Genetic susceptibility

Over the past decade, high resolution genome scans for susceptibility loci in populations enriched for either choroidal neovascularization (CNV), geographic atrophy (GA), or both suggest susceptibility loci on numerous chromosomes. A number of candidate genes and their functions are listed in Table 67.1.^2,12–15 An exhaustive discussion of the genetics and postulated mechanisms of ARMD is beyond the scope of this chapter; however, several genes deserve mention.

A significant breakthrough in the study of AMD genetics came in 2005, when it was discovered that single nucleotide polymorphisms (SNPs) in the regulation of the complement activation locus (RCA) of chromosome 1q31.3 increased the risk of AMD. The RCA locus contains the gene encoding complement factor H (CFH). The tyr402his protein polymorphism increased the risk of AMD between 2.7- and 7.4-fold and appeared to account for between 43% and 50% of the attributable risk of AMD. Additionally, it has been found that CFH genotypes may influence an individual’s response to certain treatment modalities and medications, including bevacizumab, photodynamic therapy with verteporfin, as well as AREDS (Age-Related Eye Disease Study) nutritional supplementation.^16–20 CFH is responsible for downregulating activation of the complement system, including c5b-9, known as the membrane attack complex (MAC). MAC is thought to be important in the immune-mediated damage to Bruch’s membrane and an important pathogenic component of drusen.^21,22

It is clear that both immune dysregulation and inflammation play key roles in the development and progression of AMD. Two recent studies found protective associations against the development of advanced AMD in those with genetic polymorphisms of the complement components C2 (OR 0.35–0.46) and complement factor B (CFB) (OR 0.35–0.44), while C3 (OR 2.66–3.9) was strongly associated with the development of GA.^13,23 Figure 67.3 illustrates other complement components implicated in the pathogenesis of AMD. Additionally, elevated levels of the acute phase reactant CRP have been found to correlate with neovascular AMD. It is still unclear how genotypic variations in complement lead to an increased inflammatory state and the AMD phenotype. However, clinicopathologic specimens demonstrate the presence of inflammatory cells in Bruch’s membrane, and of bioactive fragments of C3 (C3a) and C5 (C5a) in drusen of AMD eyes and elevated VEGF expression in RPE cells.^24,25 The inflammatory cascade is further characterized by retinal microglial activation and choroidal macrophage infiltration.^26,27

Fig. 67.3 AMD-associated mutations in the complement cascade. Four major activation pathways for the complement system are known, and three of these are illustrated. (The fibrinolytic-activated intrinsic pathway is not shown.) Activation of the complement system plays an important role in immunity. Inappropriate complement activation can damage tissue. Multiple complement components have been linked to AMD (green circles), including drusen, GA, and CNVs. Complement C3 (red circle) is the key point of convergence of all activation pathways.

(Reproduced with permission from Zarbin M, Rosenfeld, PJ. Review of emerging treatments for age-related macular degeneration. In: Stratton RD, Hauswirth WW, Gardner TW, editors. Oxidative stress in applied basic research and clinical practice: Studies in retinal and choroidal disorders. New York: Humana Press; 2012.)

The gene products for tissue inhibitor for metalloproteinase 3 (TIMP-3, chromosome 22) and EFEMP1 (chromosome 2, encoding an elastin-related protein, fibulin 3), which act to inhibit the stimulatory effects of VEGF on vascular cells, have been independently associated with advanced AMD in genome scans mentioned previously.^28,29 Multiple distinct alleles in exon 5 of the gene encoding TIMP-3 have been causally linked to an autosomal dominant form of exudative macular degeneration with strong phenotypic similarities to exudative AMD,^30–32 although it is not thought to account for the majority of patients with exudative AMD.³³ TIMP-3 is also found in increasing quantities and is associated with thickening of Bruch’s membrane during aging, including within drusen and the RPE.^34–36 An Arg to Trp mutation in EFEMP1 is thought to cause the abnormal accumulation of extensive drusen in malattia leventinese, an inherited macular degenerative disease with phenotypic characteristics closely correlated with advanced nonexudative AMD.^34,37 The increased frequency of genome alterations coding for these two proteins (TIMP-3 and fibulin), which are important in the regulation of the extracellular matrix, particularly Bruch’s membrane, provides support for the hypothesis proposed by Blumenkranz that generalized increased susceptibility of elastic fibers to photic degeneration is an important risk factor for choroidal neovascularization.³

Early onset macular retinal degenerations such as Stargardt disease, characterized by the accumulation of lipofuscin within the RPE, have shed light on both the pathophysiology of RPE apoptosis and potential therapeutic avenues.^38,39 While initial reports also suggested that the ABCR gene linked to Stargardt disease might also be a dominant susceptibility locus for AMD, subsequent investigators have been unable to confirm this apparent increased frequency.^40,41 Nonetheless, increased amounts of lipofuscin are present in both Stargardt disease and in the earlier stages of the nonexudative AMD, and both diseases are characterized by GA in their later stages, suggesting that an understanding of the common pathways expressed by these two different diseases may provide some pharmacologic insights. Biochemical studies on the lipofuscin found in both Stargardt disease and AMD confirm that the principal component is N-retinylidene-N-retinylethanolamine (A2E), which accumulates in cells of the RPE and results in RPE apoptosis, photoreceptor death, and vision loss.^22,42,43 This process is thought to be initiated by light activation of rhodopsin in the course of normal visual transduction as the first reactant in A2E biosynthesis. Isotretinoin (Accutane, discussed later), which slows the synthesis of 11-cis retinaldehyde and regeneration of rhodopsin, is able to slow the accumulation of lipofuscin in ABCR knockout mice and also aged wild-type mice.⁴² This suggests that compounds with mechanisms of action similar to isotretinoin, which is structurally related to vitamin A, may represent another therapeutic avenue for the slowing or prevention of AMD.^{22,39,41–43}

There is additional discussion of AMD genetic susceptibility in Chapter 64 (Pathogenetic mechanisms in age-related macular degeneration.)

Diet

The principal environmental factors thought to be associated with age-related macular degeneration include diet, history of smoking, light exposure, and use of supplemental medications. Recent AREDS reports have established a relationship between dietary intake of omega-3 long-chain polyunsaturated fatty acids and reduced risk of neovascular AMD (OR 0.61), as well as progression from drusen to GA.^44–46 A large case control study suggested that higher dietary intake of carotenoids, particularly lutein and zeaxanthin, was associated with a lower risk of AMD, with the highest quintile having a 43% reduction compared with those in the lowest quintile. In that study no reduction could be found with the use of oral vitamin A, vitamin E or vitamin C.⁴⁷ Subsequent studies suggest that the use of foods, particularly fruits rich in antioxidants and carotenoids, when consumed at the level of three servings or more per day, result in a pooled multivariate reduced relative risk of 0.64 compared with persons who consumed less than 1.5 servings per day.² However, there have been a number of studies that have demonstrated little to no benefit with dietary regimens.^{28,48–50,51}

Smoking*

Most studies have consistently implicated smoking as a statistically significant risk factor for the development of late-stage AMD.^2,54 In a meta-analysis of three pooled studies from the United States, The Netherlands, and Australia, smoking was one of only two significant associations identified with incident AMD along with total serum cholesterol. Current smoking is associated with a 4.55-fold increased risk of neovascular AMD (vs. “never” smokers) and a 2.54-fold increased risk of atrophic AMD (vs. “never” smokers).⁵⁵ Based on a pooled analysis of data, the risks of both neovascular and atrophic AMD seem to decrease once one stops smoking.⁵⁵

