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REVIEWS Ophthalmic drug discovery: novel targets and mechanisms for retinal diseases and glaucoma Kang Zhang1,2,3, Liangfang Zhang3,4 and Robert N. Weinreb2 Abstract | Blindness affects 60 million people worldwide. The leading causes of irreversible blindness include age-related macular degeneration, retinal vascular diseases and glaucoma. The unique features of the eye provide both benefits and challenges for drug discovery and delivery. During the past decade, the landscape for ocular drug therapy has substantially changed and our knowledge of the pathogenesis of ophthalmic diseases has grown considerably. Anti-angiogenic drugs have emerged as the most effective form of therapy for age-related macular degeneration and retinal vascular diseases. Lowering intraocular pressure is still the mainstay for glaucoma treatment but neuroprotective drugs represent a promising next-generation therapy. This Review discusses the current state of ocular drug therapy and highlights future therapeutic opportunities. Age-related macular degeneration (AMD). A disease process that is characterized by the degeneration of photoreceptor cells in the macula, leading to loss of central vision. Department of Ophthalmology and Molecular Medicine Research Center, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China. 2 Department of Ophthalmology and Shiley Eye Center, University of California San Diego, La Jolla, California 92093, USA. 3 Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA. 4 Department of Nanoengineering, University of California San Diego, La Jolla, California 92093, USA. Correspondence to K.Z. e‑mail: [email protected] doi:10.1038/nrd3745 Published online 15 June 2012 Corrected online 29 June 2012 1 Visual impairment is one of the most feared disabilities. The vast majority of information we acquire from the environment is dependent on our ability to see. Loss of sight affects activities of daily living and substantially reduces quality of life. The leading causes of visual impairment are age-related, and cause damage to the retina and optic nerve. These causes include age-related macular degeneration (AMD), diabetic retinopathy and glaucoma (FIGS 1,2; TABLE 1). Blindness and visual impairment represent a substantial burden to the health-care system. The total annual economic impact of major visual disorders among Americans aged 40 years or older has been estimated at US$35.4 billion1. As the lifespan of our population continues to increase and advances in health care improve longevity, there will be an increasing number of people who are at risk of developing visual impairment, and the economic impact of visual impairment will continue to grow. The unique features of the eye provide both benefits and challenges for drug discovery and delivery (FIG. 1; FIG. 2b). In contrast to other parts of the central nervous system, the eye is clinically accessible because it is located outside the cranium. With new non-invasive diagnostic technologies, such as optical coherence tomography, the retina and optic nerve can be clearly visualized and their macro- and microstructures evaluated in situ (FIG. 2a). The accessibility of these tissues also facilitates drug delivery using methods such as eye drops or local injections. Local delivery and exposure to a drug minimizes systemic toxic effects and therefore increases the therapeutic index. In addition, there are many modalities available to test visual function, thus allowing easy assessment of drug effectiveness. However, despite these many bene fits there are also challenges related to ocular barriers. For example, ocular barriers impede drug transport and lower the efficacy of many drugs. Such barriers include the blood–ocular barriers, blood flow, lymphatic clearance and tear dilution2. During the past decade, there have been considerable advances in the understanding of the pathogenesis and genetics of ophthalmic diseases. Although there were no effective treatments for wet AMD and retinal vascular diseases 10 years ago, effective therapies such as antiangiogenic agents now exist. By contrast, latanoprost was launched a decade ago for the treatment of glaucoma (and soon became the first-line treatment) but no new classes of glaucoma drugs have emerged since then. Various drugs that act by inhibiting complement activation and angiogenic pathways are currently under development for the treatment of AMD, and neuroprotective drugs are being investigated for both AMD and glaucoma3–6. However, the effective treatment of ocular diseases remains an unmet medical need, and a vigorous effort to develop new drugs and therapies is now NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 541 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Retina (DR, RD) Choroid (blood vessel layer under the retina) Pupil Light Optic nerve: goes to the brain (glaucoma) Cornea (keratitis) Lens (cataract) Macula (AMD, DME) Figure 1 | Human eye structure and diseases. Schematic diagram of the anatomy of the eye and associated diseases. AMD, age-related macular degeneration; DME, diabetic macular oedema; DR, diabetic retinopathy; RD, retinal degeneration. Nature Reviews | Drug Discovery underway. In this Review we discuss the current state of ocular drug therapy, highlight innovations within drug discovery and delivery, and discuss potential concepts for the future treatment of retinal diseases and glaucoma. Diabetic retinopathy A microvascular complication of type 2 diabetes that is characterized by increased vascular permeability in the initial stages of disease. Glaucoma A progressive disease that is characterized by optic disc cupping and peripheral visual field loss. Retinal pigment epithelium (RPE). A monolayer of pigmented cells in the retina, located between the photoreceptor cells and choroid. Macula A highly pigmented area near the centre of the retina that is responsible for detailed central vision. Geographic atrophy An advanced form of age-related macular degeneration that is characterized by the atrophy of retinal pigment epithelium and photoreceptor cells. Choroidal neovascularization (CNV). An advanced form of age-related macular degeneration that is characterized by the growth of abnormal blood vessels into the subretinal space. Ocular diseases Age-related macular degeneration. AMD is defined as an abnormality of the retinal pigment epithelium (RPE) that leads to degeneration of the overlying photoreceptor in the macula and consequent loss of central vision7,8. AMD usually occurs in individuals over 50 years of age, and is the most common cause of vision loss in elderly individuals throughout the developed world9–15. AMD represents a major public health burden, with both economic and social consequences. It is estimated that over 9 million people in the United States suffer from intermediate or advanced forms of AMD16. The aetiology and pathophysiology of AMD are poorly understood. Histologically, the disease is characterized by the accumulation of membranous debris underneath the RPE basement membrane, which forms drusen. Drusen are small, yellowish extracellular deposits of lipid, cellular debris and protein, including complement components, anaphylatoxins, modulators12,17–20,21–26 and high-temperature requirement A serine peptidase 1 (HTRA1)27. The formation of drusen can be caused by RPE dysfunction or by a change in the composition or permeability (to nutrients) of Bruch’s membrane. Therefore, early AMD is characterized by drusen (FIG. 2c) and hyper- or hypopigmentation of the RPE without loss of vision. Advanced AMD leads to loss of vision and can be classified into two categories: geographic atrophy, which is characterized by atrophy of the RPE (FIG. 2d) ; and choroidal neovascularization (CNV; also known as wet AMD) (FIG. 2e), which is due to the abnormal growth of blood vessels. It has been suggested that one or more of the following key processes is involved in AMD (FIG. 3): oxidative damage; lipofuscin accumulation and impaired activity or function of the RPE; increased apoptosis; abnormal immune system activation28; senescent loss of homeostatic control29; or abnormalities in Bruch’s membrane. Recent evidence suggests that there may be crosstalk and interaction among these pathways30. For example, oxidative stress, abnormal complement activation or inflammatory pathways may lead to RPE apoptosis or abnormal angiogenesis26,30,31. Vascular endothelial growth factor A (VEGFA) has been implicated in CNV and increased vascular permea bility that results in loss of vision32. VEGFA is a 46 kDa homodimeric glycoprotein that was first identified in vascularized tumours32–34. It is produced by many ocular cell types in response to hypoxia and has multiple functions in the eye. It stimulates endothelial cell growth, promotes vascular permeability and induces dissociation of tight junction components. Animal models of AMD have contributed to the understanding of disease pathogenesis and the development of treatments. Current models with features of dry AMD include monkeys with naturally occurring drusen, mice that have been genetically manipulated or have had high exposure to environmental risk factors for AMD (for example, a high-fat diet, exposure to cigarette smoke, and so on), and mice that have been immunized with carboxyethylpyrrole-modified mouse serum albumin35. Animal models of wet AMD include primates and rodents with laser-induced CNV, animals with mechanically induced CNV, rats that have been transfected with angiogenic factors as well as transgenic and knockout mice. Genetically modified mice are the only animal models developed to date that recapitulate the features of both dry and wet