As the eye is a highly mobile sensory organ, small defects in musculature can lead to large functional problems. Finally, the immune response within the eye is different from other areas of the body. The eye is an immune-privileged organ rendering it more susceptible to infection and altering its response to immune modifying drugs.

This chapter is focused on druggable targets for diseases of the back of the eye. The term “druggable” has widened considerably with the advent of RNA interference. This new technology allows us, in theory, to target virtually any mRNA for degradation, thereby decreasing the resultant protein. For this reason, we consider virtually any protein fair game as a potential therapeutic target. Later we describe a broad classification of ocular pathologies along with possible target proteins, followed by more detailed descriptions of various specific pathologies and therapeutic agents.

21.2  Ocular Physiology and Pathology

Anatomically, the eye is the most accessible portion of the nervous system, providing us with a variety of routes of administration targeting different tissues and regions. The eye is an out pouching of the CNS and as such has a number of structures which are homologous to CNS structures (Forrester et al. 2008). These include the outer protective cornea and sclera envelope which corresponds to the meningeal protective coverings, the uveal tract (middle vascular layer), which provides oxygen and nutrition, and the neural retina. The uveal tract, which supplies support to the retina, is divided into four layers including the iris, ciliary body, pars planum, and choroid. Of these, the first three are considered anterior structures and the choroid, a posterior structure. The vasculature which nourishes the retina may be found in the choroid layer. As the retina is the most metabolically active of tissues, it is no surprise that the blood flow of the choroid is the greatest per square millimeter of any tissue in the body. Retinal pigment epithelium (RPE) lies between the choroid and the photoreceptors of the neural retina. These cells are responsible for supplying the photoreceptors with oxygen, nutrients, and specialized survival factors and for phagocytosing rod outer segments, which are constantly being shed.

The neural retina consists of photoreceptors which synapse onto amacrine and bipolar neurons. These, in turn, synapse onto ganglion cells which project their axons to the CNS via the optic nerve. Nestled within the neural retina lay the Müller glia, the predominant form of glial cell within this tissue. These cells are associated with many forms of retinal disease thus making their function of great interest. Müller cells interact with most, if not all, neurons within the retina. They span the entire length of the retina and recent data have demonstrated that they can function as optical fibers, bringing light to the buried photoreceptor cells with minimal distortion (Franze et al. 2007). These cells also function as support cells for retinal neurons, similar to the roles of oligodendrocytes and astrocytes in the CNS. One of these functions is the secretion of neurotrophic factors such as nerve growth factor (NGF), brain derived neurotrophic factor (BNDF), ciliary neurotrophic factor

(CNTF), fibroblast growth factor (FGF), neurotrophin-3 (NT-3), and a host of others (de Melo Reis et al. 2008). These factors control differentiation and synaptogenesis in the early retina and protect retinal neurons from toxic insult in the adult retina. Glutamic acid is the most important excitatory neurotransmitter in the retina yet an overabundance of glutamate leads to toxicity and retinal degeneration. Müller cells take up excess extracellular glutamate via several transporters, the most important of which is the high affinity Na+-coupled glutamate/aspartate transporter (GLAST). In addition to these functions, Müller cells can also undergo proliferation and differentiation to replace damaged retinal neurons, at least in avian retinas and potentially in mammals (Reh and Fischer 2006).

All output from the retina travels through the optic nerve to the CNS. The optic nerve is unique in that it is the only CNS tract that may be found outside the cranium and is the only central tract that is directly visible. The beginning of the optic nerve is called the optic disc: the point where all ganglion cell axons converge. Approximately, 90% of these axons are derived from the relatively tiny macula/ fovea which covers approximately 15° of the visual field. Information from the remainder of our 200° of visual field is carried by approximately 10% of remaining axons. These axons pass from the optic disc through the retina (pars retinalis), the choroid (pars chorodalis), and the sclera (pars scleralis). Within the sclera lie a network of collagen fibers called the lamina cribrosa, through which the axons must pass. Myelination of the axons commences at this point and these axons remain myelinated throughout their projections within the CNS.

Although there are a large number of diseases that affect the structures comprising the back of the eye, many of them share the fundamental pathological processes that result in damage. It would not be a stretch to claim that all pathologies employ, in differing ratios, the processes of inflammation, neovascularization, or degeneration. This concept is shown in Fig. 21.1. These processes are not independent. Inflammation leads to the accumulation of materials such as complement, which can inhibit nutrient flow. In turn, loss of nutrients may lead to neovascularization and inhibition of survival factor function, leading ultimately to degeneration. Likewise, neuronal degeneration and neovascularization can lead to the activation of danger signals that activate an immune/inflammatory response. As we consider current therapeutic targets, later, we will find it convenient to arrange them by their actions on these processes.