Light exposure

Although there is a considerable body of compelling experimental data to suggest that increased retinal irradiance is positively correlated with an increased likelihood of advanced AMD, the epidemiological evidence supporting this hypothesis is modest.^3,56,57 In the Beaver Dam eye study, participants exposed to summer sun for more than five hours a day during selected periods of time were at increased risk for increased retinal pigment (relative risk 3.17) and early AMD (relative risk 2.14) after 10 years of follow-up compared with those exposed to less than 2 hours per day.⁵⁷

The underlying rationale for the hypothesis that increased light stress predisposes to AMD rests on the role of oxidative stress inherent with photo bleaching of the photoreceptors and, to a lesser extent, pigment epithelium. In particular, reactive oxygen intermediates (ROI) including hydrogen peroxide, singlet oxygen, and other short-lived species, which arise as the byproducts of cellular metabolism, are known to have a toxic effect on cellular membranes.⁴⁸ It is felt that the cumulative effects of chronic low levels of these species result in profound damage to the retina and pigment epithelium through lipid peroxidation, mitochondrial DNA damage and induction of apoptosis.^2,48 Experimental studies have confirmed the deleterious effect of chronic light and in particular blue and ultraviolet light on the RPE in part through the creation of A2E oxiranes, a major component of lipofuscin. The presence of natural antioxidants derived from dietary sources including lutein, zeaxanthin, lycopene, and ascorbate are thought to mitigate the effects of photo-oxidation by quenching free radicals and other intermediate species.⁴⁸ It has been hypothesized that this represents one, but not the only mechanism of action, of the antioxidants in the AREDS study.⁵⁸

Use of medications

Meta-analyses of pooled prospective studies suggest that certain medications may be associated with an increased or decreased risk of age-related macular degeneration. Antihypertensive medications, particularly beta-blockers, are associated with modest increased risk, whereas hormone replacement therapy in women, and tricyclic antidepressants, confer some relative protection.⁵⁹ These observations may be able to be further exploited in the development of new classes of drugs in addition to avoiding potentially harmful drug interactions.

The reader is referred to Chapter 63 (Epidemiology and risk factors for age-related macular degeneration).

Definition and steps in angiogenesis

Angiogenesis refers to the creation of new blood vessels from existing blood vessels, and therefore is contrasted from the process of vasculogenesis seen characteristically in utero in which vessels are created de novo. The angiogenesis cascade has been characterized as occurring in multiple sequenced steps (adapted from The Angiogenesis Foundation, www.angio.org/understanding/process.php [current as of February 15, 2012]):

Fig. 67.5 The cascade of events associated with angiogenesis are schematically diagrammed, including the release of angiogenic factors, binding by endothelial receptors, followed by endothelial cell activation, proliferation, and directed migration. With remodeling of the extracellular matrix, vascular tubes and loops form, associated eventually with the histologic appearance of neovascularization. EC, endothelial cell; BM, basement membrane; ECM, extracellular matrix; a-v, arteriovenous.

(Reproduced with permission from The Angiogenesis Foundation. http://www.angio.org/understanding/process.php [current as of 2012 Feb 15].)

VEGF and other positive and negative modulators of angiogenesis

Regardless of the inciting stimulus involved in the development of pathologic neovascularization, it is now well established that VEGF plays a principal role, including not only the neovascular forms of age-related macular degeneration, but also diabetic retinopathy, iris neovascularization, and retinopathy of prematurity. Additionally, other cytokines may play an important role as well, including fibroblast growth factor (FGF), pigment epithelial-derived factor (PEDF), the integrins, angiopoietins, and matrix metalloproteinase inhibitors.^2,30,69–72

VEGF undoubtedly represents the putative factor X first postulated by Michaelson associated with pathologic neovascularization.² First described as a vasopermeability factor associated with tumors, the molecule has since been cloned and well characterized.^73,74 VEGF has two important characteristics relevant to its role in the pathogenesis of the neovascular forms of age-related macular degeneration: (1) induction of angiogenesis through endothelial proliferation, migration, and new capillary formation, and (2) enhancement of vascular permeability.

Vascular permeability

VEGF produces important increases in hydraulic conductivity of isolated microvessels that are mediated by increased calcium influx and likely changes in levels of nitric oxide caused by induction of nitric oxide synthetase (NOS). While VEGF is both necessary and sufficient when administered exogenously to induce both these effects in vitro and in vivo, it is also well known that a number of other growth factors participate in this process. Some represent steps in a cascade initiated by VEGF while others act upstream.^75,76 The chemical structure of VEGF is that of a heparin-binding homodimeric glycoprotein of 45 kDa.⁷³ VEGF has significant homology to platelet-derived growth factor (PDGF). The human VEGF gene is organized into eight exons separated by seven introns and is localized in chromosome 6p21.3. Although there is only a single gene for VEGF-A, alternate post-translational exon splicing results in the generation of between four and six different isoforms having 121, 145, 165, 183, 189, and 206, amino acids respectively after signal sequence cleavage^72,73 (Fig. 67.6). VEGF₁₆₅ exists in both soluble and bound forms⁷³ (Figs 67.6, 67.7), and is thought to be predominantly responsible for pathologic neovascularization.

Fig. 67.6 Multiple isoforms of VEGF exist in nature and appear to participate to a differential degree in the normal events of vasculogenesis, as well as the pathologic events of angiogenesis occurring in response to local stimuli. Although there is only a single gene for VEGF, alternate splicing as well as post-transcriptional modification by plasminogen in the extracellular matrix accounts for a variety of isoforms that determine the relative balance between normal vascular homeostasis and pathologic neovascularization. The shorter amino acid length isoforms VEGF₁₂₁ and VEGF₁₄₅ are principally found in soluble form, whereas the longer spliced lengths 183, 189, and 206 are principally tissue fixed and bound by heparin. VEGF₁₆₅ is thought to stimulate pathologic neovascularization and exists both in soluble and tissue-fixed configurations.

Fig. 67.7 Pathways of VEGF expression and effects on vascular cells are demonstrated schematically. A variety of cells contribute to VEGF release, including monocytes, retinal pigment epithelial cells and neurons responding to inflammation, oxidative stress, and hypoxia respectively. These and other cell types produce predominantly VEGF-A which is expressed in various isoforms through transcriptional and post-translational steps. VEGF₁₆₅ and VEGF₁₂₁ are the predominant forms, with VEGF₁₈₉ being cleaved in the extracellular matrix to VEGF₁₁₀, a soluble form by activated plasmin. Each of these molecules, but principally VEGF₁₆₅, bind to endothelial cells through specific receptors VEGFR-1 and VEGFR-2. A sequence of events is set in motion through subsequent intracellular messaging systems as well as extracellular events that result in the loss of tight junctions between individual endothelial cells, the formation of fenestrations within endothelial cells and calcium-mediated permeability channels resulting in loss of the normal inner and outer blood–retinal barriers. Additionally matrix metalloproteinase activation occurs through its interaction with integrin receptors alpha ν beta 3 and alpha ν beta 5, which are found on the surface of endothelial cells exclusively following induction of the cell through activation with the VEGFR-1.

Although VEGF inhibitors share in common an attempt to mitigate the proliferative and permeability effects of VEGF on normal and neovascular tissue, there are likely to be differences in both efficacy and safety related to the choice of agents for several reasons. From a theoretical standpoint, since VEGF₁₆₅ is the principal isoform involved in pathologic neovascularization, it has been argued that leaving the other three principal isoforms intact (121, which is principally diffusible in soluble form, and 189 and 206 which are principally matrix-bound through high affinity heparin binding sites), avoids the possibility of interference with normal homeostatic mechanisms associated with constitutive VEGF expression. Indeed, previous animal studies suggested that blockade of VEGF₁₆₄ in the mouse (equivalent to VEGF₁₆₅ in humans) is as effective as nonselective antibody-mediated VEGF blockade in preventing pathologic neovascularization in a hypoxia-induced angiogenesis model, while not interfering with normal physiologic development of retinal vasculature.^60,77 However, it has become apparent with widespread use of bevacizumab and ranibizumab (monoclonal antibodies against multiple VEGF isoforms) that nonselective blockade has few untoward effects on normal retinal vascular physiology.

