KEY FEATURES

The term “neuroprotection” can be referred to as the process of directly modifying neurons, and/or their surrounding environment, to promote the survival and function of the neurons, especially in environments that normally would be deleterious to their health.

INTRODUCTION

In ophthalmology, although neuroprotection holds great promise, the field in many ways is still in its infancy. This is especially true from the point of view of the clinician. Despite important scientific advances, and some successes in animal models, few therapies have proceeded to clinical trials, and none to overwhelming success. However, over the next 10–20 years, this situation is likely to change significantly. In this chapter we will briefly review some of the scientific background for neuroprotection, but will focus on translational studies that show promise of bringing particular neuroprotective strategies to the clinic. Keeping with the theme of this book, we will emphasize pharmacological studies related to preservation of structure and function of photoreceptors, i.e., those related to retinal degenerative disease, but given the important parallels with retinal ganglion cell (RGC) loss in glaucoma, we will also present studies related to glaucoma where appropriate. Due to space limitations, this chapter is not intended to cover the entire field, but rather will present selected areas in an effort to provide a sense of where retinal neuroprotection is today, and where it is going. For the interested reader, relevant and more comprehensive reviews will be cited.

Over the last 20 years, there has been considerable effort aimed at elucidating the underlying mechanisms of photoreceptor and ganglion cell degeneration and identifying potential neuroprotective strate- gies.1–10 There are multiple laboratory models, both cell culture and animal-based, available for the study of both photoreceptor and ganglion cell survival. The retina has a number of advantages as a target for neuroprotection. It is easily accessible, self-contained, and visualizable, thus providing the potential for localized therapy, which minimizes the likelihood of systemic side-effects. Unfortunately, counterbalancing some of these scientific factors favoring the development of retinal neuroprotection have been certain economic issues. With the exception of age-related macular degeneration (AMD) and glaucoma, many of the retinal diseases that would seem the most appropriate for the application of neuroprotection strategies affect relatively small numbers of patients, historically limiting the interest of big pharma. However, the situation has been changing, both because a number of small biotech companies have become interested in retinal neuroprotection, partly due to the attractions of orphan drug legislation, and spinoff from the development of new AMD treatments is also attracting some big pharma interest. Thus, the field may also be poised to move forward from the economic/commercial side.

Before we begin our discussion, we would like first to define more fully our concept of neuroprotection. We will include in our discussion pharmacological agents that act directly on retinal neurons (primarily photoreceptors and ganglion cells) to prevent or delay their premature

degeneration and death due to disease.11 Antihypertensive therapies in glaucoma, for example, though certainly neuroprotective in a general sense, would not necessarily be considered retinal neuroprotectants for the purposes of this discussion. Wheeler et al. provided four general criteria to evaluate pharmacological agents as neuroprotectants, which is helpful as a conceptual framework for the neuroprotective drug discovery process.12 In order to be a clinically relevant “neuroprotective” agent, the agent must have a molecular target in the retina, achieve therapeutic concentrations in the posterior segment with clinical dosing, and demonstrate neuroprotective properties in laboratory and animal models. Finally, the agent must demonstrate efficacy in clinical trials.12 There has been much work done that satisfies the first three criteria, but a dearth of therapies have made it to trial, and few therapies have suggested clinical efficacy as retinal neuroprotectants. Our discussion will summarize the clinical work that has been performed and highlight hopeful avenues of research.

Theoretically, neuroprotective agents may be applied in multiple disease states as they act “downstream” of the primary cellular injury due to individual diseases.8 The mechanism of injury in AMD may differ from retinitis pigmentosa, for example, but a neuroprotective agent targeting downstream processes may slow disease progression in both. Ultimately, apoptosis seems to be the final common mechanism of both RGC and photoreceptor degeneration in multiple disease states.2 Diabetic retinopathy, traditionally thought of in terms of microvascular disease, is also a neurodegenerative disease, and thus can be considered a potential market for neuroprotective therapies.13 This review will focus on three broad strategies: antioxidative therapy, neurotrophic support, and antiapoptotic therapy. Many potential pharmacological agents act through multiple mechanisms, and we have tried to emphasize that where possible.