21.2.1  Ocular Inflammation

Physiologically, inflammation is the response of the body to injury or infection. Inflamed tissue swells to allow greater access for cells of the immune system. Phagocytic cells of the innate immune system such as macrophages and neutrophils home to the site of damage and begin engulfing pathogens and dead cells. They migrate to nearby lymph nodes and present antigens derived from this tissue to T cells, thus initiating the immune response. Upon recognition, T cells activate,

Autoimmune conditions such as autoimmune uveitis, rheumatoid arthritis, Behçet’s disease,Vogt-Koyanagi-Harada (VKH) disease, and systemic lupus erythematosus result in a wide variety of ocular conditions with the commonality of the activation of immune cells that infiltrate the eye and secrete substances which cause damage such as antibodies, complement, and reactive compounds evolved to kill pathogens. Importantly, the immune/inflammatory response evoked by the pathogen can be as destructive as the pathogen itself. Common infectious agents responsible for posterior uveitis include toxoplasmosis, tuberculosis, herpetic disease, cytomegalovirus (CMV), Lyme disease, endogenous endophthalmitis, and syphilis. Acute retinal necrosis (ARN) is a common cause of uveitis and is caused by varicella zoster and herpes simplex types 1 and 2. Bartonella henselae (cat scratch disease) most commonly affects children and adolescents. Patients who have experienced ocular trauma (including intravitreal injections) may show an increased risk for infections but far more frequently, the infections are systemic; caused by immunosuppression. Thus, with the rise of HIV-mediated immunosuppression, a commensurate rise in opportunistic ocular infections has been observed.

Uveitis, mediated through a noninfectious etiology, is thought to arise through immune or autoimmune processes. While it was previously believed that the eye, as an immune-privileged tissue, lacked communication with the lymphatics, it is now appreciated through tracking studies that antigens found in the eye can be transported both to the spleen and to various lymph nodes (Forrester et al. 2010). Early studies of the retina suggested that it was devoid of immune cells. However, the increase in our understanding of the roles of various myeloid cell types in the immune response along with an increase in our identification of histological markers has led us to realize that the choroid is richly endowed with CD11b+CD11c+ dendritic cells (DC) and macrophages that appear to be closely associated with both medium-sized blood vessels as well as cells of the RPE (Forrester et al. 1994). It is likely that these immune cells of the choroid, in constant contact with phagocytosed photoreceptor antigens, contribute to the autoimmune aspects of ocular inflammatory diseases. One of the first autoimmune conditions to be reported: sympathetic ophthalmia provides a model for how autoimmunity may develop. Injury to one eye leads to the release of sequestered autoantigens which are taken up by ocular antigen presenting cells (APC). These resident APC then migrate to the neighboring lymph node where the antigen is presented to T cells in a manner that breaks tolerance. A search for the antigens that activate these autoreactive T cells resulted in the discovery of several photoreceptor autoantigens (Wacker et al. 1977). These molecules now serve as initiators of experimental autoimmune uveitis (EAU) (Caspi et al. 2008). The disease process, which serves as a model for human inflammatory disease involves the priming and proliferation of an activated T cell population within 72 h of interacting with the autoantigen in the secondary lymph node rather than in the retina proper. They then home to the retina where they secrete proinflammatory cytokines such as TNFa, IFNg, and IL-17 which act to recruit and activate both pathogenic macrophages and regulatory cells.

New populations of cells with myeloid lineage are still being identified, creating the possibility of new therapeutic targets. For example, recently a small population

of MHC class II positive cells has been identified in the periphery of the retina as well as the surrounding optic nerve in EAU (Xu et al. 2007). These cells are located at sites where activated antigen-specific retinal T cells accumulate prior to disease onset and represent the major population of myeloid APC in these regions. Interestingly, the presence of these cells correlates inversely with the susceptibility of the mouse strain to EAU, suggesting that they provide a protective role.

Age-related macular degeneration (AMD), a major cause of blindness in the western world, also has an inflammatory component. The etiology of AMD is complex and likely involves the interplay of genetics and environment. The process of inflammation is implicated in this disease through the presence of drusen, which are insoluble deposits in the region of Bruch’s membrane. Although there remains some controversy as to whether drusen is causative or merely a consequence of aging, the appearance of drusen (particularly in the macula) is clearly correlated with disease progression (Klein et al. 1993; Holz et al. 1994; Sarraf et al. 1999). Analysis of drusen components has uncovered a strong similarity with other pathological deposits including plaques associated with atherosclerosis, Alzheimers disease, amyloidosis, and glomerular basement disease (Mullins et al. 2000). Should drusen play a causative role, then the components of drusen become of great importance, both as potential measures of disease and as potential therapeutic targets.