AMD35–40. Ranibizumab (Lucentis; Genentech/Roche), bevacizumab (Avastin; Genentech/Roche) and aflibercept (Eylea; Regeneron Pharmaceuticals) — all anti-VEGF agents — are currently the most common therapies for neovascular AMD. Other less commonly used treatments include photodynamic therapy, pegaptanib (Macugen; Eyetech) and retinal laser photocoagulation. Prophylactic treatments include oral vitamins and antioxidants (comprising vitamin C, vitamin E, β-carotene, zinc and copper), and are used in patients who are at a high risk of developing advanced AMD41. Diabetic retinopathy and retinal vein-occlusive diseases. Diabetic retinopathy is the leading cause of blindness and visual impairment in working-age individuals. In the United States, 4.1 million individuals have diabetic retinopathy and, of these, approximately 900,000 have sight-threatening retinopathy 42. These rates are expected to double by 2025. Retinal vein-occlusive diseases include central retinal vein occlusions, hemiretinal vein occlusions and branch retinal vein occlusions. Retinal vein-occlusive diseases affect approximately 100,000 individuals in the United States. Diabetic retinopathy and retinal vein-occlusive diseases result in loss of vision through the following major mechanisms: retinal vascular leakage and exudation, which results in macular oedema; and retinal ischaemia and secondary neovascularization, which can lead to 542 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS vitreous haemorrhage and retinal detachment. VEGFA has been implicated in increasing retinal vascular permeability and leakage, which leads to macular oedema and ocular neovascularization34,43. Current treatments for diabetic macular oedema or macular oedema arising from retinal vein-occlusive diseases include anti-VEGF therapies, focal laser therapy and steroids. Laser panretinal photocoagulation is the main therapy for neovascularization in proliferative diabetic retinopathy and retinal vein-occlusive diseases. Glaucoma. Glaucomas are a group of progressive optic neuropathies that are characterized by a slow and progressive degeneration of retinal ganglion cells (RGCs) and their axons, resulting in a distinct appearance of the optic disc (FIG. 2f) and a concomitant pattern of vision loss6. Characteristic changes to the optic nerve in glaucoma include the enlargement and elongation of the optic nerve cup, the thinning and eventual notching of the neuroretinal rim, asymmetry in cup size between the two eyes and disc haemorrhages. Changes in the visual field with perimetric testing include scotomas that correspond to local and/or diffuse loss of nerve fibres within the retina. These characteristic changes consist of arcuate scotomas, nasal steps and paracentral scotomas. The disease involves the entire visual pathway, including the brainstem and the visual cortex — not just the eye44. It has been estimated that glaucoma will affect more than 80 million individuals worldwide by 2020, with at least 6–8 million individuals becoming bilaterally blind45. Glaucoma is also the leading global cause of irreversible blindness46 and is perhaps the most prevalent of all neurodegenerative diseases. The biological basis of glaucoma is not fully understood, and the factors contributing to its progression are currently not well characterized. Intraocular pressure (IOP) is the only proven treatable risk factor, and lowering IOP is the main approach for reducing disease progression. Impaired ocular blood flow is also thought to be a risk factor 47 but the evidence for this is conflicting, perhaps largely owing to the absence of an accurate and reproducible test of ocular blood flow or the optic nerve microcirculation. With or without treatment, glaucoma can progress through a continuum48 that is initiated by an acceleration of RGC apoptosis, with the disease initially being asymptomatic but eventually resulting in blindness in some individuals, particularly those who are inadequately treated6. Intraocular pressure (IOP). The fluid pressure inside the eye, which is determined by the production and drainage of aqueous humour. Aqueous humour A clear, watery fluid produced by the ciliary epithelium that fills the anterior and posterior chambers of the eye. Current status of ocular drugs Anti-VEGF therapy. Over the past decade, considerable progress has been made in the treatment of ocular neovascular diseases owing to an increased understanding of the mechanisms of ocular angiogenesis32,33 (FIG. 4). Anti-angiogenic therapies targeting VEGFA have proven to be highly effective in treating neovascular AMD and have become the mainstay of treatment. Anti-VEGF treatment is the only therapy to date that improves vision in patients with neovascular ocular diseases, as demonstrated by several randomized, controlled Phase III studies49–51. Ranibizumab, a recombinant humanized antibody fragment that binds all known isoforms of human VEGFA, is formulated for ocular administration (via an intravitreal injection) and approved in the United States for the treatment of neovascular AMD. Bevacizumab, which was initially approved for the treatment of certain types of cancers, has also been used (off-label) to treat wet AMD52 but it is not formulated for intraocular administration. A recent study compared the safety and efficacy of these two drugs in a multicentre, randomized controlled trial51. The primary results of the 1‑year follow-up study showed that monthly administration of bevacizumab is non-inferior to monthly administration of ranibizumab. However, there were some safety concerns in the trial; a statistically higher number of serious adverse events was observed in the bevacizumab group51 (mainly hospitalizations, perhaps owing to longer systemic retention of bevacizumab compared to ranibizumab). Another therapeutic approach is the use of aflibercept, which is a fusion protein that incorporates portions of extracellular domains of the human VEGF receptor 1 (VEGFR1) and VEGFR2 that are fused to the constant region of human immunoglobulin G53,54. Aflibercept binds to all isoforms of VEGFA with a higher affinity than ranibizumab and bevacizumab. It also binds placental growth factor 1 and placental growth factor 2 (REF. 55). Two Phase III trials using aflibercept (as an intravitreal injection) to treat wet AMD have been completed and showed equivalent efficacy to ranibizumab. The drug was well tolerated and approved by the US Food and Drug Administration (FDA) for the treatment of wet AMD in 2011. The application of anti-VEGF agents in the treatment of retinal vascular diseases, including diabetic retinopathy and retinal vein occlusions, has been very successful56–58 but ranibizumab has only been approved by the FDA for treating retinal vein-occlusive diseases. IOP-lowering therapies. There are two major forms of glaucoma: open-angle glaucoma and closed-angle glaucoma. Both forms are initially treated by lowering IOP, typically with eye drops, but rapid advancement to surgical treatment is required for some types of closed-angle glaucoma. IOP is determined by the balance between the aqueous humour secreted into the eye by the ciliary body and the amount that is drained from the eye via the two major outflow pathways: the trabecular meshwork and the uveoscleral pathway (consisting of the iris root, longitudinal ciliary muscle, anterior choroid and sclera). IOP is in a steady state when the rate of aqueous inflow is the same as the rate of aqueous outflow. Pharmacological treatment of glaucoma lowers IOP by decreasing the rate of aqueous inflow and/or increasing the rate of aqueous outflow (FIG. 5a). There are five major classes of drugs (administered as eye drops) that are approved for lowering IOP in glaucoma (TABLE 2). Cholinergic agents have been used for more than 100 years to lower IOP. They act on muscarinic receptors located on the ciliary muscle to increase outflow through the trabecular meshwork59. Although the most NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 543 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS a RPE Bipolar cell Choroid Retina Cone Rod Choroid Ganglion cell Horizontal cell Vitreous Vitreous Amacrine cell Bruch’s membrane b Retina RPE Choroid Fovea ON Fovea Fovea Retina RPE Choroid Macula c d e f Nature Reviews | Drug Discovery 544 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Figure 2 | Retinal anatomy and structure in health and diseases. Imaging of the structures of an eye by optical coherence tomography. a | A schematic diagram of the retina is shown at the top of the panel. The inset shows the choroid and layers of the retina, including the retinal pigment epithelium (RPE). Optical coherence tomography images are also shown, of a normal retina (top) and a retina with pigmented epithelium detachment due to choroidal neovascularization (CNV) (indicated by the white arrows) in wet age-related macular degeneration (AMD) (bottom). b | The image shown is of a normal retina and macula. c | The image depicts a macula with confluent soft drusen (indicated by the white arrows) — a hallmark of early AMD. d | The image shows a macula of geographic atrophy (within the dashed circle). e | A macula of CNV with haemorrhage (indicated by the white arrows) is shown. f | The image illustrates an optic nerve with glaucomatous excavation and loss of neuroretinal rim between the disc margin (white dashed lines) and the border of the optic cup (black dashed lines). Images courtesy of K.Z. ON, optic nerve. Complement system A part of the innate immune system that consists of approximately 25 proteins. Three pathways activate the complement system: the classical complement pathway, the alternative complement pathway and the mannose-binding pathway. widely