antibody can impede RPE senescence [146]. TGF-b and BMP-4 may have a synergistic effect in mediating the oxidative stress-induced RPE senescence because neither TGF-b antibodies nor BMP-4 antagonist alone can completely block the expression of senescence marker genes to baseline in the oxidative stress-treated RPE cells [145].

Zhu et al. [147] reported that RPE cells induced into senescence by chronic oxidative stress secrete fourfold higher IL-8 than nonsenescent RPE cells. IL-8 promotes angiogenesis by increasing the proliferation, survival, and migration of endothelial cells and promotes inßammation by increasing neutrophil chemotaxis and degranulation. Senescent heterogeneity combined with the effects of other cytokines (e.g., TNF-a inhibition of BMP-4 expression) may drive some cells to senescence with GA and others to CNV stimulation [147].

A proposed pathogenesis (Fig. 1.4) of AMD suggests the possibility of therapeutic intervention at different points in the natural history of the disease with antioxidants, visual cycle inhibitors, anti-inßammatory agents, antiangiogenic agents, and neuroprotective agents.

1.3Treatment

Various pathway-based therapies for AMD have been reviewed extensively elsewhere [148]. Here we update some of this information.

1.3.1Antioxidants

The AREDS did not show a statistically signiÞcant beneÞt of the AREDS formulation for either the development of new GA or for involvement of the fovea in eyes with preexisting atrophy [18]. In part, this result may be due to the paucity of GA patients in the study. Carotenoids and omega-3 (w-3) fatty acids were not studied in the AREDS. Carotenoids (e.g., lutein, zeaxanthin) have potentially therapeutic biological effects: Þlter blue light (high energy) [149]; antioxidant (scavenge singlet oxygen, quench triplet state of photosensitizers, retard peroxidation of membrane phospholipids) [150, 151]; and reduce chromatic aberration [149]. They are derived from diet [152], are transported in serum on circulating lipoproteins [153], and are concentrated in the macula [154]. Some [155, 156], but not all [157], studies indicate that higher dietary intake of lutein and zeaxanthin reduce the risk of AMD. In addition, some studies indicate that dietary b-carotene increases the risk of AMD [155]. Omega-3 fatty acids are essential and are derived from diet in humans; w-3 fatty acids include a-linoleic acid (short-chain, precursor to docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA)), EPA (long-chain, precursor to DHA, antithrombotic, hypolipidemic), and DHA (long-chain, main lipid constituent of outer segment membranes) [158]. A meta-analysis of 9 studies (3 prospective cohort,

Fig. 1.4 Hypothetical scheme of AMD pathogenesis. Three major variables that affect the probability of developing AMD are time, environment, and genes. With time, there is increasing oxidative damage (e.g., lipofuscin accumulation). The major environmental risk factor for developing AMD is smoking, which is an oxidative stress (see text for details). Genetic susceptibility to AMD includes inherited and acquired mutations in mitochondrial DNA as well as inherited mutations in the complement pathway. The photoreceptor-RPEÐBruchÕs membraneÐchoriocapillaris complex is a site of chronic oxidative damage, which is most pronounced in the macula. This damage incites inßammation, mediated at least in part by complement activation, at the level of RPEÐBruchÕs membraneÐchoriocapillaris. Patients with mutations in components of the complement system are less able to modulate the inßammatory response, resulting in excessive cellular damage and accumulation of extracellular debris. These changes, which involve modiÞcation of the extracellular matrix (ECM), cause additional inßammation and cell damage. This chronic inßammatory response involves cellular components of the immune system as well as the classical and alternative pathways of the complement system. Accumulation of abnormal extracellular material (including membranous debris, oxidized molecules, ECM molecules, and components of the complement system) is thus a sign of chronic inßammatory damage, is manifest in part as drusen and pigmentary abnormalities, and fosters the development of the late sequelae of AMD in susceptible individuals, i.e., GA and/or CNVs. Oxidative damage and inßammation may impair DICER1 activity and foster a stress response with increased BMP-4 expression, both of which may contribute to the development of GA. Other aspects of RPE senescence and the inßammatory response may stimulate angiogenesis and CNV formation. Many types of treatments for AMD under investigation (red circles) are based on concepts related to this hypothesis of pathogenesis

3 case-control, and 3 cross-sectional studies), which included a total sample of 88,974 people, including 3,203 AMD cases, showed that a high dietary intake of w-3 fatty acids was associated with a 38% reduction in risk of late AMD [159]. Fish intake at least twice a week was associated with reduced risk of both early AMD and late AMD [159]. In a population-based study, the relative risk of the top decile

total zinc intake vs. the remaining population was 0.56 for any AMD and 0.54 for early AMD [155]. Those with dietary zinc intake in the top tertile (15.8 mg/day) vs. the remaining population were 46% less likely to develop early AMD and 44% less likely to develop any AMD.

