disulfide bond formation. Accumulation of misfolded protein aggregates results in endoplasmic reticulum stress and inhibition of the proteosomal degradation pathway, ultimately resulting in cell death. Chaperones defend against this by binding to hydrophobic protein surfaces and inducing refolding. Should the protein fail to fold properly, it is ubiquitinated and degraded via the proteasome. Damage ensues when this process is overwhelmed. Rhodopsin is an example of such a class of protein which aggregates when mutated. It has the curious property that almost any change in the amino acid sequence results in its inability to fold properly. Over 140 mutations of rhodopsin which cause RP have been reported (OMIM 180380, http://www.sph.uth. tmc.edu/RetNet). Rhodopsin is present in such great quantities that misfolded product easily overwhelms cellular rescue pathways leading to the unfolded protein response and ultimately, cell death. Another example is the transthyretin gene which serves as an amyloid precursor protein. Mutations in this gene cause deposition within the vitreous as well as other organs (Benson and Kincaid 2007).

Restoration of missing growth factors is currently an attractive therapeutic strategy for treating degenerative diseases. Of these only CNTF is currently being utilized. A second area that is becoming of interest is that of increasing chaperone proteins.

21.3  Current Therapies for Key Back of the Eye Disorders

21.3.1  Age-Related Macular Degeneration

21.3.1.1  Pathophysiology

Visual disturbances are present in approximately 5% of the population over 70 years of age. Examination of the eyes of this population reveals areas of depigmentation within the region of the macula. These areas are referred to as drusen. They can be discrete (hard) or diffuse (soft). Photoreceptor degeneration is often detected in these areas. In the presence of hard drusen, the degenerating photoreceptors sit over well-defined eosinophilic mounds that lie just below the RPE. In the case of soft drusen, the degenerating photoreceptors are arranged in linear bands. In a subset of patients, new capillaries begin to form within the choroid layer which can invade outward past the RPE and into the retina. These capillaries are quite leaky and result in edema and hemorrhage. In the presence of this neovascularization, the pathology is referred to as wet AMD. In the absence of neovascularization, the pathology is referred to as dry AMD.

AMD demonstrates the interplay between many of the pathological forces discussed at the beginning of the chapter. Photons have sufficient energy to produce free radicals such as hydrogen peroxide and superoxide anions. While these reactive oxygen species (ROS) are rapidly inactivated by compounds present in the eye such as superoxide dismutase and glutathione, over the course of many decades, it is thought that the buildup of retinal damage can initiate the process of AMD. The standard reaction to ROS-mediated damage is the initiation of an inflammatory response.

It is clear from the presence of complement proteins within drusen that an inflammatory process is ongoing (see Sect. 21.2.1). The buildup of drusen in conjunction with the deposition of collagen within the RPE layer along with ROS-mediated damage may place sufficient stress on photoreceptor neurons such that they atrophy. Additionally, the deposition of foreign material may be sufficient to create an ischemic environment resulting in the production of VEGF and the initiation of neovascularization.

21.3.1.2  Therapeutics Either in Current Use or in Clinical Trials

Including all clinical trials devoted to AMD in the clinicaltrial.gov database is beyond the scope of this chapter. Instead we have chosen to focus here on selected targets. A list of some of the therapeutics undergoing trials is given in Table 21.1.

AREDS formulation:  The National Eye Institute (NEI) sponsored a clinical trial in 2001 to examine the efficacy of high dose antioxidants and zinc in slowing or preventing the progression of AMD (http://www.nei.nih.gov/amd/). This trial, called AgeRelated Eye Disease Study (AREDS), found that a combination of vitamin C, vitamin E, beta-carotene, and zinc lowered the risk of vision loss by 25%. Various formulations of antioxidants with or without zinc as well as zinc alone are available as over- the-counter medications. A recent study derived from the AREDS study looked at omega-3 long-chain polyunsaturated fatty acids (PUFA) (SanGiovanni et al. 2009a; Sangiovanni et al. 2009b). Omega-3 PUFA intake was found to inversely correlate with the risk of developing neovascular AMD. Indeed, patients who took in the largest amounts of this fatty acid (0.11% of total energy intake) had a 30% decreased risk. Retinal cells have an extraordinarily high content of PUFAs which play an important role in the visual process. They serve as precursors to numerous signaling molecules including neuroprotectins and resolvins which function to provide anti-inflammatory, neurotrophic, and cytoprotective effects (SanGiovanni and Chew 2005).