used drug in this class — pilocarpine — is inexpensive and has few systemic side effects, its widespread use is largely limited by ocular side effects (such as miosis, myopia, brow ache and dimming of vision) and the inconvenience of requiring dosing four times daily 59,60. Owing to their convenient once-daily dosing, prostaglandin F receptor (PTGFR) analogues are generally administered as first-line agents and increase aqueous outflow by altering the composition of the extracell ular matrix in the ciliary muscle and trabecular meshwork60,61. These agents are the most effective of the IOP-lowering agents, and a single daily dose is effective at reducing IOP throughout the day and during the night 62. PTGFR analogues cause lengthening and thickening of eyelashes and can also cause hazel and blue irides with brown patches to become diffusely brown. β-adrenergic receptor blockers have been widely used for more than 30 years and have various strengths, including their ocular tolerability and IOP-lowering efficacy (which is surpassed only during the day by PTGFR analogues). They reduce aqueous humour secretion by inhibiting the activity of the predominant β-adrenergic receptor in the ciliary epithelium63. However, they are only effective during the day and not during the night 62, and can reduce ocular perfusion pressure. They have minimal efficacy in combination with PTGFR analogues, and for this reason the use of these agents in a combination product has not been approved in the United States. Another approach is the use of carbonic anhydrase inhibitors to inhibit the activity of carbonic anhydrase 2 in the non-pigmented ciliary epithelium, thus reducing aqueous humour formation. These drugs lower Table 1 | Leading causes of irreversible visual impairment Disease Incidence Cataract 8.4–29.7% of patients over 43 years of age Refs 183 Age-related macular degeneration 6–22% of patients over 70 years of age 184 Glaucoma 1–4% of patients over 45 years of age 6 Diabetic retinopathy 74.9–92.3% of diabetic patients over 30 years of age 185,186 Retinitis pigmentosa 1 in 4,000 187 IOP throughout the day and during the night, and are widely used as second-line agents64. α-adrenergic receptor agonists lower IOP primarily through stimulation of α2‑adrenergic receptors in the eye. Like the beta blockers, they effectively lower IOP during the day but not during the night 65. They appear to decrease aqueous inflow and increase uveoscleral outflow66. In experimental animal models they have a neuroprotective effect on RGCs67 but this has not been demonstrated convincingly in patients with glaucoma. Ongoing clinical trials Complement pathway inhibitors. Recent studies have shown that AMD is associated with certain genotypic variants of complement pathways, including complement factor H (CFH)21,68–70, complement component 2 (C2)71, complement factor B71,72, complement component 3 (C3)72,73 and complement factor I74. Owing to multiple gene associations in the complement activation pathway, several complement inhibitors are now in clinical trials. Various strategies have been exploited to treat AMD by modulating the complement system, such as the replacement of defective complement components as well as inhibition of the complement pathway (FIG. 6). Inhibition of the complement pathway disables downstream complement cascade activation, thus hindering inflammatory responses. However, the complement system is an important component of the immune system, and local or systemic complement inhibition may increase the risk of infections such as endophthalmitis. POT‑4 (also known as AL‑78898A) is a synthetic peptide C3 inhibitor that binds reversibly to C3. An appealing feature of this intravitreal drug is its gel-like preparation, which can be deposited in the vitreous at high concentrations and can last for 6 months as a sustained-delivery vesicle. A Phase I study of POT‑4 in patients with wet AMD was completed successfully without any safety concerns74. A Phase II study of AL‑78898A in conjunction with ranibizumab is currently underway. ARC1905 is a complement component 5 (C5) inhibitor that is administered by intravitreal injection. As a small-molecule aptamer-based drug it exhibits several advantages, including low activation of the immune response and the ability to formulate it in a sustainedrelease form. A Phase I trial investigating the efficacy of ARC1905 in combination with ranibizumab for treating wet AMD was performed and the agent was shown to be safe and well tolerated75. Another Phase I study investigating the efficacy of ARC1905 in dry AMD is underway. Eculizumab is a monoclonal antibody derived from a murine anti-human C5‑specific antibody. Like ARC1905, it is also intravenously administered and inhibits the cleavage of C5 to C5a and C5b. Eculizumab is already approved by the FDA for the treatment of paroxysmal nocturnal haemoglobinuria and is currently in Phase II trials known as COMPLETE (‘complement inhibition with eculizumab for the treatment of non-exudative age-related macular degeneration’) for the treatment of dry AMD. FCFD4514S is a recombinant, humanized monoclonal antibody Fab fragment directed against complement factor D, currently in a Phase I/II study to evaluate its NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 545 © 2012 Macmillan Publishers Limited. All rights reserved ▶ REVIEWS Chronic oxidative stress Antioxidants Visual cycle inhibitors Lipofuscin + Photoreceptor–RPE–Bruch’s choriocapillaris complex Anti-inflammatory agents + Chronic inflammation + + Abnormal complement activity Accumulation of extracellular and intracellular debris Drusen formation, damage to photoreceptors and RPE Geographic atrophy Neovascularization Neuroprotectants Anti-angiogenic agents Figure 3 | Proposed pathophysiology of AMD, and locations in the pathway in which different therapeutic interventions might be effective. One or more of the Nature macular Reviews degeneration | Drug Discovery following key processes is likely to have a role in age-related (AMD): oxidative damage; lipofuscin accumulation and impaired function of retinal pigment epithelium (RPE); increased cell death; abnormal immune system activation; senescent loss of homeostatic control; and abnormalities in Bruch’s membrane. VEGF, vascular endothelial growth factor. Potential strategies for the treatment of AMD are shown in orange boxes. safety, tolerability, pharmacokinetics and immunogenicity when intravitreally injected in the treatment of geographic atrophy. Small interfering RNA (siRNA). Small, double-stranded RNA molecules that interfere with gene expression by binding to and promoting the degradation of mRNA. Visual cycle inhibitors. A prominent feature of AMD is the aberrant accumulation of cellular debris or lipofuscin within the RPE. Lipofuscin is a product of autofluorescent material composed of lipids, proteins and vitamin A derivatives76–78. The rationale for the use of visual cycle modulators is to reduce the accumulation of fluorophores (for example, A2E and lipofuscin) in RPE cells (FIG. 3). Retinol is essential for vision, and its metabolism is mediated through the visual cycle (see the Webvision website). Retinol binding protein (RBP) binds to all-transretinol with a high affinity. The retinol–RBP complex serves as a substrate for the uptake of retinol by the RPE within the eye. Two visual cycle inhibitors are currently in clinical trials. N‑(4‑hydroxyphenyl)-retinamide (Fenretinide; Sirion Therapeutics) is an oral synthetic retinoid derivative that binds to RBP in the circulation and blocks its association with retinol, thereby preventing the transport of retinol to the RPE and lowering its availability in the visual cycle. Fenretinide reduces lipofuscin and A2E accumulation in the RPE of mice in which ATP-binding cassette subfamily A member 4 (ABCA4) is knocked out (ABCA4‑knockout mice) and causes modest delays in dark adaptation79. A Phase II clinical trial of this oral agent has been completed, and the drug has been shown to slow the progression of geographic atrophy and reduce the incidence of CNV. Potential side effects include night blindness and dry eye. ACU‑4429 is another orally administered visual cycle modulator. It inhibits the conversion of all-trans-retinyl ester to 11‑cis-retinol via inhibition of the isomerase retinoid isomerohydrolase (RPE65). It was shown to slow retinal degeneration in preclinical models. However, side effects of this drug include nyctalopia, dyschroma topsia and delayed dark adaptation. By modulating isomerization, ACU‑4429 slows the visual cycle in rod photoreceptors and decreases the accumulation of A2E. A Phase II study of ACU‑4429 is currently underway for the treatment of patients with AMD who have geographic atrophy. Anti-angiogenic agents. Because ocular angiogenesis is a multistep process (FIG. 4), it can be intercepted or inhibited at many steps using various approaches, and several agents are now under investigation (TABLE 3). The various strategies and related agents are discussed below. One strategy is to inhibit mammalian target of rapamycin (mTOR), which is a tyrosine kinase that has a key role in regulating cell growth and proliferation. Its activation leads to the production of hypoxia-inducible factors (for example, HIF1α), which in turn can induce the expression of the angiogenesis stimulator VEGF80. Examples of mTOR inhibitors include rapamycin (also known as sirolimus), everolimus and Palomid 529. Sirolimus is currently in Phase II clinical trials for the treatment of patients with diabetic macular oedema, and everolimus in combination with ranibizumab is in Phase II trials in patients with wet AMD. Small interfering RNA (siRNA) is another approach