VEGF receptors

VEGF exerts its effects on cells through two highly related receptor tyrosine kinases (RTKs) VEGFR-1 and VEGFR-2.⁷³ VEGFR-1 also termed FLT-1 (FMS-like tyrosine kinase) was described first, but its role remains open to debate. Like VEGF, VEGFR-1 is upregulated by a hypoxia-inducing factor (HIF)-dependent mechanism. The receptor undergoes weak tyrosine autophosphorylation in response to VEGF. It is thought not to be primarily a mitogenic stimulus but rather a “decoy” receptor, which downregulates the activity of VEGF by sequestering and rendering the factor less available to VEGFR-2 (Fig 67.7, 67.8). This factor may be most important during embryogenesis rather than during pathologic neovascularization as well as in hematopoietic bone marrow-derived cells and neural signaling.^73,78

Fig. 67.8 Intracellular signaling including induction of nitric oxide synthetase (NOS), protein kinase C activation (PKC), and expression of ICAM-1 leading to leukocyte adherence and further changes in permeability mediated by calcium flux and protein kinase C.

VEGFR-2 (KDR in humans or FLK-1 in mice)

VEGFR-2 binds VEGF with lower affinity relative to VEGFR-1, but is felt to be the major mediator of the mitogenic, angiogenic, and permeability enhancing effects of VEGF. The binding site for VEGF on VEGFR-2 has been mapped to the second and third IgG-like domains and it undergoes dimerization and strong ligand-dependent tyrosine phosphorylation resulting in a mitogenic chemotactic and pro-survival signal. It appears to have at least two separate tyrosine phosphorylation sites. VEGFR-2 is thought to be critical as a survival factor with apoptosis occurring in its absence.

A large body of evidence exists to support the critical and probably rate-limiting role of VEGF in neovascular forms of age-related macular degeneration. High messenger RNA levels and increased VEGF receptor levels are observed in areas of CNV in primates as well as in the extracted neovascular membranes of patients removed at surgery and following autopsy.^2,63 Additionally there are strong indications that elevated levels of VEGF are the proximal cause for the hyperpermeability seen not only in diabetic macular edema, but in patients with subsensory and intraretinal fluid associated with CNV as well. The permeability changes resulting from VEGFR-2 are thought to be mediated to a large extent by endothelial nitric oxide synthetase-based generation of increased nitric oxide levels, and associated changes in calcium flux (Fig. 67.8). These alterations can be reversed either by direct blockade of VEGF, the receptor, or NOS using a knockout model.^2,74,75 Inactivation of soluble VEGF by monoclonal antibodies directed against it, or inhibition of ICAM also appear to be effective as well. In addition to either the indirect inhibition of VEGF effects through modulation of ICAM, or indirect effects on nitric oxide phosphorylase, nitric oxide synthetase, or protein kinase C, another modulator of permeability, there are several other methods by which VEGF effects can be blocked in the eye and elsewhere. These include the use of a class of molecules termed aptamers, which are chemically synthesized single strand nucleic acids, either RNA or DNA, that bind to target molecules with high selectivity and affinity leaving non-targeted protein functions intact.^79,80 Other methods of direct inhibition of VEGF include inhibition of its tyrosine kinase receptors (VEGFR-1 and -2), either by systemic administration or gene transfer. Several laboratories have described the use of small-interfering RNA (siRNAs) in effectively downregulating gene regulation⁸¹ (see subsequent sections).

Other cytokines and regulators of angiogenesis

A number of cytokines have been identified that either upregulate VEGF or VEGF-associated effects, or act in an inhibitory capacity. In some instances, the same compounds may perform both functions depending upon the other extracellular signals present in the milieu as well as post-transcriptional or post-translational events, including cleavage by proteases, or binding by soluble receptors such as the family of tyrosine kinases.² Accordingly, any of this class of normally occurring compounds can be considered either in their native form, through splice or cleavage products, or chemical substitution to be potential therapeutic compounds in the treatment of pathologic neovascularization. Examples of stimulatory cytokines in addition to vascular endothelial growth factor include basic and acidic fibroblast growth factor, the angiopoietins, transforming growth factor (TGF), IL-8, and platelet-derived growth factor (PDGF). Naturally occurring inhibitory factors include interferon alpha, thrombospondin 1, angiostatin, endostatin, hemopexin-like domain of matrix metalloproteinase 2 (PEX), and pigment epithelial-derived factor (PEDF).^2,82–90 Additionally, it is likely there are many other naturally occurring compounds as well, as yet not recognized or well characterized biochemically. The relative balance between naturally occurring stimulatory and inhibitory factors is thought to contribute to the tight control of ocular vascular homeostasis, and may explain the differential ability of normal vascular as well as neovascular tissue to respond exquisitely both spatially and temporally to various stimuli.

Fibroblast growth factor and integrins

Basic fibroblast growth factor (bFGF/FGF2) has been found to be elevated in excised choroidal neovascular membranes from patients undergoing surgery for AMD, and is capable of inducing pathologic neovascularization when implanted within the eye or adjacent to the retina.^69,70 Laser models of choroidal neovascularization are accompanied by upregulation of bFGF-associated mRNA, which precedes the development of histologically identifiable new vessels. While bFGF is sufficient when inserted exogenously in creating choroidal neovascularization, it is not necessary; BFGF gene knockout animals are also capable of developing laser-induced CNV bFGF upregulation of angiogenesis is thought to be in part mediated by adhesion-mediated signals including expression of the cell membrane-associated integrins such as the alpha ν beta 3 integrin.^2,69,70 Although the alpha ν beta 3 and another integrin, alpha ν beta 5, appear to differentially represent the stimulatory effects of bFGF and VEGF respectively in laboratory studies, there is conflicting evidence as to whether these pathways are distinct. As a result, considered therapeutic approaches to integrin inhibition may be directed at downstream events such as intracellular signaling in addition to targeted inhibition of the individual receptors through monoclonal antibodies or related mechanisms.

Platelet-derived growth factor

Platelet-derived growth factor (PDGF) is a homo- or heterodimeric glycoprotein that stimulates pericyte and vascular smooth muscle cell recruitment in angiogenesis, which are essential in the maintenance and survival of new vessels. The PDGF receptors (PDGFR-A and PDGFR-B) are tyrosine kinases, and are expressed differentially on different cell types, with PDGFR-B being more extensively expressed in endothelial cells, vascular smooth muscle cells, and fibroblasts, among others.^91,92 In the absence of PDGFR-B signaling, vascular pericytes detach, and the integrity of vessel is lost. Immature vessels are highly sensitive to the withdrawal of VEGF prior to pericyte recruitment and coverage; conversely, when pericyte coverage is complete, new vessels are resistant to VEGF withdrawal. This is the so-called plasticity window, during which time vessels can regress rapidly with changes in exposure to growth factors.^93,94 PDGF blockade in animal models of corneal neovascularization has resulted in prevention and regression of new vessels.^95,96 PDGF blockade alone or combined with VEGF inhibition (taking advantage of the plasticity window) may thus serve as a potent inhibitor of neovascular AMD.