MECHANISM OF PROTECTION: APPROACHES AND CHALLENGES

ANTIOXIDATIVE THERAPY

Oxygen free radicals play an important role in many human diseases, and myriad natural and artificial compounds have been evaluated for their role in protecting both photoreceptors and ganglion cells from oxidative damage. Multiple dietary antioxidants have demonstrated neuroprotective properties in the laboratory, but the three best studied are perhaps vitamin E, vitamin A, and the omega-3 fatty acids, especially docosahexaenoic acid (DHA).10,14,15 Although still somewhat controversial, a significant advance in antioxidant therapy for retinal disease occurred with the finding from the Age-Related Eye Disease Study (AREDS) that administration of a combination of vitamin A, vitamin E, beta-carotene, and zinc decreased the probability of advancing to end-stage AMD by 25%.16 The AREDS II study (ongoing) will further evaluate the effects of lutein, zeaxanthin, and two omega-3 fatty acids (DHA and eicosapentanoic acid), all of which have demonstrated neuroprotective properties in preclinical studies.15

Vitamin E (α-tocopherol) has demonstrated beneficial effects on photoreceptors and ganglion cells in multiple models. However, clinical

trial results in glaucoma have been mixed, with two smaller, open-label studies suggesting an improvement in visual field, but a prospective, large-scale, long-term, study investigating dietary antioxidant intake failing to confirm these findings.3,17–19 DHA has demonstrated neuroprotective properties in multiple laboratory models and may inhibit photoreceptor apoptosis through modulation of the Bcl-2 family of antiapoptotic proteins in addition to its antioxidant properties. In clinical trials, fish oil (a natural source of DHA) has been studied in AMD with positive results.15 DHA is currently in phase II/III trials for retinitis pigmentosa.20 Vitamin A supplementation has also been shown to slow the rate of photoreceptor degeneration in certain forms of retinitis pigmentosa.21 There are several ongoing clinical trials in retinitis pigmentosa and retinopathy of prematurity.20

EXCITOTOXICITY

The role of excitotoxicity in glaucomatous ganglion cell dysfunction, which has been a heavily researched and controversial area, deserves some mention in the context of retinal neuroprotection. Glutamatemediated excitotoxicity has been hypothesized to be important in RGC damage in glaucoma, and based on this hypothesis efforts have been made to reduce RGC injury by blocking the effect of glutamate. A variety of animal studies with different glutamate antagonists have been reported, but the results have been mixed.14,22 The best-studied member of this class of drug is memantine, which is approved for the treatment of Alzheimer’s disease and is an uncompetitive N-methyl-d- aspartic acid (NMDA) receptor antagonist and partial agonist, which allows it to exert its inhibitory effect in the presence of excess glutamate, but have minimal effect in the normal state.23 The interest in memantine as a retinal neuroprotectant resulted from positive results reported in laboratory and animal models of RGC death.3 In addition, in monkeys with experimental glaucoma, oral memantine was reported to protect against structural and functional glaucomatous changes, compared with placebo controls.24,25 However, in a large-scale, randomized, prospective phase III clinical trial, memantine failed to demonstrate a significant improvement over placebo.

In the excitotoxicity paradigm, many common medications modulate cellular neural activity (including glutamate release) and have been explored as potential neuroprotective drugs. Voltage-gated sodium channels begin the process of ionic destabilization by depolarizing the cell, and sodium channel antagonists have been explored to inhibit the influx of sodium during periods of cellular stress.3 Beta-receptor antagonists have also received attention as potential neuroprotective agents. Betaxolol, which has been reported to have neuroprotective properties in several small clinical trials, appears to have more neuroprotective action than metipranolol, timolol, and nipradilol, though all have shown some benefit in laboratory models, and all may act through different mechanisms.3,26 Betaxolol modulates sodium and calcium channel activity, upregulates brain-derived neurotrophic factor mRNA, and prevents RGC death in multiple models of retinal injury.3,26–28 EGb 761, better known as ginkgo biloba and a well-known dietary antioxidant, has been reported to demonstrate neuroprotective activity in rat models of ischemia and ocular hypertension.3 In a small-scale, prospective, randomized, double-blind, crossover trial, ginkgo biloba improved visual field damage in some patients with normal-tension glaucoma.29 However, these results are controversial and need to be confirmed in larger studies.