Photodynamic Therapy (PTD):  Laser coagulation, developed in the 1990s, represented a first crude attempt to physically suture leaking vessels and stop neovascularization. Recurrence was common and resulted in even greater loss of visual acuity. PTD represents the next step in refining this technology. The technique relies on the compound verteporfin to preferentially bind to plasma lipoproteins and be taken up by low density lipoprotein (LDL) receptors. Vascular endothelium comprising new vessels express high levels of LDL receptors causing them to preferentially take up the compound. Upon exposure to a specific wavelength of light, a photosensitizer that is part of the verteporfin molecule initiates a reaction that produces large amount of free radicals. These free radicals damage the vascular endothelium and induce clotting and occlusion of the pathological vessels. The TAP (treatment of AMD with PTD) study (Bressler 2001) and the VIP (verteporfin in PTD) study (Verteporfin-In- Photodynamic-Therapy-Study-Group 2001) demonstrated a clear decrease in vision loss as compared to controls. The technique was approved by the FDA in 2000 and is often used for comparison in more recent studies.

512

Table 21.1  (continued)

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PTD is also being tested as an adjuvant therapy along with anti-VEGF formulations (described below) in several current trials (for example, NCT00413829 and NCT 00433017). Several recent clinical trials have examined the combination of verteporfin with the corticosteroid: TA (Chan et al. 2007; Chaudhary et al. 2007; Gilson et al. 2007; Weigert et al. 2008; Katome et al. 2009; Maberley 2009). Although visual acuity was not improved by the presence of the corticosteroid, the number of times the patient needed to be re-treated was significantly reduced. A similar strategy has been envisioned for non-steroidal anti-inflammatory drugs (NSAIDs), which have a better safety profile as compared to corticosteroids. Unfortunately, a trial examining diclofenac sodium in this context did not show benefits (Boyer et al. 2007). A similar trial examining orally administered celecoxib was completed by the NEI in recent years (Chew et al. 2010). The trial indicated that patients receiving celecoxib were more likely to have a reduction in fluorescein leakage compared to the placebo group. However, no large visual function benefits were observed when compared to standard laser treatment. It is possible that local sustained delivery of celecoxib might be more beneficial in reducing vascular leakage and possibly visual function (Amrite et al. 2006).

VEGF signaling:  VEGF signal transduction currently represents the primary target for antiangiogenic therapeutic interventions. Currently, three VEGF inhibitors have received FDA approval and are in clinical use. Pegaptanib and ranibizumab have been approved for the treatment of the neovascular (wet) form of AMD. Bevacizumab is used to treat AMD as an off label application.

Pegaptanib (Macugen; Eyetech Pharmaceuticals, Inc.) is a pegylated RNA aptamer specific for the VEGF165 isoform of VEGF-A, thought to be the major ligand for VEGFR2. It was approved by the FDA in December 2004 for intravitreal treatment of subfoveal neovascular AMD. Pegaptanib binds close to the heparin binding domain within VEGF-A and blocks the ability of VEGF to associate with its receptor. Preclinical studies demonstrated that pegaptanib decreased vascular permeability, VEGF-induced corneal angiogenesis, and leukocyte adhesion (Eyetech-Study-Group 2002; Ishida et al. 2003). The VISION study, a combination of two concurrent clinical trials found that intravitreous pegaptanib injection (0.3 mg) provided modest protection with little to no safety concerns (Gragoudas et al. 2004; Chakravarthy et al. 2006). The endpoint for this study was the loss of less than 15 letters of visual acuity (three lines of the ETDRS chart at a distance of 2 m). By 12 months approximately 70% of patients were within this endpoint group. Both pegaptanib and sham operation groups deteriorated. However, the pegaptanib group experienced deterioration at a slower rate over a 2-year period.