Angiopoietins

Angiopoietins, which are also highly specific for endothelial cells, perform a variety of other regulatory activities related to supporting cells and the extracellular matrix. A laboratory study suggests that two different isoforms, Angiopoietin 1 and Angiopoietin 2, appear to have differential and counteracting effects on the vasculature. Angiopoietin 2, which is upregulated by both hypoxia and VEGF, binds to an endothelial cell receptor TIE2 and enhances VEGF-mediated retinal neovascularization, but does not by itself stimulate endothelial cells or proliferation in vitro.^2,97 Angiopoietin 1 appears to play a maturation role and is associated with nonleaky behavior and may have potential therapeutic benefit through its inhibition of inflammatory pathways.²

Matrix metalloproteinases and tissue inhibitors of metalloproteinases

Expression of matrix metalloproteinases (MMPs) and their associated RNA, particularly MMP-2 and MMP-9, have been associated with pathologic neovascularization. Both matrix metalloproteinases, which are bound to the integrin receptors alpha ν beta 3 and alpha ν beta 5 on endothelial cells, are activated by VEGF and other regulatory cytokines and act to digest the extracellular matrix and thereby facilitate the spreading and chemotaxis of actively proliferating endothelial cells as they aggregate to form new capillaries.² Matrix metalloproteinases (MMPs) appear to be modulated and principally downregulated by naturally occurring tissue inhibitors of metalloproteinases (TIMPs) of which TIMP-1, TIMP-2, and TIMP-4 are thought to be soluble, and TIMP-3 principally extracellular matrix-bound. TIMP-3 is thought to play an important and possibly critical role in the natural modulation of matrix metalloproteinase regulation in Bruch’s membrane, and is found in increased amounts in drusen and thickened basement membranes associated with age-related macular degeneration.^36,98 Mutations in exon-5 of the TIMP-3 molecule are associated with Sorsby’s fundus dystrophy,^31,32,35 and an experimental model that either delivers increased amounts of TIMP-3 or induces overexpression of TIMP-3 by gene therapy demonstrates potent antiangiogenic effects for this molecule.^99,100

Pigment epithelial-derived factor

Pigment epithelial-derived factor (PEDF), which is secreted by the pigment epithelium in vivo as well as in cell culture, has been shown to be biochemically identical to the product of the wild type of retinoblastoma tumor suppressor gene (RB) and is thought to induce differentiation of retinoblastoma cells, inhibit microglial growth, and other important regulatory functions in addition to its effects on angiogenesis.⁵⁶ Its relevance to AMD is likely related to its role in counterbalancing VEGF. In a study of donor human eyes with and without AMD, relative VEGF and PEDF levels were measured using immunoreactivity assays. Interestingly, VEGF levels were not significantly different between the two groups of eyes, even in the presence of advanced exudative change. In contrast, PEDF levels were much lower in the RPE and choroid of eyes with AMD, suggesting that it is lower levels of PEDF in the ocular milieu that may contribute to the formation of choroidal neovascularization.¹⁰¹ Results from in vitro studies have suggested that the role of PEDF is regulation or inhibition of VEGF receptor signaling, rather than direct VEGF antagonism.¹⁰²

Other cytokines

Another interesting class of regulatory cytokines that appear to have an inhibitory effect on angiogenesis relate to the class of aminoacyl-tRNA synthetases, which are associated with the first step of protein synthesis. Tryptophanyl-tRNA synthetase (TrpRS, a homolog of TyrRS) has no effect in its native form on angiogenesis or angiogenesis signaling. In contrast, an alternatively spliced fragment reported to be stimulated by interferon-alpha and lacking a portion of the amino terminal fragment, has potent antiangiogenic effects in both in vitro and in vivo.¹⁰³

Thrombospondin 1 (TSP-1) has been described as both an up- and downregulator of VEGF. Under specified conditions TSP-1, which is produced by platelets and monocytes, enhances simulated VEGF release and is dependent upon binding to alpha ν beta 5 and alpha ν beta 1 integrins. In separate studies, TSP-1 has also been noted to inhibit endothelial cell proliferation, migration, and angiogenesis. VEGF and TSP-1 appear to participate in a feedback loop and its potential role as a therapeutic agent remains uncertain.² Angiostatin, is a 38 kDa internal fragment of plasminogen that encompasses the first four cringles of the molecule. It shows inhibitory effects on vascular endothelial proliferation in vitro and in vivo, particularly within tumors.^90,104 Endostatin, a cleavage product of collagen XVIII, is structurally related to and shares homology with angiostatin, and inhibits tumor-associated angiogenesis.⁸⁹ Each of these classes is further discussed in the following section as they relate to preclinical or clinical studies.

Antioxidants, vitamins, and cofactors

The rationale for the use of antioxidants, multivitamins and other cofactors is based upon several lines of evidence, including the essential role of vitamin A in the visual transduction cycle, the known and predicted effects of oxidative stress resulting in reactive oxygen intermediates, and the requirement of certain critical metal ions including zinc in the functioning of critical proteases and other naturally occurring defense mechanisms against oxidative injury. Prior to the release of the data from the AREDS study, there remained some controversy as to whether antioxidant multivitamin therapy was efficacious, with some studies suggesting a protective effect, and others not.^49,50,105

Age-Related Eye Disease Study (AREDS) and related supplements

The AREDS was a large, well-designed multicenter randomized clinical trial, which evaluated the effect of high-dose vitamin C and E, beta-carotene, and zinc supplements on AMD progression and visual acuity. A total 4757 persons were enrolled and stratified into one of four categories of increasing severity of disease. Due to the low rate of progression of category 1, consisting of drusen area of less than five small drusen of 63 µm or less, statistical analysis was only performed on the remaining 3640 patients with categories 2, 3, 4. Only the latter two (categories 3 and 4) were ultimately found to be at more than a trivial risk for progression. Patients in category 2 with at least one intermediate size druse, but not extensive drusen, had only a 1.3% probability of progression to advanced AMD by year 5 and no meaningful inference could be drawn regarding their risk with or without intervention. Patients in category 3 had either extensive intermediate drusen, large drusen, or non-central geographic atrophy, and patients in category 4 had visual acuity of less than 20/32 due to AMD in one eye related either to geographic atrophy involving the center of the macula or choroidal neovascularization in the fellow eye, but not the study eye, which had visual acuity 20/32 or better. The average follow-up of the 3640 enrolled study participants aged 55–80 was 6.3 years, and by comparison with placebo, patients treated with antioxidants plus zinc demonstrated a statistically significant odds reduction for the development of advanced AMD (OR 0.72, 99% CI 0.52–0.98). The odds ratio for zinc alone and antioxidants alone were 0.75 and 0.8, respectively, and the odds reduction estimates increased when only patients with category 3 or category 4 drusen were included, reducing to 0.66, 0.71, and 0.76 for antioxidants plus zinc, zinc alone, and antioxidants alone respectively. These data are further reflected in Fig. 67.9.

Fig. 67.9 Repeated-measures estimates of the probability of loss in the visual acuity score of at least 15 letters in at least one study eye of participants within categories 3 and 4.

(Reproduced with permission AREDS report no. 8: from A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss. Arch Ophthalmol 2001;119:1417–36.)

Based upon these findings, and extrapolation of the US population at risk, it has been estimated that of the 8 million persons in the United States at least age 55 or greater with AMD, if those at highest risk including categories 3 and 4 were to receive therapy with the formulation employed in the AREDS trial, as many as 300 000 of the 1.3 million at highest risk for advancement might avoid progression to the severe forms of the disease.¹⁰⁶ Additionally, based upon recent epidemiologic data, exclusion of beta-carotene is recommended for persons with a current history of smoking, or a long smoking history based upon a theoretical increased risk of lung cancer.

