controversia con respecto al hallazgo de poros inter y transcelulares en la pared interna del canal de Shlemm; puesto que algunos autores que analizan mediante microscopia electrónica de barrido la monocapa endotelial sostienen que su número aumenta con la elevación de la presión; otros, concluyen que su número se reduce en los ojos glaucomatosos. Donde sí parecen estar de acuerdo las últimas publicaciones es en destacar el dinamismo de este sistema de drenaje, y postulan que los cambios encontrados en su ultraestructura pudieran hablarnos a favor de un sistema sensible y cambiante con los cambios de presión intraocular. En física se denomina tensión superficial de un líquido a la cantidad de energía necesaria para aumentar su superficie por unidad de área. Las fuerzas intermoleculares en los líquidos, junto con las fuerzas existentes entre distintas superficies dan lugar a esta resistencia y condicionan la capacidad de capilaridad del líquido al contactar con una superficie sólida. Las fuerzas que afectan a cada molécula son diferentes en el interior del líquido y en la superficie. Así en el seno de un líquido cada molécula esta sometida a fuerzas de atracción que en promedio se anulan, permitiendo tener una energía bastante baja pero en la superficie existe una fuerza neta hacia el interior. Los agentes surfactantes al colocarse en la interfase entre dos sistemas actúan disminuyendo la tensión superficial y facilitando así la continuidad entre los mismos. Las moléculas más conocidas en este campo son los fosfolípidos y actúan como agentes anfipáticos al constar de una parte hidrófila y otra hidrófoba. Su acción es clave en la superficie alveolar evitando la atelectasia alveolar en la fase final de la espiración, y por el contrario su deficiencia precipita en Síndrome de Distress respiratorio del lactante. La malla trabecular y el tejido conectivo yuxtacanalicular contiene matriz extracelular y es conocido que la composición de glucosaminoglicanos y ácido hialurónico en esta estructura está alterada en el glaucoma. La disminución del contenido en ácido hialurónico tanto en la malla trabecular como en tejido yuxtacanalicular puede dificultar la capacidad de drenaje del humor acuso al elevarse su tensión superficial, incrementándose así las uniones intercelulares en la capa endotelial y finalmente colapsar los espacios trabeculares existentes en condiciones normales. Esto conlleva irremediablemente a la elevación de la presión intraocular; pero paradójicamente, puede elevarse también de forma mecánica tal y como se observa típicamente en la cirugía de la catarata tras la introducción de sustancias viscoelásticas compuestas de ácido hialurónico en cámara anterior que bloquean los espacios trabeculares. El desequilibrio en la proporción de glucosaminoglicanos mediante la inyección exógena sustancias viscoelásticas o de condritin sulfato en cámara anterior puede elevar significativamente la presión intraocular y permite desarrollar así un modelo de glaucoma experimental. El análisis proteómico de humor acuoso de pacientes con glaucoma mediante electroforésis ha demostrado diferencias cualitativas en su composición respecto a controles jugando un papel relevante en el glaucoma pseudoexfoliativo. En el único estudio hasta la fecha que analizan la tensión superficial del humor acuso se ha observado una disminución de la misma en los ojos afectos de glaucoma primario de ángulo abierto respecto a los ojos seleccionados como controles, siendo estos últimos os pacientes sometidos a cirugía de catarata. En este estudio la tensión superficial se analizó mediante la observación del tamaño y ángulo de contacto de una gota en suspensión siguiendo la técnica de Bashford-Adams. Desafortunadamente, al seleccionar un grupo de pacientes afectos de glaucoma excesivamente heterogéneo (tipos distintos de glaucoma y sometidos previamente a diversos fármacos antiglaucomatosos) y errores en la metodología en el diseño del estudio (recogida de material, condiciones biofísicas durante la conservación y posterior análisis) los resultados son contradictorios y no concluyentes. De todos modos, se postula que el humor acuoso de los ojos afectos de glaucoma al tener una menor tensión superficial, esto pueda interferir negativamente en la formación de vacuolas en su vía de drenaje hacia el canal de Schlemm. Estos mismos autores animan a proseguir futuros trabajos en esta misma línea de investigación y enfocarse a su vez en el análisis y la composición lipídica del humor acuoso en el glaucoma, al ser otro de los puntos clave para la formación de vesículas pinocítocas y su posterior drenaje