There has been much recent interest in the finding that there is an association between particular complement factor H (CFH) alleles and the risk of developing AMD, which, like many genetic risk factors, appears to be modified by oxidative stress.30 This association appears to be robust and has been confirmed by a large number of independent studies. As we begin to understand the genetic heterogeneity of risk factors for both rare retinal diseases (e.g., retinitis pigmentosa) and more common diseases (AMD), and their environmental interactions, the efficiency of “downstream” neuroprotective therapy will hopefully become clearer. Persons with high-risk CFH profiles, or perhaps retinitis pigmentosa patients with particular mutations, may one day be able to reduce their disease progression risk by taking prophylactic antioxi-

dative therapy. Better yet, we may find that modification of risk is possible with dietary change alone, a thus far unproven hypothesis.

NEUROTROPHIC FACTORS

Neurotrophic support as a therapeutic paradigm has received much interest in the last decade since basic fibroblast growth factor first demonstrated structural and functional neuroprotective effects on photoreceptor degeneration in a rat model of inherited retinal dystrophy.21,31 Since then, a number of additional factors have been identified that also demonstrate neuroprotective properties in animal models. To date, human clinical trials have been initiated with two of these factors, pigment epithelium-derived factor (PEDF) and ciliary neutrotrophic factor (CNTF). PEDF acts on multiple pathways in the eye, including modulating glutamate excitotoxicity, protecting against oxidative damage, and inhibiting apoptosis. Intraocular injection of PEDF increased the number of photoreceptors surviving in a light-induced retinal injury model, and slowed degeneration in two mouse models of inherited retinal degeneration.10 PEDF also demonstrates anti-VEGF activity, and a phase I trial with adenoviral-mediated PEDF administration in wet AMD has been completed, with encouraging results.32

CNTF has been one of the most promising molecules in retinal degeneration, demonstrating beneficial effects on both ganglion and photoreceptor cells in multiple laboratory models.2,3 In a recent phase I safety trial, Sieving and colleagues reported that CNTF-encapsulated retinal implants (NT-501) were well tolerated by 10 patients and provided sustained release of CNTF throughout the trial period, suggesting that encapsulated cell technology might be an effective vehicle for delivering neurotrophic factors to the retina in chronic neurodegenerative diseases.33 NT-501 is currently in phase II/III trials for retinitis pigmentosa and AMD.34 In another model, NTC-201, genetically engineered RPE cells designed to overexpress CNTF, has been successfully implanted into rat and canine models of hereditary photoreceptor degeneration.35 Despite the encouraging results with CNTF, some have raised concerns because in some animal models CNTF can cause dose-dependent adverse effects on ERG and on photoreceptor gene expression.36 Another promising molecule is glial-derived neurotrophic factor, which has been reported to have impressive antioxidant and antiapoptotic activity in a rat model of oxidative injury without inducing changes in ERG.37,38 However, it has not yet been tested in clinical trials.

There are several potential delivery mechanisms for neurotrophic factors, some of which are described in more detail elsewhere in this book. As evidenced by the recent demonstration of the apparent safety of RPE-65 gene therapy, adeno-associated viral (AAV) vectors can deliver gene therapy directly to the retina and may represent an ideal treatment vehicle for neurotrophic gene delivery.39 Alternatively, small interfering RNA molecules (siRNAs) have received much attention for their ability to interfere with specific RNA transcripts, preventing deleterious gene expression. O’Reilly et al. recently reported the ability to suppress disease and wild-type rhodopsin with siRNAs and replace them with mutation-free versions, allowing a single therapy to treat multiple genetically heterogeneous forms of AD retinitis pigmentosa.40 Polyamide nucleic acids, which are DNA–protein chimeric molecules that can be tailored to maximize cell penetration and RNA specificity, may also prove useful.41 This is currently a fruitful area of research that may yield answers to some of the practical difficulties with biologic drug delivery to the retina.