The macular carotenoids thought to be the principal contributors to the yellow coloration of the macula include lutein and zeaxanthin. One study suggested that a high dietary intake of these carotenoids protected against AMD, resulting in a 43% reduction in risk for AMD when the upper quartile of use was compared with the lowest quartile.⁴⁷ Recent AREDS reports have supported these findings, showing in cohort studies that those with a high level of dietary intake of lutein and zeaxanthin were less likely to have advanced AMD, as well as large or extensive intermediate drusen.¹⁰⁷ High dietary intake of ω-3 long-chain polyunsaturated fatty acids were also found by the AREDS investigators to be inversely related to progression to both GA as well as neovascular AMD.^44–46 The critical dependence of the naturally occurring antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) as well as the matrix metalloproteinases, on copper, zinc and manganese, provides a potential mechanism by which AREDS supplementation of both copper and zinc could be beneficial. The naturally occurring antioxidants such as vitamin E and vitamin C might have a beneficial effect on the progression of AMD as well.⁴⁸ At present no definitive recommendations can be made regarding dietary supplementation with lutein and zeaxanthin, as well as ω-3 long-chain polyunsaturated fatty acids. However, the AREDS2 randomized multicenter phase III clinical trial (NCT00345176) is currently underway, and is designed to address: (1) the role of lutein (10 mg)/zeaxanthin (2 mg) and ω-3 long-chain polyunsaturated fatty acids (350 mg docosahexaenoic acid (DHA)/650 mg eicosapentaenoic acid (EPA)) in the prevention of development of GA or CNV; and (2) the effect of possible deletion of β-carotene and lowering the daily zinc oxide dose from 80 mg to 25 mg. The AREDS study is discussed also in Chapter 65 (Age-related macular degeneration: Non-neovascular early AMD, intermediate AMD, and geographic atrophy).

Visual cycle inhibitors*

Visual cycle modulators are intended to reduce the accumulation of toxic fluorophores such as A2E in RPE cells. Fenretinide (ReVision Therapeutics, Inc.) treatment causes a dose-dependent, reversible reduction in circulating retinal binding protein (RBP) and retinol by displacing retinol from RBP, as well as interfering with RBP binding to transthyretin (TTR), thus facilitating their elimination via glomerular filtration. In ABCA4^−/− mice, fenretinide reduces RPE lipofuscin and A2E accumulation, although it causes modest delays in dark adaptation.¹⁰⁸ Notably, however, despite low blood retinol levels, RPB^−/− mice acquire normal vision by 5 months of age when given a vitamin A-sufficient diet.^109,110 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 the RPE during long-term administration unless dietary vitamin A is restricted also. Patients in a phase IIb clinical trial of fenretinide received placebo, 100 mg, or a 300 mg daily dose for 24 months (NCT00429936). At the conclusion of the two-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, compared with 15 (9.2%) of 164 patients receiving fenretinide at either dose (P = 0.039). 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 affect dark adaptation^111–118 and ERG readings,^119–122 and is associated with symptoms of dry eye, ^116,117 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-retinal by retinol dehydrogenase and also reduces lipofuscin accumulation in ABCA4^−/− mice.^42,123 This oral agent is associated with a high incidence of nyctalopia.¹²⁴ 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 I clinical trial (NCT00942240) in 46 healthy volunteers was completed successfully.¹²⁵ The most common adverse events were vision-related, occurring in 50% of patients receiving the medication, which included dyschromatopsia (32%), unspecified visual disturbance (29%), night blindness (18%), blurred vision (11%), and photophobia (8%). 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. A dose escalation phase II study is underway in patients with GA (NCT01002950).

Complement modulators*

In general, complement modulating agents work by replacing a defective complement component; by inhibiting the activation of convertases; by promoting the decay of convertases; or by blocking effector molecules (Fig. 67.10).¹²⁶ The following examples illustrate some of the promise as well as the challenges associated with manipulation of the complement pathway.

Fig. 67.10 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 modified. The complement pathway illustrated is adapted from Donoso et al.¹²⁶

(Reproduced from Zarbin MA, Rosenfeld PJ. Pathway-based therapies for age-related macular degeneration: an integrated survey of emerging treatment alternatives. Retina 2010;30:1350–67 with permission of Springer Science+Business Media.)

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 affinity (KD = ~700 pM) and prevents cleavage to C5a and C5b. ARC1905 is in a phase I 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. Eculizumab is FDA-approved for the treatment of paroxysmal nocturnal hemoglobinuria and is in phase II 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 both advantages and disadvantages over direct C5a inhibition. C5a receptor blockade might inhibit some important inflammatory pathways,²⁴ although without disabling membrane attack complex formation.

Factor D is the rate-limiting enzyme in the activation of the complement alternative pathway. It plays an important role in the positive feedback loop that results in the amplification of proinflammatory effectors.¹²⁷ FCFD45145 (Genentech/Roche) is a monoclonal antibody fragment (Fab) directed against Factor D. A 108-patient placebo-controlled phase I 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 II study (NCT01229215) is underway.

Replacement of complement factor H should inhibit inflammation in AMD patients with risk-enhancing mutations in CFH. 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. Replacement of defective CFH is being developed by Alexion. TT30 is a recombinant fusion protein comprising complement receptor type 2 and the regulatory domain of factor H. The receptor domain of TT30 binds iC3b/C3d-coated cells, delivering the regulatory domain of CFH to areas where complement regulation is necessary.¹²⁷ Preclinical testing is underway. Alexion is also exploring factor B inhibition using a humanized antibody fragment (TA106).

The differential expression of complement components in different ocular tissues presents difficulty in determining the optimal route of administration of these agents. For example, factor D is deposited diffusely in the human retina as well as in the choroid,¹²⁸ while factors B, H, C3, C5, and C5b-9 are found primarily in the choroid, Bruch’s membrane, and/or subjacent to the RPE.^128,129 Thus, intravitreal injection may not be the ideal route of delivery for all the complement inhibitors under study.

Another approach to complement modulation is the silencing of genes by preventing mRNA expression, as deletion of genes closely related to CFH (i.e., CFHR1 and CFHR3) appears to be strongly protective against AMD.¹³⁰ However, short-interfering RNA (siRNA) therapies in the eye may be toxic, 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.¹³¹ See below for further discussion of siRNAs and other complement inhibitors for exudative AMD.

VEGF inhibitors

A variety of methods are now available to directly inhibit the VEGF₁₆₅ molecule as well as its various other isoforms. These include the use of monoclonal antibodies directed at one or more isoforms, oligonucleotides with chain-specific sequences corresponding to the VEGF protein, and molecules directed at one or more of the VEGF receptors, including native decoys, tyrosine kinase inhibitors, fusion proteins, and TIE2 receptors. Finally, because VEGF is thought to initiate a cascade of intracellular signals initially, and subsequently extracellular events, it is possible to inhibit VEGF effects either through prevention of secretion of the molecule, direct inhibition of the molecule in the extracellular space, blockade of the receptors, or through interruption in the downstream intracellular signaling pathways leading to both intra- and extracellular events.¹³² In addition to the sections that follow, the interested reader is referred to Chapter 66 (Neovascular (exudative or “wet”) age-related macular degeneration).