that is used to inhibit VEGF expression. PF‑655 is a synthetic siRNA that inhibits the expression of DNA damageinducible transcript 4 protein (DDIT4; also known as RTP801), which has been shown to be involved in pathological retinal neovascularization in animal models81. It is currently in Phase II trials for diabetic macular oedema and wet AMD. Bevasiranib is a naked, 21‑nucleotide-long siRNA that specifically targets VEGF. However, clinical trials of bevasiranib were discontinued because they did not meet their end point (stabilization of vision), which was defined as a loss of less than three lines of vision on the ETDRS (Early Treatment for Diabetic Retinopathy Study) vision chart. However, in hindsight, the failure of these trials is not surprising as siRNAs need to be formulated to achieve cell permeation in order to cause bona fide RNA interference. They 546 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS • Sirolimus • RAD001 • Palomid 529 • PF4523665 Smooth muscle cell Angiogenic factor production Release PEDF • Aflibercept • Ranibizumab • Bevacizumab Sirna-027 Binding to endothelial cell receptor Sonepcizumab Tube formation Directional migration • JSM6427 • Volociximab Endothelial cell proliferation Endothelial cell Blood vessel Fosbretabulin α5β1 integrin Intracellular signalling E10030 Pazopanib Vascular stabilization Figure 4 | Mechanism of action of ocular angiogenesis inhibitors in clinical care or development. Schematic diagram of new capillary blood vessel growth. Several classes of angiogenic factors, including vascular endothelial growth Nature Reviews | Drug Discovery factor (VEGF), platelet-derived growth factor (PDGF) and pigment epithelium-derived factor (PEDF), bind to specific receptors on vascular endothelial cells to induce specific signalling pathways that activate the cells. Angiogenesis can be inhibited by blocking these signalling pathways as well as through various other approaches. Tyrosine kinase inhibitors (sirolimus, RAD001, Palomid 529 and PF4523665) prevent the production of angiogenic factors. Bevacizumab, ranibizumab and aflibercept bind to and sequester VEGF, thereby inhibiting its interaction with VEGF receptors. Sirna‑027 inhibits the synthesis of VEGF receptor 1. JSM6427 and volociximab both bind to α5β1 integrin and block the migration of endothelial cells. The active metabolite of fosbretabulin (combretastatin A4) inhibits microtubule polymerization in proliferating endothelial cells, inducing vessel regression. also need to be modified to avoid off-target effects and recognition of their nucleotide structure by the innate immune system82–84. In contrast to VEGF, pigment epithelium-derived factor (PEDF) has an antagonistic effect on endothelial cell proliferation and migration, and its levels are decreased in AMD85–87. A Phase I clinical trial has been initiated using an adenoviral vector (AdGVPEDF.11D) to deliver PEDF via intravitreal injections. Many agents, such as ranibizumab or bevacizumab, have now been developed to inhibit the binding of VEGF to its receptor. Aflibercept is another example of an agent that prevents receptor binding by trapping VEGF in the extracellular space. AAV2‑sFLT01 is an adeno-associated virus (AAV)88 that produces a soluble VEGFR1 that can bind to free VEGF89,90 to interrupt VEGF signalling. VEGFA binds primarily to VEGFR1 and VEGFR2, both of which are tyrosine kinases91. A siRNA (AGN211745) designed to inhibit VEGFR1 synthesis has been tested in a Phase II trial, but this was terminated presumably because AGN211745 was not formulated for cell permeation. Another approach that is currently undergoing testing involves the blockade of chemokine CC motif receptor 3, which mediates angiogenic signalling initiated by eotaxins92. VEGF binds and activates its receptors VEGFR1, VEGFR2 and VEGFR3. Phosphorylation of the tyrosine residue of the kinase domain of VEGFRs is crucial for receptor activation. Small-molecule tyrosine kinase inhibitors therefore have considerable potential in targeting the kinase domain and blocking the action of VEGFR1, VEGFR2 and VEGFR3. Such inhibitors are usually also active against platelet-derived growth factor receptor (PDGFR), KIT and fibroblast growth factor receptor 1 (FGFR1). Some non-specific tyrosine kinase inhibition and cross-inhibition may be beneficial. As these growth factors are also involved in ocular angiogenesis, they can potentially provide synergistic effects on CNV inhibition. Pazopanib is a tyrosine kinase inhibitor that inhibits VEGFR1, VEGFR2, VEGFR3, PDGFR, KIT and FGFR1. Pazopanib is effective in preclinical models of CNV93. It is administered topically and has shown promising results in a Phase II trial for CNV94. Vatalanib is an oral tyrosine kinase inhibitor that has been tested previously as a treatment for CNV in Phase I and Phase II clinical studies. AL39324 is an intravitreally administered tyro sine kinase inhibitor that is in a Phase II clinical trial in combination with ranibizumab. TG100801 is a tyrosine kinase inhibitor that binds and inhibits VEGFR and PDGFR. It is administered as an eye drop and has been found to suppress CNV and retinal oedema95. A Phase I trial was completed successfully but a Phase II trial was discontinued because of corneal toxicity. NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 547 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Lens Anterior chamber Cornea Iris Inflow • β-adrenergic receptor blockers • α-adrenergic receptor agonists • Carbonic anhydrase inhibitors Trabecular meshwork outflow • Cholinergic agents • Latrunculins • ROCK inhibitors • siRNA β-adrenergic receptor antagonist Uveoscleral outflow • Prostaglandin analogues • α-adrenergic receptor agonists • Prostaglandin EP2 agonists • Nitric oxide-donating prostaglandin F2α analogue • Drug-eluting punctal plug with latanoprost Ciliary processes Ciliary muscle Sclera Current treatments Investigational drugs Figure 5 | Agents that lower intraocular pressure. Current and investigational drugs for lowering intraocular pressure, decreasing aqueous inflow and/or increasing aqueous Nature Reviews | Drug Discovery outflow are indicated on the figure. Aqueous humour is secreted into the posterior chamber by the ciliary processes and eventually circulates into the anterior chamber angle, where it drains from the eye via one or both outflow pathways: the trabecular meshwork or the uveoscleral outflow route. ROCK, RHO-associated protein kinase. An alternative approach to inhibiting the action of VEGF involves preventing the activation of the nicotinic acetylcholine receptor (nAChR) pathway in the vasculature, which inhibits VEGF-induced angiogenesis in endothelial cells96. ATG‑3 is an eye drop formulation of mecamylamine, an antagonist of the nAChR pathway 97. It is the first anti-angiogenic eye drop that has been studied in humans. ATG‑3 has successfully completed two Phase II clinical trials, one in patients with diabetic macular oedema98 and the other in patients with wet AMD who were receiving anti-VEGF treatments. Sphingosine-1‑phosphate (S1P) is the extracellular ligand for S1P receptor 1 (also known as EDG1), a G protein-coupled lysophospholipid receptor, and it is a pro-angiogenic and profibrotic mediator 99,100. Intra vitreous injection of an anti‑S1P monoclonal antibody inhibited CNV formation and subretinal collagen deposition in a preclinical model of laser-induced CNV and in a preclinical model of diabetic retinopathy 101,102. A humanized S1P monoclonal antibody (iSONEP) has completed a Phase I clinical trial in patients with wet AMD. iSONEP was well tolerated, and no drug-related serious adverse events were observed in any of the patients. iSONEP also exhibited positive biological effects (including lesion regression, reduction of retinal thickness and resolution of pigment epithelium detachment). The mechanisms responsible for the positive effects exerted by S1P appear to be independent to those of anti-VEGF agents, indicating the potential of S1P antibodies to serve as a monotherapy (or an adjunct therapy to anti-VEGF agents) for the treatment of neovascular AMD. Endothelial cell migration involves interactions between integrins (transmembrane heterodimeric proteins) and extracellular matrix ligands. For example, α5β1 integrin is expressed on the surface of vascular endothelial cells and mediates cell migration103. JSM6427 and volociximab both bind to α5β1 integrin and block the migration of endothelial cells. Both agents have been shown to inhibit CNV formation in preclinical models104,105. Fosbretabulin is a novel vascular disrupting agent, and its active metabolite is combretastatin A4 (CA4)106. CA4 binds to tubulin and prevents microtubule poly merization in proliferating endothelial cells, thus inducing vessel regression107. In animal models, CA4 has been demonstrated to inhibit retinal neovascularization and suppress the development of CNV108,109. Phase II trials are underway to evaluate the safety and efficacy of fosbretabulin in patients with CNV and vasculopathy. Pericyte recruitment is a crucial step in vascular maturation and stabilization. PDGF has a key role in this process110,111. With this in mind, E10030 — a pegylated aptamer that inhibits PDGF-β — was developed. Inhibition of PDGF results in the stripping of pericytes from endothelial cells, which increases the sensitivity of mature CNV to VEGF inhibition. PDGF inhibition may therefore synergize with VEGF inhibition in CNV treatment. Indeed, in a Phase I dose-escalating study, some CNV regression was observed in over 90% of patients receiving the combination therapy of E10030 and ranibizumab, compared to about 10% of patients receiving ranibizumab alone112. A Phase II study is currently