AREDS2 (NCT00345176) is a randomized multicenter phase 3 clinical trial to assess the following: (1) the role of lutein (10 mg)/zeaxanthin (2 mg) and w-3 longchain polyunsaturated fatty acids (350 mg docosahexaenoic acid [DHA]/650 mg eicosapentaenoic acid [EPA]) in prevention of development of GA or CNVs, (2) the possible deletion of b-carotene and lowering the daily zinc oxide dose from 80 to 25 mg.

Despite the abundant evidence that oxidative damage may play a role in the development and progression of AMD, a meta-analysis of supplementation trials indicated that there was insufÞcient evidence to support the use of antioxidant supplements in AMD prevention [160]. However, it may be that use of better targeted agents, different agents (e.g., molecular chaperones such as Hsp70, Hsp60, or a-crystallin), or targeting speciÞc agents to patients with speciÞc genetic abnormalities [21] will yield more effective outcomes.

1.3.2Visual Cycle Modulators

Visual cycle modulators are intended to reduce the accumulation of toxic ßuorophores (e.g., A2E) and lipofuscin in RPE cells. Retinol binding protein (RBP) possesses a high-afÞnity binding site for all-trans-retinol. The binding of retinol to RBP, in turn, creates a high-afÞnity binding site for transthyretin (TTR). Binding of TTR to the RBP-retinol complex creates a large molecular size complex that resists Þltration in the kidney and permits a high steady-state concentration of retinol in the circulation, which facilitates delivery of retinol to extrahepatic target tissues such as the eye. Unlike other extrahepatic tissues, the eye demonstrates a unique preference for uptake of retinol when it is presented in the RBP-TTR complex. N-(4- hydroxyphenyl)retinamide (Fenretinide, ReVision Therapeutics, Inc.) displaces all- trans-retinol from RBP in blood. Fenretinide possesses a bulky side chain on its terminal end that prevents interaction of the complex with TTR. In the absence of TTR binding, the RBP-fenretinide complex is eliminated through glomerular Þltration (excreted in urine) due to its relatively small size. Thus, fenretinide treatment causes a dose-dependent, reversible reduction in circulating RBP and retinol. The unique requirement of the eye for retinol delivered by RBP renders the eye more susceptible to reductions in serum RBP-retinol compared to other tissues. Consequently, during chronic fenretinide administration, levels of retinol within the eye will be dramatically reduced while other extrahepatic tissues will obtain retinol from alternate sources. Fenretinide reduces lipofuscin and A2E accumulation in the RPE of ABCA4−/− mice and causes modest delays in dark adaptation [161]. We note, however, that RPB−/− mice acquire normal vision by 5 months of age when given a vitamin A sufÞcient diet even though blood retinol levels remain low [162, 163]. Thus, it is not clear that blockade of vitamin A transport to RPE by inhibition

of vitamin A binding to RBP will block vitamin A uptake by RPE during long-term administration unless dietary vitamin A is restricted also. A phase 2b clinical trial of this oral agent is complete (NCT00429936). Patients received placebo (n = 82), 100 mg (n = 80), or a 300 mg (n = 84) daily dose for 24 months. Interim analyses reported at scientiÞc meetings indicate that fenretinide reduced the incidence of CNVs by ~50% in patients with GA. Patients receiving fenretinide also demonstrated a trend for reduced GA lesion growth rates. At the conclusion of the 2-year study, an exploratory and ad hoc analysis of the data revealed that 15 (18.3%) of 82 patients in the placebo arm progressed to CNV, while 15 (9.2%) of 164 patients receiving fenretinide at either dose developed CNV (P = 0.039). Preclinical studies show that fenretinide reduces the expression of VEGF isoforms and upregulates the expression of complement factor H. In the 300 mg fenretinide dose cohort, analysis of GA lesion growth by color fundus photography showed a trend for slowing of lesion growth, particularly among patients who had RBP and retinol levels reduced by more than 50%. Although fenretinide can have effects on dark adaptation [164Ð 171] and the ERG [172Ð175] and can be associated with symptoms of dry eye [164Ð166, 169, 170], it was generally well tolerated in this study.