Direct VEGF inhibitors

Monoclonal antibody: bevacizumab (Avastin)

Bevacizumab is a humanized monoclonal antibody (IgG1) against human VEGF-A that selectively inhibits all isoforms and bioactive proteolytic breakdown products of VEGF-A (Fig. 67.11).¹³³ Bevacizumab is an immunoglobulin G molecule that is comprised of amino acid sequences which are about 93% human and 7% murine. Preclinical studies in animal models of various tumor cell lines as well as different forms of ocular neovascularization indicated that the fully sized antibody had excellent efficacy against the primary permeability and proliferative effects of VEGF isoforms. Following extensive clinical testing, it was found to be effective in slowing tumor growth through its effects on angiogenesis.⁷³ Bevacizumab was FDA-approved in 2004 as a treatment for metastatic colorectal cancer when given intravenously at a dose of 5 mg/kg and infused every 2 weeks in combination with 5-fluorouracil. Bevacizumab was shown to increase survival, response rate, and duration of response compared with conventional therapy consisting of irinotecan, fluorouracil, and leukovorin for metastatic colorectal cancer.¹³⁴ Additional phase III clinical trials with bevacizumab have since resulted in FDA-approval for the treatment of lung, kidney, and brain cancers.

Fig. 67.11 Relationship between ranibizumab and bevacizumab. Ranibizumab is a recombinant humanized monoclonal IgG1 kappa-isotype antibody fragment (with a molecular weight of about 48 kD). It is produced in an Escherichia coli expression system (and thus is not glycosylated) and is designed for intraocular use. Bevacizumab is a recombinant humanized monoclonal IgG1 antibody (with a molecular weight of about 149 kD). It is produced in a Chinese-hamster-ovary mammalian-cell expression system (and thus is glycosylated) and is designed for intravenous infusion. Both the antibody fragment and the full-length antibody bind to and inhibit all the biologically active forms of vascular endothelial growth factor (VEGF) A and are derived from the same mouse monoclonal antibody. However, ranibizumab has been genetically engineered through a process of selective mutation to increase its affinity for binding and inhibiting the growth factor. The Fab domain of ranibizumab differs from the Fab domain of bevacizumab by six amino acids, five on the heavy chain (four of which are in the binding site) and one on the light chain. Not all the intermediate Fabs between the mouse monoclonal antibody and ranibizumab are shown.

(Reproduced with permission from the Massachusetts Medical Society from Steinbrook R. The price of sight – ranibizumab, bevacizumab, and the treatment of macular degeneration. N Engl J Med 2006;355:1409–12.)

In 2004, a study investigating the use of systemic bevacizumab in patients with exudative AMD (Systemic Avastin for Neovascular AMD – SANA) was initiated at the Bascom Palmer Eye Institute. This study was the first demonstration that bevacizumab was effective for this disease. In this open-label, uncontrolled study, 18 patients underwent two to three intravenous infusions of bevacizumab (5 mg/kg) to evaluate the potential safety and efficacy of systemic bevacizumab over 6 months.^135,136 Although clinical trials using intravitreal pegaptanib and ranibizumab for CNV were underway, the prior use of bevacizumab for ocular disease in humans using any route had previously been untested. While no serious adverse events were observed, and only a mild transient increase in blood pressure was identified, the SANA study was too small to establish the safety of bevacizumab. However, significant improvements in visual acuity and OCT central retinal thickness measurements were observed comparable to the changes observed in the early phase Lucentis trials.

While systemic bevacizumab (5 mg/kg) was shown to reduce leakage from CNV, decrease OCT central retinal thickness measurements, and significantly improve vision in exudative AMD, the intravenous use of bevacizumab for exudative AMD was never widely adopted, because the intravitreal approach used up to 500-fold less drug, is much less expensive, and is perceived to be safer due to the smaller dose of drug. In the first-reported case of intravitreal bevacizumab,¹³⁷ a patient with recurrent CNV secondary to AMD, who had previously been treated with verteporfin photodynamic therapy (PDT) in combination with triamcinolone acetonide followed by treatment with pegaptanib injections, was shown to improve with a decrease in OCT central retinal thickness, subretinal fluid, as well as improvement of subjective visual distortion within 1 week of a single injection of 1.0 mg bevacizumab.

The bevacizumab used for this injection was the commercially available form available as 100 mg and 400 mg preservative-free, single-use vials in volumes of 4 ml or 16 ml (25 mg/ml). The 100 mg product is formulated in 240 mg α(alpha),α(alpha)-trehalose dihydrate, 23.2 mg sodium phosphate (monobasic, monohydrate), 4.8 mg sodium phosphate (dibasic, anhydrous), 1.6 mg polysorbate 20, and was diluted in water prior to intravenous infusion. For off-label intravitreal use, bevacizumab was not diluted, but rather dispensed into individual syringes for intravitreal injection and the volume (dose) of injection ranged from 0.05 ml (1.25 mg) to 0.1 ml (2.5 mg).

Initially, there was concern that a molecule as large as the 149 kDa bevacizumab may not penetrate the retina and effectively treat CNV compared with ranibizumab, which is one-third the size. However, it became apparent that bevacizumab could penetrate the retina based upon the empiric observations of decreased subretinal fluid on OCT examination and improved visual acuities in treated patients. Subsequently, Han et al. were the first to show that a full-length immunoglobulin the size of bevacizumab was capable of penetrating the rabbit retina after an intravitreal injection.¹³⁸ Sharar et al. used qualitative immunofluorescence to confirm that intravitreal bevacizumab was able to completely penetrate the retina by 24 hours and was essentially absent at 4 weeks after an injection.¹³⁹ Moreover, Dib et al. demonstrated bevacizumab molecules in the subretinal space of all six eyes studied two hours after an intravitreal bevacizumab injection of 0.05 ml (1.25 mg), confirming the initial observations that the molecule could rapidly diffuse through the retina.¹⁴⁰

The ocular pharmacokinetics of various other monoclonal antibodies injected into the vitreous cavity of monkeys and rabbits demonstrated a half-life of about 5.6 days.^141,142 Studies of bevacizumab in rabbits and monkeys suggest a half-life in the range of 4–6 days.^143–145 A human study reported that a single dose of intravitreal bevacizumab had a half-life of 3 days and was likely to provide complete intravitreal VEGF blockade for a minimum of 4 weeks.¹⁴⁶ One human study suggested a half-life of 6.7 days and yet another study has reported a half-life of as long as 9.8 days.^147,148 It is likely that human ocular pharmacokinetics of bevacizumab varies from patient to patient, depending on factors such as the extent of vitreous liquefaction and the phakic status of the eye.

Since the first case of intravitreal bevacizumab for the treatment of exudative AMD was reported, numerous retrospective studies and a few small prospective studies using a dose range between 1.0 and 2.5 mg have been published, all demonstrating clinically significant improvement in mean visual acuity, reduction in fluorescein angiographic leakage, and resolution of OCT-visualized edema in up to 90% of bevacizumab-treated patients. These reports also supported the apparent clinical safety of bevacizumab. As of the time of writing (February 2012), despite the subsequent approval of ranibizumab in 2006, the off-label use of intravitreal bevacizumab has become the most common treatment for exudative AMD in the United State.^149,150 In addition, it has also become frequently used for wide range of other forms of macular new vessels including pathologic high myopia, multifocal choroiditis, and ocular histoplasmosis; it has also been used successfully for treatment of clinically significant macular edema from diabetic retinopathy, retinal venous occlusions, and retinopathy of prematurity.^151–159 It is being explored for treatment of other macular conditions not associated with new vessels or known elevated VEGF levels, including central serous choroidopathy and uveitides, although its efficacy in those conditions is not yet firmly established. Prior to the release of the Comparison of Age-Related Macular Degeneration Treatments Trial (CATT, discussed below), most of the early pilot studies were small (less than 100 patients) and uncontrolled, with different retreatment criteria and outcome measures; most physicians have frequently extrapolated the results from the phase III ranibizumab trials for these indications to bevacizumab.¹⁶⁰ However, two randomized, controlled studies deserve mention.