underway. Pharmacologic vitreolysis. Pharmacological interventions for retinal diseases, such as macular oedema and macular holes, that are associated with the vitreoretinal interface (VRI) may soon include one for pharmacologic vitreolysis. The goals of an intravitreal pharmacologic vitreolysis agent are to cleanly cleave the VRI at the internal limiting membrane, removing the potential for fibro cellular traction, and to liquefy the vitreous by altering its molecular organization and structure203. Ocriplasmin (ThromboGenics, Belgium) is currently being investigated as a pharmacologic vitreolysis agent. A stable, truncated form of human plasmin retaining proteolytic activity, ocriplasmin (molecular weight 27.2 kDa) is manufactured using recombinant DNA technology204. Preclinical studies suggest that ocriplasmin targets substrates that are important to both the vitreous structure and the VRI, including collagen fibrils, fibronectin and laminin204. Ocriplasmin was recently studied in two Phase III pivotal clinical trials as a single intravitreal injection for treatment of patients with symptomatic vitreomacular adhesion (VMA), including macular holes. Results from both trials indicated that treatment with ocriplasmin met the primary end point of VMA resolution as well as secondary end points, including visual acuity improvement and closure of full-thickness macular holes in a subset of patients with VMA when compared to placebo. Most adverse events were considered mild and were either transient or temporary in nature, with a majority occurring within the first 7 days after 548 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Table 2 | Available classes of IOP-lowering eye drops for glaucoma Class Drugs Mechanisms of action Notes Aqueous humour outflow (conventional pathway) Cholinergic drugs Pilocarpine, carbachol •Increase trabecular meshwork outflow through ciliary muscle contraction59 •Highly effective but their use is compromised by dim vision (from small pupil) and discomfort59,60 Aqueous humour outflow (uveoscleral pathway) Prostaglandins PGF2α analogues : latanoprost, travoprost, bimatoprost and tafluprost •Increase outflow by •Highly effective and well tolerated: increasing matrix generally used as first-line therapy62 metalloproteinase expression and altering the extracellular matrix in the ciliary muscle and trabecular meshwork61,192 Aqueous humour formation β-adrenergic receptor blockers Timolol, betaxol, carteolol and levobunolol •Decrease inflow by regulating •Few ocular side effects but occasional aqueous humour formation in systemic effects include fatigue and the ciliary processes63 bradycardia62 α-adrenergic receptor agonists Apraclonidine and brimonidine •Decrease inflow by inactivating adenylyl cyclase in ciliary processes and mediating noradrenaline release via activation of pre-synaptic α2-adrenergic receptors; may also increase uveoscleral outflow65 Carbonic anhydrase inhibitors Dorzolamide, •Decrease aqueous humour brinzolamide, formation by inhibiting acetazolamide* and carbonic anhydrase and methazolamide* HCO3 production in the ciliary epithelium64 •Sedation is minimized by occluding nasolacrimal tear duct for up to 2 minutes •These drugs have also been suggested to have neuroprotective effects67 •Allergy is not uncommon, particularly with the 0.2% formulation •Eye drops are better tolerated than orally administered drugs193 Combination of existing classes of IOP-lowering drugs β-adrenergic receptor blockers plus carbonic anhydrase inhibitors Timolol and brinzolamide Decrease inflow196 – β-adrenergic receptor blockers plus α-adrenergic receptor agonists Timolol and brimonidine Decrease inflow and increase outflow197 •Combination product has less hyperaemia than brimonidine alone β-adrenergic receptor blockers plus prostaglandins Timolol and PGF2α analogues Decrease inflow and increase outflow198–202 •Not available in the United States IOP, intraocular pressure; PGF2α, prostaglandin F2α receptor. *Available as oral medications. treatment205. Ocriplasmin is currently under review by the FDA. Other indications may include: vitreous liquefaction and lysis to facilitate complex surgical cases such as paediatric vitrectomy, proliferative vitreoretinopathy and proliferative diabetic retinopathy; relief of vitreous traction on retinal breaks in pneumatic retinopexy; and treatment of macular oedema due to vitreomacular traction. Novel IOP-lowering drugs. In addition to the five major drug classes currently being used to lower IOP (FIG. 5), several novel drugs are being evaluated in clinical trials (FIG. 5; TABLE 4) but results of these studies have not been published. Approved ocular hypotensive drugs with new delivery systems, including drug-eluting punctal plugs (for example, with a prostaglandin analogue), are also being investigated for sustained release and to improve patient adherence. Existing drug classes can be separated into those that lower habitual IOP throughout the day and during the night when administered topically (such as prostaglandin analogues62,113 and carbonic anhydrase inhibitors64), and those that are effective only during the day and not during the nocturnal period (such as beta blockers64 and α-adrenergic receptor agonists65). The relationship of IOP lowering (throughout the day and during the night) to glaucoma progression is unknown and under investigation114. The latrunculins are macrolides from marine sponges that inhibit actin polymerization115. Studies in nonhuman primates and post-mortem analyses of eyes from patients have demonstrated that latrunculins increase trabecular meshwork outflow via a novel mechanism that disrupts the actin cytoskeleton in the trabecular meshwork116. However, their performance in clinical trials has so far been marginal and they have poor solubility. It has been suggested that their efficacy may be improved by changes in their delivery system117. NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 549 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Classical pathway IgM, IgG immune complexes or CRP Lectin pathway Alternative pathway Mannose-binding lectin complex Endogenous or foreign pathogens C3 (H2O) C1 CFB CFH C1 complex + + C2 CFI C3 (H2O)-B CFI + C4 C3 C3 convertase (C4b2a) C3b C5 C3a C6, C7, C8 and C9 C5 convertase C5a C5b Terminal membrane attack complex Figure 6 | Complement pathways, their association with AMD and drug targets. Schematic diagram of classical, alternative and lectin complement activation pathways. Circled proteins have corresponding gene variants that are associated with age-related Nature Reviews | Drug Discovery macular degeneration (AMD). C1, complement component 1; CFB, complement factor B; CFH, complement factor H; CFI, complement factor I; CRP, C-reactive protein; IgG, immunoglobulin G. RHO-associated protein kinase (ROCK) is a downstream effector of RHO (a small guanine triphosphatase) in the RHO-dependent signal transduction pathway. ROCK inhibitors induce changes in the actin cytoskeleton and the cellular motility of the outflow pathways, and they can also lower IOP118. Several drugs in this class have been tested in clinical trials. However, it is still unknown whether the efficacy of these agents will outweigh their side effects, so the use of this class of compounds may be limited by conjunctival hyperaemia and subconjunctival haemorrhages119,120. One strategy for addressing these side effects is to use a pro-drug that is converted into a more active compound once it passes through the cornea and is present in the anterior chamber 121. Such an example is ATS907; the pro-drug form of ATS907 is less active and highly permeable across the cornea. After it passes through the cornea, ATS907 is then converted into a more active ROCK inhibitor in the aqueous humour of the anterior chamber. ATS907 is currently in Phase I/II clinical trials. Other ROCK inhibitors that are currently in Phase II glaucoma trials include AR‑12286 and K‑115. AR‑12286 is currently in a Phase II clinical trial to evaluate its 24‑hour efficacy in patients with open-angle glaucoma or ocular hypertension. K‑115 has completed two Phase II clinical trials, which demonstrated the ability of the compound to lower IOP in patients with primary open-angle glaucoma or hypertension122,123. Amakem also has a ROCK inhibitor (AMA0076) in preclinical development. It is currently unknown whether ROCK inhibitors will be effective when used only once a day or whether they will require administration at least twice a day. In addition, it has not yet been determined whether ROCK inhibitors will be effective at lowering IOP during the night or whether they will be used as primary — rather than adjunctive — therapy. Drugs in this class are also being experimentally evaluated for their neuroprotective potential124. The only ROCK inhibitor that is approved and clinically used is fasudil (Eril; Asahi Kasei). Fasudil is used in Japan for neuroprotection in subarachnoid haemorrhage. ROCK inhibitors can also be useful in trabeculectomy surgery to reduce fibrosis125. Another potential use of ROCK inhibitors is as anti-inflammatory agents, which may be beneficial at ameliorating ocular surface disease and inflammation. Other classes of drugs are also being studied for their ability to lower IOP. Adenosine receptor agonists increase outflow by increasing trabecular meshwork outflow 126. Several drug companies are pursuing strategies involving modulation of the adenosine receptor pathway by targeting different receptors. Examples of such drugs include ATL313 (an adenosine A2A receptor agonist), INO8875 (an adenosine A1 receptor agonist) and OPA‑6566. However, these drugs may have limited use owing to desensitization of their