Preformulation Characterization And Formulation Development of Δ9-Tetrahydrocannabinol Prodrugs For Potential Treatment Of Glacuoma

Relationship between smoking marihuana and a drop in intraocular pressure (IOP) was first reported in 1971. Research efforts since then have identified a number of constituents that could be linked to this observation. Delta-9-Tetrahydrocannabinol (THC) was one of the ingredients identified. However, four decades and numerous research efforts since the first observation, it has still not been concluded whether THC is effective in the treatment of glaucoma or not. Current therapy for IOP control in glaucoma, though effective, cannot prevent vision loss completely. Thus, identification of agents that can lower IOP as well as protect the retinal ganglion cells from apoptosis could yield a new class of anti-glaucoma molecules. Protection of retinal ganglion cells against apoptosis could yield a new class of antiglaucoma agents. Numerous investigations have demonstrated that THC also acts as a neuroprotective agent making it a very promising lead compound. However noninvasive delivery of THC to the targeted intraocular tissues is challenging due to its poor physiochemical properties. The aim of the current project was to effectively deliver THC to the intraocular tissues. THC prodrugs were synthesized to improve physicochemical properties. Preformulation characterization and formulation development for the THC prodrugs was undertaken. A high throughput in vitro model to screen the various formulations for transcorneal permeability was also developed. Promising formulations were instilled in vivo in anesthetized rabbit model and tissue concentrations were determined. Earlier formulations reported in the literature, used for determining efficacy were not observed to deliver THC to the targeted intraocular tissues. The formulations developed in this study were able to deliver significantly higher concentrations to the intraocular tissues. Future studies need to investigate if these improved formulations of THC demonstrate pharmacological activity against glaucoma

Penetration enhancers in ocular drug delivery

There are more than 100 recognized disorders of the eye. This makes the development of advanced ocular formulations an important topic in pharmaceutical science. One of the ways to improve drug delivery to the eye is the use of penetration enhancers. These are defined as compounds capable of enhancing drug permeability across ocular membranes. This review paper provides an overview of anatomical and physiological features of the eye and discusses some common ophthalmological conditions and permeability of ocular membranes. The review also presents the analysis of literature on the use of penetration-enhancing compounds (cyclodextrins, chelating agents, crown ethers, bile acids and bile salts, cell-penetrating peptides, and other amphiphilic compounds) in ocular drug delivery, describing their properties and modes of action

Efficacy of 180 versus 360 Degrees of Selective Laser Trabeculoplasty on Lowering Intraocular Pressure

The purpose of this study was to determine if the application of 360° of selective laser trabeculoplasty (SLT) to the trabecular meshwork (TM) is more effective than 180° in the lowering of intraocular pressure (IOP) in glaucoma. This issue was addressed in the form of a retrospective chart review of patients treated consecutively with SLT for primary open angle glaucoma, pseudoexfoliation glaucoma or ocular hypertension. Patients were treated with either 180° of SLT by Dr. Wand or 360° by Dr. Martone using a 532nm, Q-switched, frequency doubled Nd:Yag laser. Only the initial treatment with SLT of a given eye was analyzed. There were 108 patients per group. Patient age, type of glaucoma, history of previous argon laser trabeculoplasty (ALT), number of medications, lens status and visual acuity pre-and post-treatment and the number and power of laser spots were compared between the treatment groups. The mean IOP from three consecutive visits prior to treatment was compared with post-operative IOPs measured at 1 hour, 6 weeks, and 3 months. Patients were classified as responders if the three month postoperative IOP was reduced by more than or equal to 3 mmHg compared to baseline. At three months post-op the 360° group had a response rate of 60% and a mean IOP drop of 3.6 mmHg (17.8%). The 180° group had a response rate of 29% with a mean IOP drop of 1.5 mmHg (7.5%). The number of pre- and post-treatment medications, eye treated, gender, phakic status, number of spots delivered and pre-op IOP were equivalent for the two study groups. The 180° group subjects had more overall energy delivered during treatment and had had previous ALT more frequently than the 360° group. The 180° group also was significantly older and was less likely to have non-open angle glaucoma. This study suggests that with a 2.1 mmHg greater drop in IOP and 31% greater response rate at three months post-op 360 degrees of SLT is more effective in lowering intraocular pressure than 180 degrees

Study on erythropoietin subconjunctival administration in a glaucoma animal model