Some of the major components of drusen are proteins that take part in the complement cascade. The complement system is made up of a great many different proteins which function to promote lysis or phagocytosis of pathogens, extravasation and degranulation during inflammation, and finally, clearance of immune complexes. In the classical pathway, antibodies bind to the surface of the pathogen. Complement component C1 binds to at least two antibodies. A complex then begins to form through the sequential binding and proteolytic activation of C4 and C2 followed by C3, and finally C5. The C5 fragment, C5b, then goes on to nucleate the assembly of the membrane attack complex consisting of C5b, C6, C7, C8, and C9. The formation of the membrane attack complex results in the lysis of the pathogen. Fragments of complement components during this activation process, such as C3a, C4a, and C5a, among others, serve to coat bacteria to enhance macrophage phagocytosis and bind to cellular receptors to further activate the inflammatory response.

The first observation of complement in drusen was made by Mullins et al. (2000). They found antigenic evidence for both C5 and other components of the membrane attack complex. Complement is not associated with healthy RPE, but rather, with swollen RPE (Anderson et al. 2002). These data have led the authors to propose that RPE are targets of pathogenic complement attack and that this process is central to the formation of drusen and ultimately, AMD disease progression. In addition, these complement components have also been found in eyes affected by diabetic retinopathy (Gerl et al. 2002). Here they are located in the choriocapillaris immediately underlying the Bruch membrane and densely surrounding the capillaries.

In addition to complement components, acute phase proteins have been found in drusen (Hageman et al. 1999; Mullins et al. 2000). C reactive protein, in particular, is of interest in that it can serve as an opsonin, leading to increased phagocytosis and an increased inflammatory response. Additionally, CRP can directly activate complement.

Acute phase proteins are primarily made in the liver. However, there is some evidence for the presence of mRNA encoding acute phase proteins inthe eye (Anderson et al. 1999; Mullins et al. 2000). Vitronectin, another acute phase protein found in drusen, has been shown to be upregulated in RPE cells upon complement stimulation (Wasmuth et al. 2009).

Aggregates of vacuolar material have also been identified in drusen. These ­fluorescent aggregates are thought to be caused by the buildup of abnormal proteins that cannot be properly cleaved and processed by vacuolar digestive enzymes. Oxidative damage is the most likely cause. These bodies are collectively referred to as lipofuscin.

Other components of drusen include ApoE, a cholesterol transport protein which is also found in atherosclerotic plaques and Alzheimer’s disease. RPE are particularly rich in ApoE mRNA suggesting that this cell type might be the source of ApoE within drusen (Anderson et al. 2001; Rudolf et al. 2008). Interestingly, in ApoE transgenic mice, lipid deposits accumulated in the RPE layer as well as Bruch’s membrane and VEGF levels were elevated indicating that AMD was progressing (Lee et al. 2007). ApoE also binds to a receptor of the LDL-related (LDLR) family (Bu 2009). This binding event affords a degree of neuroprotection and has been used as a therapeutic target in animal models of Alzheimer’s disease and multiple sclerosis. ApoE receptors are present in retinal ganglion cells and their importance will be considered in a subsequent section later.

The list of druggable targets for inflammation is large and continuing to grow. Classic targets include the glucocorticoid receptor and COX2. Biological therapeutics such as antibodies have been developed to block the activity of a host of inflammatory signaling molecules such as TNFa and IL-1. Emerging targets include elements of the complement pathway and transcription factors, such as NF-kB, which broadly regulate the inflammatory process.

Glucocorticoids represent a heavily used therapeutic for numerous ocular condition and so deserves some additional discussion. Although this drug class represents the most powerful anti-inflammatory therapy at our disposal, it causes a significant number of side effects limiting its usefulness. Systemic side effects include Cushing syndrome (osteoporosis, fat redistribution, limb muscle wasting, and thinning of the skin), along with CNS effects such as alterations of mood. Ocular side effects include increased intraocular pressure (IOP) and clouding of the lens leading to glaucoma and cataract, respectively. One especially interesting hypothesis is that the mechanism by which glucocorticoids mediate much of their anti-inflammatory effects may be separable from the mechanism by which glucocorticoids mediated their adverse effects.

Glucocorticoids bind to the glucocorticoid receptor (GR) which functions as a transcription factor. There exist two major isoforms derived from splice variants: GRa and GRb (Lu and Cidlowski 2004). At present, it is unclear how the different isoforms contribute to GR-mediated anti-inflammatory action or to GR-mediated side effects. The powerful anti-inflammatory and immunosuppressive action of GR comes primarily from its ability to mediate the repression of transcription of proinflammatory genes [reviewed in (Beck et al. 2009)]. Activation of transcription involves the binding of liganded GR to DNA response elements (GREs), often