receptors. The angiotensin II type 1 receptor antagonist olmesartan is being tested in Japan for its ability to lower IOP by increasing outflow 127. Cannabinoid receptor agonists128 are also being tested. The evidence for their use in glaucoma is based on the observation that smoking marijuana transiently lowers IOP129. In a non-human primate model of glaucoma, topical application of a cannabinoid receptor agonist that is selective for cannabinoid receptor 1 lowered IOP by decreasing aqueous humour flow 128. A cannabinoid receptor-selective agonist that lowers IOP might be better tolerated and have a longer ocular hypotensive duration of action than marijuana. SYL040012, a siRNA targeting the β2‑adrenergic receptor, is currently in clinical trials to investigate its effect on IOP in patients with ocular hypertension130. PF‑04217329 is a selective agonist of prostaglandin E receptor 2 that has completed Phase II clinical trials. The results showed that PF‑04217329 significantly reduces IOP in patients with primary open-angle glaucoma and ocular hypertension, but doses higher than 0.015% were associated with a higher incidence of photophobia and iritis131,132. BOL‑303259‑X (latanoprostene bunod) is a nitric oxide-donating prostaglandin F2α analogue. A Phase II clinical trial is currently underway to compare the safety and efficacy of BOL‑303259‑X with latanoprost in patients with glaucoma or ocular hypertension. The L-PPDS (Latanoprost Punctal Plug Delivery System) study 133 by QLT recently completed a Phase II clinical trial and latanoprost was found to be well tolerated; adverse events observed were similar to those reported for punctal plugs (for example, tearing). In addition, the L‑PPDS was shown to significantly lower IOP133. 550 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Table 3 | Anti-angiogenic drugs in clinical trials Drug Company Description Target Clinical phase ClinicalTrials.gov identifiers Sirolimus (also known as rapamycin) MacuSight/ Santen Tyrosine kinase inhibitor mTOR Phase I/II NCT01445548 Phase I/II NCT00766649 Phase II NCT00656643 RAD-001 (everolimus) Novartis Tyrosine kinase inhibitor mTOR Phase II NCT00304954 Palomid 529 Paloma Tyrosine kinase inhibitor mTOR Phase I NCT01033721 Phase I NCT01271270 PF‑655 (also known as PF‑04523655) Pfizer Synthetic siRNA DDIT4 Phase II NCT01445899 AdGVPEDF.11D GenVec Adenovirus vector containing PEDF Endothelial cells Phase I NCT00109499 (completed) AAV2-sFLT01 Genzyme Adeno-associated virus (AAV) vector carrying VEGFR VEGF Phase I NCT01024998 Pazopanib GlaxoSmithKline (United States) Tyrosine kinase inhibitor VEGFRs Phase II NCT01362348 Phase II NCT01134055 PTK787 Novartis Oral tyrosine kinase inhibitor VEGFRs Phase I NCT00138632 (completed) AL-39324 Alcon Tyrosine kinase inhibitor VEGFRs Phase II NCT00992563 (completed) ATG‑3 CoMentis Eye drop formulation of mecamylamine Nicotinic acetylcholine receptor Phase II NCT00536692 (completed) Phase II NCT00607750 (completed) Monoclonal antibody Sphingosine‑1phosphate Phase II NCT01414153 Phase I NCT01334255 iSONEP (sonepcizumab) Lpath JSM6427 Jerini Ophthalmic Monoclonal Antibody α5β1 Integrin Phase I NCT00536016 Volociximab Ophthotech Corporation Monoclonal Antibody α5β1 Integrin Phase I NCT00782093 Fosbretabulin OXiGENE Combrestatin A4 phosphate Microtubule assembly Phase II NCT01423149 (completed) E10030 Ophthotech Corporation Pegylated aptamer Platelet-derived growth factor-β Phase II NCT01089517 DDIT4, DNA damage-inducible transcript 4 protein; mTOR, mammalian target of rapamycin; PEDF, pigment epithelium-derived factor; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor. Neuroprotective agents. Both retinal diseases and glaucoma are neurodegenerative disorders in which a common end point is the apoptosis of retinal neurons. Therefore, the use of agents that confer neuroprotection is a very appealing therapeutic approach. Stimulation of glutamate receptors, interleukin‑1 receptors (IL‑1Rs), JUN receptors and tumour necrosis factor receptors (TNFRs) triggers retinal neurons to undergo apoptosis through a cascade of cellular signalling events134. These signals include activation of the pro-apoptotic proteins BCL‑2 antagonist of cell death (BAD), BH3‑interacting domain death agonist (BID) and BCL‑2‑associated X protein (BAX), which then promote the release of cytochrome c and activate the caspase pathways. The anti-apoptotic mechanisms can also be stimulated through neurotrophin signalling pathways, including pathways involving brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial cell-derived neurotrophic factor (GDNF), resulting in the inhibition of the pro-apoptotic cascade through signalling molecules such as AKT, B cell lymphoma 2 (BCL‑2), BCL-XL as well as X‑linked inhibitor of apoptosis (XIAP)134. To date, only a few clinical trials (TABLE 5) have been carried out to investigate the efficacy of neuroprotective agents in glaucoma (for example, memantine (Namenda; Forest/Lundbeck) and brimonidine (Alphagan; Allergan)) and in AMD (for example, brimonidine, AL‑8309B, CNTF and RN6G). These are discussed in more detail below. AL‑8309B is a topical selective agonist of the 5‑hydroxy tryptamine 1A receptor (5‑HT1A). 5‑HT1A agonists have been shown to have neuroprotective effects against NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 551 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Table 4 | Other novel glaucoma drugs and possible mechanisms of action Drugs Companies Latrunculins Mechanisms of action Clinical phase Clinical trial identifiers Refs Wisconsin Increase outflow by altering actin Alumni Research cytoskeleton and increasing Fund (WARF) trabecular meshwork outflow Preclinical NA 116 SYL040012 Sylentis β-adrenergic receptor antagonist; small interfering RNA that reduces aqueous humour inflow Phase II NCT01227291 130 PF‑04217329 (also known as taprenepag) Pfizer Prostaglandin E receptor 2 agonist; increases outflow Phase II NCT00934089 (completed); NCT00572455 (completed) 194 BOL‑303259‑X NicOx (with Bausch & Lomb) Nitric oxide-donating prostaglandin F2α analogue; increases outflow by increasing uveoscleral outflow Phase II NCT01223378 195 Latanoprost Punctal Plug Delivery System (L-PPDS) QLT Sustained delivery of latanoprost by the plug increases outflow Phase II NCT01481051 133 INO‑8875 Inotek Pharmaceuticals Adenosine receptor agonist; increases outflow via trabecular meshwork Phase I/II NCT01123785 126 OPA‑6566 Acucela/Otsuka Adenosine receptor agonist; increases outflow via trabecular meshwork Phase I/II NCT01410188 126 ATL313 Santen Adenosine receptor agonist; increases outflow via trabecular meshwork Preclinical NA 126 Angiotensin II type 1 receptor antagonists (olmesartin, candesartin and losartin) Santen Increase outflow and may also have neuroprotective effects on retinal ganglion cells Preclinical NA 127 Cannabinoid receptor agonist Investigational Decrease aqueous humour outflow Preclinical NA 128 Aerie ROCK inhibitor; alters actin cytoskeleton and cellular motility of outflow pathways Phase II NCT01330979 118 Phase II NCT01474135 ROCK inhibitors AR‑12286 ATS907 Altheos ROCK inhibitor; alters actin cytoskeleton and cellular motility of outflow pathways Phase I/II NCT01520116 118 AMA0076 Amakem ROCK inhibitor; alters actin cytoskeleton and cellular motility of outflow pathways Preclinical NA 118 K‑115 Kowa ROCK inhibitor; alters actin cytoskeleton and cellular motility of outflow pathways Phase II JapicCTI‑101015 (completed) 118 Phase II JapicCTI‑090708 (completed) NA, not available; ROCK, RHO-associated protein kinase. excitotoxic neuronal damage in animal models of central nervous system injury 135,136. However, the mechanisms underlying the neuroprotective properties of 5‑HT1A agonists remain elusive. Several putative mechanisms that have been identified include inhibition of caspase 3 and activation of the mitogen-activated protein kinase (MAPK) signalling pathway 137, which leads to increased expression of anti-apoptotic proteins (for example, XIAP, BCL‑2 and BCL-XL)138. AL‑8309B exhibited neuroprotective effects in animal models of excitotoxic neuronal damage and light damage, in which it reduced the incidence of neuronal death139. A 2‑year, multicentre, randomized, double-masked, placebo-controlled Phase III clinical trial (the Geographic Atrophy Treatment Evaluation (GATE) trial) is currently underway to investigate the effects of topical AL‑8309B administration (at doses of 1.0% and 1.75%) in AMD patients with geographic atrophy; the trial is expected 552 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS Table 5 | Neuroprotective agents in clinical trials Drugs Company Description and mechanism of action Indication (clinical phase) ClinicalTrials. gov identifier Refs AL‑8309B Alcon Selective 5-HT1A agonist; neuroprotective against excitotoxic neuronal damage Geographic atrophy (Phase III) NCT00890097 (completed) 139 Brimonidine tartrate (intravitreal implant) Allergan α-adrenergic receptor agonist; promotes the production of neurotrophic factors such as CNTF, and protects photoreceptors in animal models of retinal degeneration caused by light damage of photoreceptor cells Geographic atrophy (Phase II) NCT00658619 140 RN6G Pfizer Monoclonal antibody; prevents accumulation of amyloid-β40 and amyloid-β42, thus preventing damage to retina AMD (Phase I) NCT01003691 141,142 CNTF Neurotech Neurotrophic factor; protects photoreceptors from degeneration, slowing progression of vision loss Geographic atrophy (Phase II) NCT01408472 94, 143–150 Memantine Allergan NMDA glutamate receptor antagonist; protects retinal ganglion cells and their target (the relay neurons) in the lateral geniculate body Primary open-angle glaucoma (Phase