Tese de Doutoramento em Ciências Veterinárias na Especialidade de ClinicaGlaucoma is the number one cause of irreversible vision loss worldwide. Death of retinal ganglion cells (RGC), which results in the progressive loss of visual function, occurs in glaucoma and other ocular diseases caused by hypoxia and ischemia. Although glaucoma is a multifactorial neurodegenerative disease, the only currently method of treatment involves reduction of intraocular pressure and, at the present, there is no effective treatment to prevent RGC apoptosis. Notably, it has been reported that erythropoietin (EPO), a cytokine hormone produced in response to hypoxia, has significant neuroprotective and neuroregenerative properties in several types of ocular disorders. Pre-clinical studies in glaucoma animal models involving EPO have yielded very promising results. All studies involving EPO ocular administrations have used systemic, intravitreal or retrobulbar administration to reach retinal desired EPO concentrations. However, EPO chronic systemic administration can lead to adverse side effects related with haematopoiesis stimulation, while intravitreal or retrobulbar administrations are invasive procedures that can induce several complications such as endophthalmitis, retinal detachment, vitreitis, retinitis, choroiditis or cataracts. This thesis aims to clarify if EPO’s neuroprotection could be achieved by a non-invasive and safe periocular administration route without adverse effects. Being so, this work evaluates the subconjunctival route as an alternative for EPO administration in glaucoma disease. After the first in vitro study, where the permeation of EPO across the periocular tissues was quantified, all work was developed in in vivo models. EPO’s ocular permeation after subconjunctival administration was tested, both in physiological and glaucomatous conditions. Furthermore, both retinal morphological and physiological effects of EPO administered by this route were assessed in glaucomatous animals. Results showed that EPO, when administered subconjunctivally, can permeate the main ocular barriers and reach RGC layers, in both physiological and glaucomatous conditions, without significant local or systemic side effects. More than showing that EPO can reach the retina by this route, results also concluded that subconjunctival EPO administration seems to have structural and functional beneficial effects on the retina after glaucoma induction in rats.RESUMO - Estudo da administração subconjuntival de eritropoietina num modelo animal de glaucoma. - O glaucoma é a principal causa de cegueira irreversível no mundo. A morte das células ganglionares da retina (RGC) causada por hipóxia e isquémia resulta numa progressiva perda de visão. Apesar do glaucoma ser uma doença neurodegenerativa multifatorial, as únicas opções terapêuticas visam o controle da pressão intraocular, não havendo atualmente um tratamento eficaz para prevenir a apoptose das RGC. Estudos recentes demonstraram que a eritropoietina (EPO), uma glicoproteína sintetizada maioritariamente em resposta a estados de hipóxia, tem ação neuroprotetora e neuroregenerativa em várias doenças oculares, tendo revelado resultados promissores em vários modelos animais de glaucoma. Nos estudos experimentais em que a EPO foi utilizada como substância neuroprotetora, foi administrada pelas vias sistémica, intravítrea ou retrobulbar para se obterem concentrações terapêuticas na retina. No entanto, a administração sistémica prolongada de EPO pode produzir efeitos secundários adversos relacionados com o aumento da hematopoiese, enquanto que as administrações intravítrea ou retrobulbar são procedimentos invasivos suscetíveis de causar várias complicações tais como endoftalmite, descolamento de retina, vitreite, retinite, coroidite ou catarata. Esta tese teve por objetivo estudar uma via de administração periocular de EPO não invasiva, segura, eficaz e sem efeitos adversos. Assim, este trabalho avaliou a via subconjuntival como uma alternativa para a administração ocular de EPO em condições de glaucoma. No primeiro estudo in vitro, quantificou-se a permeação da EPO nos tecidos perioculares. O restante trabalho, desenvolvido em modelos in vivo, testou a permeação ocular da EPO após administração subconjuntival, tanto em condições fisiológicas como de glaucoma. O estudo contemplou ainda os efeitos morfológicos e fisiológicos da EPO ao nível da retina em animais glaucomatosos. Os resultados obtidos demostraram que a EPO, quando administrada pela via subconjuntival, pode permear as principais barreiras oculares e atingir as RGC, em ambas as condições testadas, fisiológicas e de glaucoma, sem efeitos adversos locais ou sistémicos apreciáveis. Além disso, revelaram que a administração subconjuntival de EPO parece ter efeitos benéficos estruturais e funcionais na retina após a indução de glaucoma experimental em ratos.N/