Ranibizumab (Lucentis; Genentech, Inc.) is a humanized antibody Fab fragment that binds to all isoforms of VEGF-A. It was approved by the FDA in June 2006 for the treatment of wet AMD. Humanization was achieved by identifying the six complementarity determining regions of the mouse antihuman VEGF antibody (muMAb VEGF A.4.6.1) and cloning them into a human immunoglobulin framework (Presta et al. 1997). Ranibizumab binds to a conserved region of VEGF found in all isoforms (Kim et al. 1992; Chen et al. 1999), giving it an expanded range of activity as

compared to pegaptanib which only binds to the VEGF165 isoform. Two pivotal clinical trials established ranibizumab as a revolutionizing therapy for AMD. The MARINA trial recruited 716 subjects that were randomized to receive intravitreal injections of either 0.3 mg ranibizumab, 0.5 mg ranibizumab, or sham injection (Rosenfeld et al. 2006). At 12 months over 94% of patients remained in the group that had lost fewer than 15 letters. By 2 years, 90% of ranibizumab treated patients retained this level of acuity while only 52.9% of sham-operated patients retained vision at this level. Remarkably, approximately one-third of ranibizumab treated patients achieved an improvement of 15 letters of vision by 12 months as compared to 5% in the sham-operated group. By 12 months the average acuity in the ranibizumab treated group was 17 letters improved over the sham-operated group and this improvement increased to 20 letters at 2 years. A second clinical trial (the ANCHOR trial) compared intravitreal ranibizumab injection to PDT in 423 subjects (Brown et al. 2006). Patients received either monthly injections of ranibizumab (0.3 or 0.5 mg) along with sham PTD or else monthly sham injections and standard PDT. By 12 months, greater than 35% of the ranibizumab injected group showed a 15 letter improvement as compared to 5.6% of the PDT group. The ranibizumab group, as a whole, experienced a mean improvement of 8.5 and 11.3 letters of acuity (for the 0.3 mg and 0.5 mg dose, respectively) while the PDT group, as a whole, experienced a mean loss of 9.5 letters of acuity. At 2 years, average visual acuity was improved by 8.1 and 10.7 letters, respectively, for the ranibizumab groups and had declined by 9.8 letters in the PDT group (Brown et al. 2009).

Bevacizumab (Avastin; Genentech, Inc.) is a full length humanized monoclonal antibody against VEGF, derived from the same mouse monoclonal as ranibizumab. The FDA approved bevacizumab for use in treating metastatic colorectal cancer in February 2004. It is widely used off label for intravitreal injections for the treatment of wet AMD. Its use in AMD stems from the timing of its availability. Bevacizumab became available over 2 years before the FDA approved ranibizumab. Two uncontrolled clinical trials established that bevacizumab, administered systemically, was efficacious in the treatment of AMD (Michels et al. 2005; Moshfeghi et al. 2006). Due to the development of increased blood pressure in a number of study participants, the same group attempted an intravitreal injection of bevacizumab in a single subject who was not responding well to pegaptanib (Rosenfeld et al. 2005). Strikingly, within a week of administration there was a decrease in subretinal fluid on optical coherence tomography and improved visual acuity. This result propelled intravitreal bevacizumab into the clinic where it remains today, despite the subsequent approval of ranibizumab. A trial comparing ranibizumab with bevacizumab head to head for the treatment of AMD is currently in progress (NCT00710229).

Bevasiranib represents yet another VEGF inhibiting therapeutic which is unique in that it is a small interfering RNA (siRNA) rather than a protein. As with other siRNAs, bevasiranib functions by targeting a specific mRNA (in this case VEGF) for degradation. Bevasiranib has been examined in several small clinical trials (NCT00303904, NCT00557791, and NCT00259753); however, no data have yet been reported. Interestingly, one group reported on a nonspecific antiangiogenic

property of siRNAs through engagement of TLR3 in mice (Kleinman et al. 2008). It is unclear if this will adversely affect the use of siRNA therapeutics in human trials as phase I studies have established a good safety record for these compounds.

Aflibercept (VEGF-Trap Eye; Regeneron) is a fusion of the VEGF ligand binding domains of human VEGFR 1 as well as VEGFR 2 to the Fc portion of human IgG1 (Holash et al. 2002). It was found to bind with high affinity to many members of the VEGF family including placental growth factors 1 and 2. In animal models, aflibercept was found to have a longer half life than ranibizumab after intraocular injections. Systemic administration decreased vascular edema in patients with CNV secondary to AMD but also induced hypertension and proteinuria (Nguyen et al. 2006). A formulation for intravitreal injection (VEGF Trap-Eye, Regeneron) has been produced and is under investigation in a number of clinical trials (http://clinicaltrial.gov/).