III) NCT00168350 (completed) 44 5-HT1A, 5-hydroxytryptamine 1A receptor; AMD, age-related macular degeneration; CNTF, ciliary neurotrophic factor; NMDA, N-methyl-d-aspartate. to be completed in 2012. The primary efficacy end point is the rate of progression to geographic atrophy. Another promising approach is the use of brimon idine tartrate (delivered through an intravitreal implant), which is an α2‑adrenergic receptor agonist that was originally designed to lower IOP in the treatment of glaucoma. Although the mechanism of action of the compound is largely unknown, brimonidine was shown to promote the production of neurotrophic factors (such as CNTF) and protect photoreceptors in animal models of retinal degeneration induced by light damage140. Brimonidine tartrate has been formulated as an intravitreal implant for delivering brimonidine to the retinal tissue over a period of 3 months. A Phase II study to evaluate the safety and efficacy of the compound is expected to be completed in 2012. The primary end point is the change from the baseline in the surface area of geographic atrophy after 1 year. RN6G (also known as PF‑4382923) is a humanized monoclonal antibody that is under development for the treatment of Alzheimer’s disease. It targets the carboxyl termini of amyloid‑β40 and amyloid‑β42. Intravenous treatment with RN6G is intended to prevent the accumulation of amyloid‑β40 and amyloid‑β42, and to prevent their cytotoxic effects141. Amyloid-β is present in the eyes of patients with AMD; systemic treatment with RN6G decreased the amount of amyloid-β in the eyes of a mouse model of AMD and prevented damage to the retina142. A Phase I clinical trial has been successfully completed. CNTF, which protects photoreceptors from apoptosis during retinal degeneration143, also has potential as a treatment for geographic atrophy. Although the intrinsic function of CNTF is not fully understood, exogenous CNTF affects the survival and differentiation of various cells in the nervous system, including retinal cells144,145. The CNTF study used an implanted semipermeable capsule that encapsulates genetically modified RPE cells that overexpress CNTF. RPE cells are encapsulated within a semipermeable membrane with small pores, which allows the release of CNTF into the vitreous yet prevents the allogeneic rejection of RPE cells. CNTF effectively protected photoreceptors in 12 animal models of photoreceptor degeneration94,146–149. A multicentre, 1‑year, double-masked, sham-controlled dose-ranging study found that CNTF delivered by the encapsulated cell technology (ECT) implant appears to slow the progression of vision loss in patients with geographic atrophy, especially in patients with 20/63 vision or better vision on an eye chart at the baseline150. Degeneration and functional loss of the glaucomatous optic nerve was once thought to be primarily due to diffuse RGC injury; however, it has recently been proposed that this form of neurodegeneration appears to be compartmentalized, thus affecting neuronal processes well before the RGC body in the inner retina150. This has important implications for the development of neuroprotective therapies for glaucoma, as functional changes or axonal dystrophy may be more amenable to treatment than the replacement of lost RGC bodies or long stretches of their axons in the optic nerve or their projections into the brainstem151. Excessive activation of the NMDA (N‑methyld‑aspartate) receptor signalling cascade leads to excitotoxicity, wherein intracellular calcium overloads RGCs and other neurons, causing cell death through apoptosis. Oral administration of memantine to non-human primates with experimentally induced glaucoma conferred protection of RGCs and their target — the relay neurons in the lateral geniculate body 44. A Phase III clinical trial has been completed to investigate the efficacy of the NMDA glutamate receptor antagonist memantine in glaucoma NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 553 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS neuroprotection (protection of the structure and function of the optic nerve to halt glaucoma progression independently of lowering IOP). However, memantine failed to reach the primary efficacy end point of reducing visual field loss in patients at a high risk of developing glaucoma. Data from this study have not yet been reported. The efficacy of topical brimonidine in preserving visual field loss in glaucoma has also been investigated. Brimonidine is approved by the FDA for lowering IOP in glaucoma, and systemic administration of the drug has been reported to protect RGCs in an experimental rat model of ocular hypertension152. In the low-tension glaucoma treatment study, patients who were randomized to receive topical brimonidine monotherapy had less visual field loss than those who were randomized to receive topical timolol (Timoptic; Merck & Co.), even though both drugs invoked similar IOP-lowering effects153. Although it has been suggested that this result is consistent with the neuroprotective effects of topical brimonidine, there are several important limitations of the study, including the considerably small sample size, the large number of dropouts in the brimonidine group and the possibility that the patients who were treated with brimonidine were unmasked as they often had conjunctival hyperaemia. Therefore, although the results are intriguing, they do need corroboration. The brimonidine tartrate intravitreal implant that is being investigated for AMD might also be useful for treating glaucoma, particularly if it is used for intracameral drug delivery to lower IOP and provide neuroprotection in glaucoma, but such studies have not yet been undertaken. Potential future therapeutic approaches Target discovery through genomic research. A crucial role of genetic variation in the occurrence and development of primary open-angle glaucoma has been affirmed by the varying prevalence of primary open-angle glaucoma observed in different populations, family aggregation trends154,155 and in twin studies. Some populations have a higher prevalence of primary open-angle glaucoma than others (for example, the prevalence of the disease in individuals of African ancestry is several-fold higher than in those of European ancestry). This suggests that genetic background may be a primary cause of the disease. A growing number of studies, including genomewide association studies, have identified glaucoma susceptibility loci for monogenic and common primary open-angle glaucoma (reviewed in REFS 156,157). Genes that are involved in cell cycle control and transforming growth factor-β (TGFβ) pathways have emerged as the main risk loci for primary open-angle glaucoma158,159. However, it is not clear at present how these findings could be translated into therapies. There is increasing evidence that genetic susceptibilities contribute to the development of diabetic retinopathy. For instance, the frequency of diabetic retinopathy varies among different ethnicities and its prevalence is higher in individuals with a positive family history 160–162. Identifying genes (such as erythropoietin)163 that are associated with diabetic retinopathy could lead to the development of targeted treatment strategies in the near future. AMD is an excellent example in which novel target discoveries have been achieved through genomic research. More than ten genes have been identified for AMD, which point to several new pathways that may be involved in the pathogenesis of AMD and could be used to predict the risk of developing the disease164. These genes encode proteins such as hepatic lipase and apolipoprotein E (which are involved in lipid metabolism)165,166, proteases such as HTRA1 (REFS 27,40) as well as components of the complement pathway. Owing to multiple gene associations in each of these pathways, various drugs targeting these pathways (for example, the alternative complement pathway) are now in clinical trials. Gene therapy. Gene therapy has been an active area of clinical investigation. It can provide long-term, sustained effects with a single injection; however, reversibility needs to be addressed as long-term continuous blockade may not be desirable. Various ocular gene therapy trials have been highly successful in treating retinal degeneration167–169. An AAV2‑mediated gene transfer of the MER receptor tyrosine kinase (MERTK) gene rescued retinal degeneration resulting from a MERTK mutation in RCS rats170, and a Phase I clinical trial is underway in patients with recessive MERTK mutations. TT30 is a recombinant CFH fusion protein that is under development for replacement therapy of defective CFH (see the Taligen Therapeutics website). Knockdown or silencing of CFHrelated 1 (CFHR1) by preventing its mRNA expression might be useful for the treatment of AMD because deletion of CFHR1 protects against AMD independently of CFH modulation itself 171,172. Stem cell biology and therapy. More recently, there has been increasing interest in the potential use of stem cell therapy to achieve ocular tissue repair and/or regeneration173,174. The eye is a particularly attractive target organ for stem cell therapy as it is in a contained intraocular space and is therefore associated with minimal systemic safety concerns and immune reactions. The effect of stem cell therapy can also be monitored through non-invasive diagnostic tools such as optical coherence tomography. These stem cell advancements include our considerably improved ability to manipulate and control stem cell fate and function in a more precise and defined fashion, as well as new approaches to reprogramme somatic cells towards various tissue-specific lineages173,175. For example, RPE can