The ABC trial compared intravitreal bevacizumab (three loading doses every 6 weeks, followed by additional injections at 6-week intervals as needed) to standard therapy, defined at the time of recruitment as verteporfin PDT for predominantly classic CNV, or pegaptanib injection or sham injection for minimally classic or occult CNV. A total of 131 patients were recruited and randomized 1 : 1 to receive either bevacizumab or standard therapy. A significantly higher proportion of the bevacizumab group improved by 15 letters compared with the standard therapy group (32% vs 3%, P < 0.001), irrespective of lesion type. Additionally, more patients lost fewer than 15 letters in the bevacizumab group (91% vs 67%, P < 0.001), and a larger proportion of bevacizumab patients achieved 6/12 or better vision by week 54 of the study. By study end, mean change in vision was +7.0 letters in the bevacizumab group, versus −9.4 letters in the standard care group. Serious ocular and nonocular adverse events were rare in all groups, with no serious intraocular infections in any of the intervention groups. Two myocardial infarctions, one fatal, occurred in the bevacizumab group.¹⁶¹ This study represented the first level 1 evidence of the efficacy of bevacizumab for the treatment of AMD.

A small randomized study conducted at the Veterans Affairs Boston Healthcare System Hospital compared bevacizumab and ranibizumab directly. A total of 28 patients were randomized in a 2 : 1 ratio to receive bevacizumab or ranibizumab, of which 22 patients completed one year of follow-up. Injections were given monthly for 3 months, followed by additional injections given on the basis of OCT findings primarily, or vision or clinical exam. Visual and anatomic results were similar between the two groups: patients receiving bevacizumab improved an average of 7.6 letters, compared with 6.3 letters in the ranibizumab group (P = 0.74). The change in central macular thickness as determined by OCT was −50 µm in the bevacizumab group, versus −91 µm in the ranibizumab group (P = 0.29). The number of ranibizumab injections administered was significantly lower than the number of bevacizumab injections (4 vs 8, P = 0.001). The authors hypothesized two possible reasons: (1) tachyphylaxis to bevacizumab; or (2) a more robust early anatomic response to ranibizumab, leading to a higher number of bevacizumab injections given on an as-needed basis determined primarily by OCT findings.¹⁶² This study, although small in size, was the first head-to-head trial of bevacizumab and ranibizumab, and demonstrated the similar efficacy of bevacizumab to ranibizumab.

Antigen binding fragment: ranibizumab (Lucentis)

Ranibizumab is a humanized anti-VEGF-A recombinant Fab fragment that has been affinity matured to increase its binding affinity for VEGF-A (see Fig. 67.11). Ranibizumab binds within the VEGFR-binding domain of all biologically active isoforms of VEGF-A. Two randomized, double-masked, pivotal phase III clinical trials have demonstrated that monthly intravitreal injections of ranibizumab are an effective and safe treatment for subfoveal CNV in AMD patients.^163,164

The MARINA study assessed the response of minimally classic or occult CNV to ranibizumab.¹⁶³ Patients (n = 716) were assigned randomly to receive sham injection (n = 238), 0.3 mg (n = 238), or 0.5 mg (n = 240) ranibizumab. After 4 months of follow-up, approximately 90% of ranibizumab-treated patients had lost less than 15 letters on the Bailey – Lovie (ETDRS) chart as compared to 53% of the sham-injected patients. This treatment response was independent of lesion size, initial visual acuity, or whether the lesion was classified as minimally classic or occult with no classic CNV on fluorescein angiography. Vision improved by at least 15 letters in approximately 33% of patients receiving 0.5 mg ranibizumab at 24 months of follow-up versus 4% in the sham-injected patients (Fig. 67.12).¹⁶⁵ Approximately 1% of patients receiving intravitreal ranibizumab developed endophthalmitis. The risk of cataract was approximately 0.2%. There were no cases of retinal detachment among patients receiving intravitreal therapy. Despite the theoretical risk of systemic vascular complications, there was no imbalance among treated and control groups regarding hypertension, and the risk of myocardial infarction among sham-injected, 0.3 mg, and 0.5 mg cohorts was 1.7%, 2.5%, and 1.3%, respectively. The risk of stroke among the three groups was 0.8%, 1.3%, and 2.5%, respectively. The risk of nonocular hemorrhage was 5.5%, 9.2%, and 8.8% in each of the three cohorts, respectively. None of these differences was statistically significant, although the authors recognized that the study was not powered to detect small differences.

Fig. 67.12 Mean change from baseline in best-corrected visual acuity by month for (A) MARINA, (B) ANCHOR, (C) PIER, (D) EXCITE, (E) PrONTO, (F) SUSTAIN.

(Panel A Copyright © 2006 Massachusetts Medical Society. All rights reserved. Panel B reproduced with permission from Brown DM, Michels M, Kaiser PK, et al. Ranibizumab versus verteporfin photodynamic therapy for neovascular age-related macular degeneration: 2-year results of ANCHOR Study. Ophthalmology 2009;116:57–65, Copyright 2009, with permission from Elsevier. Panel C reproduced with permission from Regillo, et al. Ranibizumab (Lucentis) in treatment of neovascular age-related macular degeneration (AMD): 2-year results of PIER study, poster PO459 presented at the AAO 2007. Panel E reproduced with permission from Fung AE, Lalwani GA, Rosenfeld PJ, et al. An optical coherence tomography-guided, variable dosing regimen with intravitreal ranibizumab (Lucentis) for neovascular age-related macular degeneration. Am J Ophthalmol 2007;143:566–83, Copyright 2007, with permission from Elsevier. Compilation reproduced with permission from Mitchell P, Korobelnik JF, Lanzetta P, et al. Ranibizumab (Lucentis) in neovascular age-related macular degeneration: evidence from clinical trials. Br J Ophthalmol 2010;94:2–13.)

The ANCHOR study assessed the response of patients with predominantly classic CNV as determined by fluorescein angiography.¹⁶⁴ Patients (n = 423) were randomly assigned to receive verteporfin-PDT plus sham injection (n = 143) or sham PDT plus injection of either 0.3 mg (n = 140) or 0.5 mg (n = 140) ranibizumab. At 12 months follow-up, approximately 95% of ranibizumab-treated patients lost less than 15 letters of vision versus 64% in the verteporfin active-treatment control group. Forty percent of patients treated with 0.5 mg ranibizumab gained at least 15 letters vision versus 6% in the verteporfin treatment cohort. At month 24, the visual benefit from ranibizumab remained statistically (P < 0.0001 vs PDT) and clinically significant: 89.9–90.0% of ranibizumab-treated patients had lost <15 letters from baseline (vs 65.7% of PDT patients); 34–41.0% had gained ≥15 letters (vs 6.3% of PDT group); and, on average, visual acuity was improved from baseline by 8.1–10.7 letters (versus a mean decline of 9.8 letters in the PDT groups).¹⁶⁶ Changes in lesion anatomic characteristics on fluorescein angiography also favored ranibizumab (all comparisons P < 0.0001 vs PDT). Overall, there was no imbalance among groups in rates of serious ocular and nonocular adverse events. In the pooled ranibizumab groups, 3 (1.1%) of 277 patients developed presumed endophthalmitis in the study eye (rate per injection = 3/5921, or 0.05%). Retinal detachment was observed in one patient in both the verteporfin (0.7%) and 0.3 mg ranibizumab (0.7%) cohorts. There were no cases of lens injury. The risk of hypertension was the same in all cohorts. The risk of myocardial infarction among verteporfin, 0.3 mg ranibizumab, and 0.5 mg ranibizumab cohorts was 0.7%, 0.7%, and 2.1%, respectively. The risk of stroke or cerebral infarction was 0.7% in each of the three cohorts. The risk of nonocular hemorrhage was 2.1%, 5.1%, and 6.4% in each of the three cohorts. Immunoreactivity to ranibizumab before treatment was 1.5%, 3.2%, and 0.8% among the verteporfin, 0.3 mg ranibizumab, and 0.5 mg ranibizumab cohorts, respectively. Immunoreactivity was present in 1.6%, 1.6%, and 3.9% of patients in each of these cohorts, respectively, after 12 months of treatment. (The concern regarding immunoreactivity is that patients who develop an immune response might exhibit increased intraocular inflammation following intravitreal injection and consequently might not respond to the medication as well as patients who do not exhibit such a response. In practice, most retina surgeons have not observed this phenomenon and the drug has been minimally immunogenic in the doses tested.¹⁶⁷)