Anti-inflammatory therapeutics:  The presence of inflammatory components in drusen clearly indicates that an inflammatory response occurs during the progression of AMD. To address this issue, Alimera Sciences is examining the use of a FA intravitreal insert to provide a long term anti-inflammatory therapy locally to the region of pathology (clinical trial identifier: NCT00695318). The patients being recruited have geographic atrophy. As described above, systemic corticosteroids produce serious side effects upon long-term usage including osteoporosis, poor wound healing, muscle wasting, and a redistribution of body fat (collectively referred to as Cushing’s syndrome). Ocular corticosteroid use leads to glaucoma. The degree to which this intravitreal insert will induce glaucoma remains to be seen in this ongoing study.

The other major class of anti-inflammatory therapeutic agents being considered is NSAIDs. In certain ocular diseases such as cystoid macular edema, prostaglandins are thought to play an important role in disease etiology (Miyake and Ibaraki 2002). One cause of macular edema is ocular surgery. Prostaglandin release, due to tissue injury, results in the degradation of the blood-retinal barrier. Recently, perioperative use of NSAIDs is being used to counter this phenomenon (Colin 2007). These drugs are primarily being considered in combination with more established therapeutics. One attractive aspect of NSAID use is that they may be delivered by the transscleral route, a method that is far less invasive than intravitreal injections and thus less likely to cause adverse events (Amrite et al. 2010). Peter Francis at the Oregon Health and Science University in collaboration with Genentech is examining the combination of Bromfenac ophthalmic drops with ranibizumab intravitreal injections for patients with neovascular (wet) AMD. The trial (NCT00805233) is ongoing. In a separate trial, the National Eye Institute has examined the use of celecoxib in combination with PDT (NCT00043680).

TNFa is another important inflammatory signaling molecule which can be successfully inhibited by anti-TNFa antibodies and related molecules. The primary applications of this class of therapeutic have been in the treatment of rheumatoid arthritis and Crohn’s disease (Feldmann et al. 2010). Small pilot studies have found that TNFa blockade does improve neovascularization secondary to AMD (Markomichelakis et al. 2005; Theodossiadis et al. 2009). Two larger randomized clinical trials have been initiated examining infliximab in AMD. One, sponsored by

the National Eye Institute (NCT00304954), is examining infliximab in comparison to sirolimus and daclizumab. The study was recently completed. However, no results have been posted as of this writing. The second trial, sponsored by the Retina Research Foundation (NCT00695682) is currently recruiting for a safety study.

Neurotrophic factors for photoreceptor survival:  One of the functions of the RPE is to take up all-trans-retinol that is generated in the photoreceptors and, through a series of steps, convert it to 11-cis-retinal. This is taken back up by the photoreceptors and can then be used to regenerate visual pigments (Lamb and Pugh 2004). Fenretinide, a potent inhibitor of the retinoid cycle was found to block the production of lipofuscin in mice (Maeda et al. 2006). This and similar studies has propelled Sirion Therapeutics to investigate the efficacy of fenretinide in the treatment of geographic atrophy. The study (NCT00429936) has been completed but the results are not available as of this writing.

Photoreceptors, given their neuronal origin, are programmed to respond to certain neurotrophic factors that provide survival signals. One such factor being considered by Neurotech Pharmaceuticals is CNTF (Dutt et al. 2010). They have genetically engineered human NTC-201 (genetically engineered ARPE-19) cells to produce CNTF and have encapsulated these cells such that they can be safely implanted into the eye. This ongoing study (NCT00447954) will examine the efficacy of these implants on the treatment of atrophic macular degeneration.

Newer antiangiogenic targets:  Angiogenesis, as discussed extensively earlier, is regulated by a complex web of signals. One additional signal not yet mentioned is acetylcholine. Vascular EC express nicotinic acetylcholine receptors and it has been demonstrated that engagement of these receptors provides an angiogenic signal (Heeschen et al. 2001). CoMentis has initiated a clinical trial (NCT00607750) combining ATG003 (topical mecamylamine) with anti-VEGF therapy. The study is ongoing.