Accutane (13-cis-retinoic acid) inhibits the conversion of all-trans-retinyl esters (in retinosomes) to 11-cis-retinol and the conversion of 11-cis-retinol to 11-cis-ret- inal by retinol dehydrogenase and also reduces lipofuscin accumulation in ABCA4−/− mice [176]. This oral agent may be associated with a high incidence of nyctalopia [177]. Another drug known as ACU-4429 (Acucela) is an orally administered compound that inhibits conversion of all-trans-retinyl ester to 11-cis-retinol via blockade of RPE65. ACU-4429 also reduces lipofuscin and A2E accumulation in the RPE of ABCA4−/− mice. A phase 1 clinical trial (NCT00942240) in 46 healthy volunteers was completed successfully [178]. The most common adverse events were vision-related (50% ACU-4429; 0% placebo) and included dyschromatopsia (32%), unspeciÞed visual disturbance (29%), night blindness (18%), blurred vision (11%), and photophobia (8%). Dyschromatopsia was observed in all study participants who received 60 or 75 mg of ACU-4429. Since the effect of ACU-4429 was limited to the rod ERG, the dyschromatopsia reported with high doses may indicate a collateral rod effect on cone pathways [179]. All adverse events were mild or moderate in intensity and were transient in nature, resolving within a few days after onset. There was dose-dependent suppression of the ERG b-wave as expected (Fig. 1.5). A dose escalation phase 2 study is underway in patients with GA (NCT01002950).

1.3.3Anti-Inflammatory Agents

Corticosteroids have numerous antiangiogenic effects and have been used previously as sole treatment and as part of combination treatment for CNVs [180]. Iluvien¨ (Alimera Sciences) is a nonbioerodible polyimide tube containing 180 mg of the corticosteroid ßuocinolone acetonide. It is inserted via a 25-gauge intravitreal injector, which creates a self-sealing wound. A phase 2 study (NCT00695318) is

Fig. 1.5 Rod and cone response on day-2 after treatment with ACU-4429. Prebleach amplitude after 40 min of dark adaptation on day-2 is shown. Amplitudes are expressed as a percentage of the prebleach amplitude for each dose on the 2 pretreatment days. Unlike rod amplitudes, cone amplitudes remain within 20% of the pretreatment amplitudes for all doses of ACU-4429 (reproduced with permission from Kubota et al. [178])

underway involving 40 patients with bilateral GA. The study eye is randomized to high (0.5 mg/day) or low (0.2 mg/day) dose Iluvien¨, and the primary outcome is a difference in the enlargement rate of GA in treated vs. untreated eyes. The fellow eye serves as a control.

Agents that modulate different parts of the complement system are in clinical trials (Fig. 1.6). In general, these agents work either by replacing a defective complement component (e.g., TT30 (Taligen), which is a factor H recombinant fusion protein that could provide normal factor H to patients with Y402H mutations) so that complement activation can be modulated properly, by inhibiting the activation of convertases (e.g., FCFD45145 (Roche)), which binds factor D (the rate-limiting step in alternative pathway activation), by promoting the decay of convertases (e.g., anti-properdin antibody, which destabilizes C3 convertase), or by blocking effector molecules (e.g., AL-78898A (Potentia/Alcon), which inhibits C3). Several examples will be discussed as they illustrate some of the challenges associated with manipulation of the complement pathway. Many of these compounds are administered intravitreally by injection. In some cases (e.g., factor D), the complement component is deposited diffusely in the human retina as well as in the choroid [98]. In other cases (e.g., factor B, factor H, C3, C5, C5b-9), deposition of the complement component(s) is primarily in the choroid, BruchÕs membrane, and/or subjacent to the RPE [52, 98]. Thus, it is not clear that intravitreal administration is the best route of delivery for all the complement inhibitors under study.

The classical, lectin, Þbrinolytic, and alternative pathways all generate bioactive fragments C3a and C5a as well as the membrane attack complex (C5b,6,7,8,9) via C3 cleavage. Thus, C3 inhibition should block complement activation arising from

Fig. 1.6 Numerous compounds that modulate the complement pathway are in development for or in clinical trials for AMD treatment. Red circles indicate the parts of the complement pathway that are being modiÞed. The complement pathway illustrated is adapted from Donoso et al. [273]. The Þgure is adapted from Zarbin and Rosenfeld [148]