ANTIAPOPTOPIC THERAPY

If apoptosis is the final common pathway in neuronal cell death, then pharmacological inhibition of apoptosis could be the ultimate neuroprotective strategy. In reality, many hypothetical and practical obstacles exist to the realization of an effective antiapoptotic therapy in human ocular diseases. Unfortunately, apoptosis is turning out to be a far more complex, and diverse, process than initially thought. In addition, there is some concern that once apoptosis is initiated, the cell may already be too sick to recover fully. Nonetheless, some progress has been made to

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Diseases Retinal and Neuroprotection• 42 chapter

prove to be effective and safe for the treatment of retinal disease remains to be seen.

Intracellular calcium concentrations play a pivotal role in cell survival, and abnormalities in calcium homeostasis can lead to loss of cellular integrity and activation of several apoptotic mechanisms. There has been much interest, therefore, in the role of calcium channel modulation in the preservation of neurons. d-diltiazem, nilvadipine, and nicardipine have all demonstrated mixed results in various rat models of inherited retinal degenerations, with some studies suggesting a neuroprotective effect.10 Flunarizine, flupirtine, lomerizine, and nifedipine have also been reported to promote RGC survival in laboratory and/ or animal models, and several calcium channel blockers have been reported to improve visual function in some patients with normaltension glaucoma. However, these studies have been controversial, and the mechanism is thought by some to involve improved blood flow, rather than direct neuroprotection.3

Though not directly antiapoptotic, Schwartz and colleagues have reported a relationship between T-cell function and RGC survival, suggesting that the immune system may play a role in RGC repair after injury, perhaps preventing apoptotic cell death.3,43,44 In addition to T cells, microglia have also been reported potentially to play a role in both photoreceptor and ganglion cell loss. Therapeutically, this raises the possibility that the immune system might be “primed” for neuroprotection through an immunologic vaccine. Following disappointing results with immunization targeted at myelin-associated protein, attention was turned to glatiramer acetate (copolymer-1), a random oligopeptide that is a low-affinity antigen that cross-reacts with self-reactive T cells, and has been approved by the Food and Drug Administration for the treatment of multiple sclerosis.3 Immunization with glatiramer acetate has been reported to increase RGC survival in mouse and rat models of RGC injury.45 Copaxone is currently in phase I, II, and III trials for AMD.

THE FUTURE OF RETINAL

NEUROPROTECTION

The field of retinal neuroprotection holds much promise for future clinical advances, but much work clearly remains to be done. Many agents demonstrate neuroprotective properties in some laboratory or animal models, but not others, complicating the decision of which to pursue in clinical trials.10 Moreover, it is uncertain how well animal models can accurately predict safety and efficacy in humans, as demonstrated by the disappointing results of the memantine clinical trial. In terms of future clinical application, in addition to identifying the most promising agents, development of improved drug delivery approaches for potential therapies will also be of prime importance. This is particularly true because many of the most promising neuroprotective agents that are now being developed are not the traditional small molecules of the past, but rather are large-molecule “biologics,” such as proteins and genes, that require special modes of administration.

SUMMARY AND KEY POINTS

Despite the numerous obstacles to ophthalmological drug development in neuroprotection, there are several encouraging reasons to suggest that there will be significant advances in the coming years. The last decade has heralded several fundamental revolutions in eye care delivery. The safety and effectiveness of intravitreal injection proved that medications could be efficiently delivered directly to the retina as needed. The development of implantable steroid devices and encapsulated cell technology for CNTF demonstrated that pharmacologic and biologic therapies could be delivered over long periods of time with tolerable side-effects. Moreover, the ongoing development of small molecules optimized for retinal penetration may further simplify drug delivery and allow more drug development and clinical testing. Finally, at the organizational level, there is increasing infrastructure being estab-

lished to facilitate the translation from basic science to clinical trials for orphan and other retinal diseases. As one example, the National Eye Evaluation and Research network, established by the National Neurovision Research Institute, was developed to coordinate and facilitate the translation of promising retinal degeneration research from the lab to the clinic. As another indication of the promise that lies ahead, Table 42.1, obtained from the national clinical trials database,20 summarizes the currently ongoing clinical trials that are relevant to the theories and pharmacologic options described in this chapter. The diversity of treatment approaches and applicable disease states highlights the research interest in (and the potential market of) neuroprotective therapies for retinal diseases. Hopefully, studies such as these, and others that are just being conceived, will some day soon lead to the development of new and more effective neuroprotective approaches for the treatment of disabling retinal diseases that affect millions around the world.

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