Fig. 21.2  Mechanisms of the anti-inflammatory actions of glucocorticoids. (a) Glucocorticoids (yellow) bind to an intracellular receptor which functions as a transcription factor (GR). GR binds to specific sites on DNA called glucocorticoid responsive elements (GREs). The binding of GR to its GRE helps recruit an active transcription complex which then transcribes an anti-inflammatory gene. (b) Proinflammatory gene transcription is activated by the transcription factor; NF-kB. Glucocorticoids activate the transcription of an inhibitor of NF-kB called IkB. IkB then sequesters NF-kB thus blocking inflammation. (c) NF-kB-mediated transcription can also be blocked by GR through the ability of GR to interact with components of the transcription complex. Interestingly, in this scenario, GR does not bind to DNA. Rather, GR binds to NF-kB and to components of the transcription complex. This process (which is fundamentally different from (a) and (b)) is termed “transrepression”

found in the promoters of glucocorticoid responsive genes. RNA polymerase can then enter a transcription complex and become activated (Fig. 21.2a). Transcriptional repression is mediated through a more complicated process. GR is capable of setting up conditions leading to the sequestration of proinflammatory transcription factors such as NF-kB and AP-1 (important regulators of many proinflammatory genes). One way in which GR mediates this is by interacting physically with these transcription factors (Diamond et al. 1990; Jonat et al. 1990; Lucibello et al. 1990; Schule et al. 1990; Yang-Yen et al. 1990; Caldenhoven et al. 1995; Ray et al. 1995; Scheinman et al. 1995b). Another means by which GR promotes the sequestration of NF-kB is by inducing the transcription of its endogenous inhibitor; IkB (Auphan et al. 1995; Scheinman et al. 1995a) (Fig. 21.2b). IkB holds NF-kB in the cytoplasm until inflammatory signals activate a kinase cascade that results in the phosphorylation, ubiquitination, and degradation of IkB thus releasing NF-kB (Basseres and Baldwin 2006). Furthermore, by physically interacting with the integrating

co-activator proteins p300/CBP (part of the core promoter complex), GR can limit the ability of transcriptional activators to interact with the core transcription complex (Sheppard et al. 1998). Finally, GR can integrate into the DNA bound transcription complex and alter the phosphorylation state of DNA polymerase rendering it less active (Nissen and Yamamoto 2000) (Fig. 21.2c). What is unique about this system, is that the majority of genes affected by glucocorticoid treatment which contribute to the inflammatory state do not contain GREs. Examples include the inhibition of cytokines such as IL-2 and IL-6, the downregulation of cell adhesion molecules such as E-selectin and ICAM-1. There do exist some anti-inflammatory genes which are activated in the classic sense by GR. One example is lipocortin. This protein functions to inhibit phospholipase A2 leading to decreased prostaglandin synthesis. Conversely, many of the genes which contribute to glucocorticoidmediated side effects, such as osteoporosis and IOP, contain GREs and are regulated in the classic fashion by GR.

Currently used glucocorticoids include prednisone, prednisolone, dexamethasone (Dex), triamcinolone acetonide (TA), and fluocinolone acetonide (FA). These drugs are often first line therapies for inflammatory ocular conditions. All of these compounds have highly similar structures with a shared steroid backbone. Interestingly, despite their structural similarly, these steroids are capable of eliciting subtly different effects. For example, a study comparing Dex with TA and FA found that all three bound GR with a similar affinity and activated a reporter gene to a similar extent (Nehme et al. 2009). This would suggest that the gene populations activated or repressed by GR when bound to any of these three glucocorticoids would be indistinguishable. However, this did not turn out to be the case. Using several human trabecular meshwork cell lines, the authors performed gene array analyses in the absence of steroid and then in the presence of a saturating concentration of each of the three steroids. Surprisingly, in all cell lines, a significant number of genes whose expression was altered by steroid treatment were unique for each of the three steroids used. For example, TM93 was a cell line derived from a 35-year-old donor. Treatment with a saturating concentration of FA for 24 h resulted in the altered expression of 4,483 genes. Of these 2,294 genes were unique (i.e., they were not altered by treatment with either Dex or TA). Conversely, of the 3,523 genes whose expression was altered by a similar treatment with Dex, 745 were unique. In the case of TA, of 2,430 genes, 555 were unique. For TM93, 1,150 genes were altered similarly by all three steroids. What this study illustrates is that each steroid confers upon GR a subtly different shape. These shapes, in turn, determine which protein DNA complexes will form and upon which genes they will form. While in the early stages of development, this sort of fine control may allow us to design steroids that target inflammatory genes but spare the genes which cause IOP and cataract formation.

Side effects are a serious issue with this drug target. Cataract and IOP are common effects of intravitreal triamcinolone injections. For example, a sustained-release implant of fluocinolone which releases for a period of 30 months allows for the tapering of systemic glucocorticoids but in phakic eyes, will induce cataract formation in almost all patients over time with a 60% risk of glaucoma as well (Callanan et al. 2008).