many of the currently described complement pathway mutations (Fig. 1.3), which enables targeting a relatively large population of AMD patients. This feature of C3 inhibition may be a therapeutic advantage, but this degree of complement inhibition may create risks such as an increased risk of intravitreal injection-associated endophthalmitis. In a murine model, it seems that C3 deÞciency does not increase the risk of Staphylococcus aureus endophthalmitis [181]. On the other hand, in a guinea pig model, complement depletion with cobra venom factor does seem to increase the risk of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa endophthalmitis [182, 183]. POT-4 (AL-78898A) (Alcon/Potentia Pharmaceuticals), a cyclic peptide of 13 amino acids that is a derivative of Compstatin, is a C3 inhibitor, and is administered by intravitreal injection. Gel-like deposits form in the vitreous when POT-4 is injected at high concentrations (>0.45 mg dose). These deposits last as long as 6 months, thus providing a sustained-release delivery system. It is not known whether the doses administered intravitreally will have systemic effects or not, but a phase 1 study of POT-4 in AMD eyes with CNV was completed successfully without any safety concerns at doses up to 1.05 mg (NCT00473928). A phase 2 study is underway (NCT01157065).

Inhibition of C5 blocks terminal complement activity, but proximal complement functions remain intact, e.g., C3a anaphylatoxin production, C3b opsonization, and immune complex and apoptotic body clearance. ARC1905 (Ophthotech Corp.) is an anti-C5 aptamer delivered by intravitreal injection. It binds C5 with high afÞnity (KD = ~700 pM) and prevents cleavage to C5a and C5b. ARC1905 is in a phase 1 trial (NCT00950638) for patients with GA. Eculizumab (SOLIRIS, Alexion

Pharmaceuticals) is a humanized monoclonal antibody that binds to and prevents cleavage of C5 and is administered intravenously. To synthesize this molecule, human IgG2/4 heavy chain constant regions were used to eliminate the ability of the antibody to bind Fc receptors and activate complement. Eculizumab is already FDA-approved for the treatment of paroxysmal nocturnal hemoglobinuria and is in phase 2 trials (NCT00935883) for treatment of nonexudative AMD, including patients with high-risk drusen or GA. C5a receptor blockade, e.g., JPE1375 (Jerini); PMX025 (Arana Therapeutics); Neutrazimab (G2 Therapies), might have an advantage or a disadvantage over direct C5a inhibition. C5a receptor blockade might inhibit some important inßammatory pathways [57] without preventing membrane attack complex formation.

Factor D is the rate-limiting enzyme in the activation of the complement alternative pathway. FCFD45145 (Genentech/Roche) is a monoclonal antibody fragment (Fab) directed against Factor D. A 108-patient placebo-controlled phase 1 clinical trial (NCT00973011) of intravitreal therapy for GA is complete, and the medication was well tolerated up to a 10 mg dose. Patients were treated monthly or every other month during an 18-month period. A phase 2 study (NCT01229215) is underway.

Replacement of complement factor H should inhibit inßammation in AMD patients with risk-enhancing mutations in CFH. It is not clear that patients with other mutations will beneÞt from this therapy. This approach, which probably would require genetic screening prior to treatment, involves restoration of complement homeostasis so there is no increased risk of infection with therapy. The recombinant human form of the full-length complement factor H protein in its ÒprotectiveÓ form is known as rhCFHp (Ophtherion, Inc.). This protein can be administered intravenously or intravitreally. In preclinical models, intravitreal adenoviral vector delivery of the CFH gene has been effective and offers the promise of a sustained delivery system. (Our understanding is that Ophtherion, Inc. is not going to continue with its rhCFHp program.) Replacement of defective CFH also is being developed by Taligen/Alexion. TT30 is a recombinant fusion protein comprising complement receptor type 2 and factor H. TT30 binds iC3b/C3d coated cells and restores CFH activity. Preclinical testing is underway. Taligen/Alexion was also exploring Factor B inhibition using a humanized antibody fragment (TA106).

Silencing genes by preventing mRNA expression might be useful for AMD treatment since deletion of genes closely related to CFH (i.e., CFHR1 and CFHR3) seems to be strongly protective against AMD [99]. However, short-interfering-RNA therapies in the eye may be toxic [184], and it seems that the deletion of CFHR1 and CFHR3 protect against development of AMD at least in part because the deletion tags a protective haplotype and does not occur in association with the Y402H single nucleotide polymorphism [185].

Sirolimus (rapamycin, Macusight/Santen) is a macrolide fungicide that targets mTOR (mammalian target of rapamycin) and is anti-inßammatory, antiangiogenic, and antiÞbrotic; mTOR is a protein kinase that regulates proliferation, motility, survival, and protein synthesis. Rapamycin can be administered subconjunctivally and is in phase 1/2 studies in patients with GA (NCT00766649). Glatiramer acetate (Copaxone, TEVA) induces glatiramer acetate-speciÞc suppressor T-cells and