be derived from patients with geographic atrophy who have a high genetic risk genotype, and this RPE can be subjected to an in vitro high-throughput screen to identify small molecules that can prevent the death of RPE cells or enhance their survival173. Ultimately, it is possible that conventional small-molecule or biological therapeutics may be developed that could enable one’s own cells to regenerate in vivo to alleviate damage caused by diseases and injuries. Advanced Cell Technology has produced human embryonic stem cell-derived RPE cells and has received FDA approval for their use in clinical trials. Two combined, multicentre, open-label Phase I/II trials are currently underway, using RPE cell subretinal transplantation 554 | JULY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS to treat patients with Stargardt macular dystrophy and geographic atrophy. These trials are prospective openlabel studies to determine the safety and tolerability of transplanted RPE cells. Preclinical studies in animals showed that transplanted RPE cells preserved overlying photoreceptors, slowed the progression of degeneration and improved visual functions176. StemCells has recently filed an investigational new drug application with the FDA to initiate a Phase I/II multicentre study using adult-derived neural stem cells to treat patients with geographic atrophy. Ocular drug delivery Despite advances made in the discovery of novel targets and an understanding of their mechanisms of action, drug delivery to these targets — especially when it is to the posterior eye — is continually challenged by anatomical and physiological constraints, particularly the blood– ocular barriers and an array of efflux transporters. As a result, therapeutic agents that are systemically administered are inevitably associated with adverse systemic exposure and unsatisfying efficacy 177. Although using intraocular or periocular injections can substantially lower systemic exposure when delivering therapeutic entities to the retina, poor patient compliance and the high risk of complications (including retinal detachment, endophthalmitis and increased IOP) have limited the frequency of administration via these routes178. For example, anti-VEGF therapies are highly effective in treating wet AMD, diabetic macular oedema and retinal vein-occlusive diseases, but a monthly intraocular injection is not an ideal drug delivery platform and it is hard to maintain patient compliance. Overall, these challenges have hindered the use of many novel therapeutic agents, thus emphasizing the urgency to develop novel drug delivery strategies for the eye. Among various approaches to optimize drug delivery, ECT and applications of nanomedicine can have essential roles. Nanotechnology The understanding and control of matter at dimensions between approximately 1 nm and 100 nm. It involves imaging, measuring, modelling and manipulating matter at this length scale. Nanoparticle drugs Nanometre-scale particles that are used to carry and transport pharmaceutical agents to improve therapeutic efficacy, drug safety and patient compliance. Encapsulated cell technology. ECT is a cell-based delivery system that is used to treat chronic disorders of the eye179. In this technology, specific cells are first genetically engineered to overexpress desired therapeutic proteins, and are then encapsulated into semipermeable capsules that facilitate the diffusion of nutrients and proteins but prevent attack by the host immune system. For treatment, the capsules are surgically implanted into target sites, where the cells will survive and release proteins. As any gene encoding a therapeutic protein can now be engineered into cells, ECT has a broad range of applications. The implantation of cell-loaded capsules also allows for controlled, continuous and long-term administration of therapeutic proteins to the proximity of the eye in situ with no systemic exposure. The implant can also be retrieved, providing an added level of safety 180. ECT has advanced into the clinic as a result of an improved understanding of the mechanisms of protein functions related to visual impairments, coupled with advances in genetic engineering and implant technology 181. In particular, encapsulated human cells that are genetically modified to secrete CNTF (for the treatment of geographic atrophy associated with dry macular degeneration and retinitis pigmentosa) have advanced to Phase II clinical trials, with promising results150. ECT can also be extended to other ophthalmic diseases, depending on the secreted protein. For example, the delivery of alginate-encapsulated cells producing glucagon-like peptide 1 (GLP1) resulted in the intraocular production of GLP1 without causing the obvious cytotoxic effects in animal studies182. Meanwhile, a Phase I clinical trial that is currently being planned by Neurotech involves the implantation of encapsulated human cells that are genetically modified to deliver a structural antagonist of VEGF for the treatment of wet AMD150. Nanomedicine. Nanomedicine, which exploits nanotechnology to solve medical challenges, has offered numerous exciting possibilities in health care. In particular, nanoparticle drugs have become established therapeutics by formulating nanometre-scale carriers composed of various therapeutic entities (including small-molecule drugs, peptides, proteins and nucleic acids), together with excipients such as lipids and polymers that assemble with these therapeutic entities183,184. Although nanoparticle therapeutics were initially developed for cancer treatment, their application has expanded into ophthalmology owing largely to their unique capability of enhancing drug efficacy — through improved drug encapsulation, sustained or triggered drug release, and preferential targeting to disease sites185. Some nanoparticle therapeutics have been clinically approved for treating ophthalmic diseases. For example, pegaptanib — an anti-VEGF aptamer conjugated with branched polyethylene glycol — has been approved for the treatment of AMD186. In addition, nanomedicine has provided new opportunities for overcoming obstacles in treating ophthalmic diseases. For example, by combining synthetic chemistry with a basic understanding of protein–polymer interactions, nanotechnology has led to the efficient encapsulation and controlled delivery of proteins within biological fluid under mild conditions187. These technologies are particularly promising as the therapeutic potential of proteins and monoclonal antibodies is increasingly becoming realized in ophthalmology, particularly for halting retinal angiogenesis188. In addition, nanotechnology has resulted in the formation of sophisticated carriers that are particularly suitable for the delivery of therapeutic agents to the cytoplasm for bio activity 189. Technology for intracellular delivery, if available, can be used in siRNA-based therapeutics to selectively silence gene expression for the treatment of ophthalmic diseases. Moreover, nanotechnology has provided new opportunities for drug delivery, with the goal of attaining prolonged ocular drug retention. In particular, approaches that are under active investigation include the incorporation of bioadhesive components to formulate multifunctional nanoparticle drug reservoirs that can be attached to the corneal tissue with a high affinity 190. Furthermore, the recent development of polymeric nanoparticles that NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | JULY 2012 | 555 © 2012 Macmillan Publishers Limited. All rights reserved REVIEWS are camouflaged by the red blood cell membrane allows man-made delivery vehicles to share functionalities that have been engineered and perfected by nature, hence taking another step towards bridging synthetic materials with natural components191. These biomimetic nanoparticles — that have excellent biocompatibility and well-controlled drug release kinetics — will have many applications in the treatment of ophthalmic diseases. Conclusions Currently, anti-VEGF therapies are highly effective in treating wet AMD, diabetic macular oedema and retinal vein-occlusive diseases, and have become the first-line treatment for these conditions. However, there are still several common blinding ophthalmic disorders, such as geographic atrophy, for which no effective therapies are currently available. 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Shaw for their helpful comments. K.Z. is supported by grants from the Chinese National 985 Project to Sichuan University and West China Hospital, the National Eye Institute and the US National Institutes of Health, VA Merit Award, Research to Prevent Blindness, King Abdulaziz City for Science and Technology (through the University of California San Diego Center of Excellence in Nanomedicine centre grant) and the BWF (Burroughs Wellcome Fund) Clinical Scientist Award in Translational Research. L.Z. is supported by the National Science Foundation (NSF) grants CMMI1031239 and DMR1216461; R.N.W. is supported by grants from the National Eye Institute and the US National Institutes of Health (EY019692) and an unrestricted grant from Research to Prevent Blindness, New York, USA. We apologize for the omission of references owing to page limitations. Competing interests statement The authors declare competing financial interests: see Web version for details. FURTHER INFORMATION Kang Zhang’s homepage: http://eyesite.ucsd.edu/retina/KZL/index.html Taligen Therapeutics website: http://www.taligentherapeutics.com/pipeline/index.html Webvision website: http://webvision.med.utah.edu ALL LINKS ARE ACTIVE IN THE ONLINE PDF VOLUME 11 | JULY 2012 | 559 © 2012 Macmillan Publishers Limited. All rights reserved