Over 25 years ago, Judah Folkman discovered a class of steroid that had no classic DNA binding activity (steroid mechanism is discussed previously) but instead, interacted with the basement membrane of new blood vessels to inhibit their growth (Folkman and Ingber 1987). He called these molecules angiostatic steroids. Anecortave acetate represents a more recent synthetic molecule designed to enhance this angiostatic property. A large number of clinical trials have either been completed, or are currently underway, examining the efficacy of anecortave acetate in various forms of AMD. In one published study, AMD patients with choroidal neovascularization (CNV) showed similar visual acuity improvements to the control group receiving PDT (Slakter et al. 2006). Although approved for clinical use in Australia, its efficacy was inadequate for regulatory approval in the USA.

Pigment epithelial growth factor (PEDF) is a protein found in the ECM of ocular tissues (Becerra 2006). It has been shown to be downregulated in several neovascular disease states including diabetic retinopathy, AMD, glaucoma, and RP. PEDF has antiangiogenic properties and thus has been examined as a therapeutic agent in numerous ocular animal models as well as in humans. The receptor for PEDF is not yet clearly identified. Indeed, it is thought that PEDF may bind to multiple targets including sulfated and nonsulfated glycosaminoglycans and collagens (Filleur et al. 2009).

Notari and colleagues employed a yeast 2-hybrid screen strategy to discover a PEDF receptor and identified a novel transmembrane protein (PNPLA2) with phospholipase A2 activity from human RPE cells (Notari et al. 2006). A second group performed a similar 2 hybrid screen using a human skeletal muscle library and identified both PNPLA2 as well as the nonintegrin laminin receptor 67LR (Bernard et al. 2009). They additionally demonstrated interaction through co-immunoprecipitation and surface plasmon resonance assays. After mapping the 67LR interaction domain of PEDF to a 34 amino acid peptide (PEDF 46–70), they demonstrated that this peptide was sufficient to block EC tube formation as well as ex vivo retinal angiogenesis. Intravitreal injection of PEDF protein or viral PEDF gene transfer has provided protection in models of OIR as well as CNV (Mori et al. 2001; Duh et al. 2002; Mori et al. 2002a; Mori et al. 2002b; Saishin et al. 2005). Intravitreal injection of PEDF has been shown to delay retinal degeneration in animal models of retinitis (Cayouette et al. 1999; Cao et al. 2001). Similarly, introduction of PEDF by viral gene transfer has also shown to be protective (Miyazaki et al. 2003; Imai et al. 2005). A phase I clinical trial of adenovirus-mediated introduction of PEDF to patients with advanced AMD (NCT00109499) demonstrated some efficacy and minimal toxicity (Campochiaro et al. 2006). Structurally, while PEDF is a member of the serpine (serine protease inhibitor) family, it does not retain protease inhibitor function and the serpine motif does not appear to play a role in PEDF’s therapeutic properties (Amaral and Becerra 2010).

Squalamine is an aminosterol originally isolated from the liver of the dogfish shark and found to have antibiotic properties (Savage et al. 2002). More recently, the compound was found to have antiangiogenic properties and received fast track status by the FDA for the treatment of AMD. Squalamine works at multiple levels to block angiogenesis including inhibition of VEGF and integrin expression as well as cytoskeletal formation (Connolly et al. 2006). Several clinical trials were initiated by Genaera Corporation but subsequently terminated.

During the processes underlying the breakdown of the retinal vascular permeability barrier, fibrous material is also produced. In late stage AMD, the contraction of this material can contribute to retinal detachment. The contribution of this material to decreased visual acuity during wet AMD is currently under investigation in a recently initiated clinical trial (NCT00996684). Microplasmin is a stable fragment of plasmin. It functions by digesting the connections between the vitreous and the retina, effectively performing a nonsurgical posterior vitreous detachment. It is thought that by isolating the retina from the vitreous, the forces of the vitreous will no longer play upon the retina, contributing to decreased visual acuity.

The VEGF receptor is a tyrosine kinase. A VEGFR tyrosine kinase inhibitor, developed by GlaxoSmithKline, has recently been identified and called pazopanib (Podar et al. 2006). As a small molecule, it is expected that ocular accessibility will be improved over the antibody inhibitors of VEGF. Pazopanib has been approved by the FDA for the treatment of renal cell carcinoma. A number of clinical trials testing pazopanib in AMD have been initiated or recently completed but as of this writing no study results have been made available.

In addition to the VEGF blocking agents currently in use, some groups are examining methods to inhibit VEGF secretion. The strategy is to create an intracellular