In both the MARINA and ANCHOR trials, visual acuity improvement seemed to reach a plateau by the 4-month time point (Fig. 67.12). Because monthly injections were considered inconvenient and daunting for some patients and their family members, and entailed risk as well as considerable expense, studies were undertaken to evaluate alternative treatment regimens. The PIER and EXCITE studies provided useful information in this regard.^168,169 In PIER, a phase IIIb multicenter, randomized, double-masked trial, patients with subfoveal CNV were randomly assigned to sham injection (n = 63), 0.3 mg ranibizumab (n = 60), or 0.5 mg ranibizumab (n = 61). Patients received sham or ranibizumab injections every 4 weeks for three doses followed by additional treatment every 3 months. By month 12, mean changes from baseline visual acuity were −16.3, −1.6, and −0.2 letters for the sham, 0.3 mg, and 0.5 mg groups, respectively (P ≤ 0.0001, each ranibizumab dose vs sham). Ranibizumab arrested CNV growth and reduced leakage from CNV. The treatment effect declined, however, in the ranibizumab groups during quarterly dosing. At month 3, for example, the mean changes from baseline vision had been gains of +2.9 and +4.3 letters for the 0.3 mg and 0.5 mg doses, respectively. During study year 2, eligible sham-group patients crossed over to 0.5 mg ranibizumab quarterly. Later in year 2, all eligible randomized patients rolled over to 0.5 mg ranibizumab monthly dosing. The ranibizumab-treated patients showed an improvement in mean visual acuity during the first three months of the study, but this improvement was not sustained. Nonetheless, the 0.5 mg ranibizumab cohort had a mean visual acuity change that was 16 letters better than that of the sham cohort by month 12 (P < 0.0001). By month 24, visual acuity had decreased an average of 21.4, 2.2, and 2.3 letters from baseline in the sham, 0.3 mg, and 0.5 mg groups (P < 0.0001 for each ranibizumab group vs sham). The visual acuity of sham patients who crossed over to ranibizumab decreased over time, with an average loss of 3.5 letters 10 months after crossover. The visual acuity of patients in the 0.3 mg and 0.5 mg cohorts who rolled over to monthly ranibizumab injections increased for an average gain of 2.2 and 4.1 letters, respectively, 4 months after transition. These data indicate that injecting patients with ranibizumab every 3 months (after an induction phase of three monthly injections) does not produce the same chance for visual benefit as monthly injection, at least during the first 12 months of therapy. Ranibizumab appeared to provide additional visual benefit to treated patients who rolled over to monthly dosing, but not to patients who began receiving ranibizumab after >14 months of sham injections.

The EXCITE study, was a 12-month, multicenter, randomized, double-masked, active-controlled, phase IIIb study designed to demonstrate the noninferiority of a quarterly treatment versus monthly intravitreal ranibizumab treatment regimen in patients with AMD-associated CNV.¹⁷⁰ Patients with primary or recurrent subfoveal CNV secondary to AMD (353 patients), including those with predominantly classic, minimally classic, or occult (no classic component) lesions, were enrolled. Patients were randomized (1 : 1 : 1) to 0.3 mg quarterly, 0.5 mg quarterly, or 0.3 mg monthly doses of ranibizumab. Treatment comprised three consecutive monthly injections followed by a 9-month maintenance phase (either monthly or quarterly injection). In the per-protocol population (293 patients), visual acuity increased from baseline to month 12 by 4.9, 3.8, and 8.3 letters in the 0.3 mg quarterly (104 patients), 0.5 mg quarterly (88 patients), and 0.3 mg monthly (101 patients) dosing groups, respectively. Similar results were observed in the intent-to-treat (ITT) population (353 patients). The mean decrease in CRT from baseline to month 12 in the ITT population was −96.0 µm in the 0.3 mg quarterly, −105.6 µm in the 0.5 mg quarterly, and −105.3 µm in the 0.3 mg monthly group. At month 12, the visual acuity gain in the monthly treatment cohort was higher than that of the quarterly regimens. The noninferiority of a quarterly regimen was not achieved with reference to 5.0 letters.

The SUSTAIN study is a 12-month, phase III, multicenter, open-label, single-arm study involving 513 ranibizumab-naive patients with AMD-associated CNV.¹⁷¹ In this study, patients received three initial monthly injections of ranibizumab (0.3 mg) and thereafter pro re nata (PRN) retreatment for 9 months based on prespecified retreatment criteria. Patients switched to 0.5 mg ranibizumab after approval in Europe. The average number of retreatments from months 3 to 11 was 2.7. Mean best-corrected visual acuity increased steadily from baseline to month 3 to reach +5.8 letters, decreased slightly from month 3 to 6, and remained stable from month 6 to 12, reaching +3.6 at month 12. The mean change in CRT was −101.1 µm from baseline to month 3 and −91.5 µm from baseline to month 12.

In summary, data from the ANCHOR, MARINA, PIER, EXCITE, and SUSTAIN studies indicated that after a loading dose period of three monthly injections, subsequently monthly ranibizumab injections give superior visual acuity outcomes compared to quarterly or PRN injections (Fig. 67.13).

Fig. 67.13 Mean change in visual acuity from baseline at the end of the loading phase (•) and at 12 months (arrowhead) against the number of injections during 9 months of the maintenance phase (ranibizumab 0.5 mg data unless indicated).

(Reproduced with permission from Mitchell P, Korobelnik JF, Lanzetta P, et al. Ranibizumab (Lucentis) in neovascular age-related macular degeneration: evidence from clinical trials. Br J Ophthalmol 2010;94:2–13.)

In the PrONTO (Prospective OCT Imaging of Patients with Neovascular AMD Treated with Intraocular Lucentis) study, patients (n = 40) received 0.5 mg ranibizumab at entry, month 1, and month 2.¹⁷² Optical coherence tomography (OCT) measurements were obtained at baseline and at least monthly after injection (more frequently during the first two months after entry). Fluorescein angiograms were obtained at baseline and every 3 months thereafter. Retreatment with ranibizumab was done only if one or more of the following conditions was observed: (1) OCT central thickness (CRT) increased 100 µm; (2) ≥5 letter visual loss associated with subretinal fluid (as judged with OCT); (3) new onset classic CNV; (4) new macular hemorrhage; (5) persistent subretinal or intraretinal fluid was present 1 month after the previous injection. One day after the first injection, there was a decrease in the mean OCT thickness of 47 µm. By month 12, the mean visual acuity improved by 9.3 letters (P <0.001), and the mean central thickness decreased by 178 µm compared with baseline (P <0.001). Mean visual acuity improvement was 9.3 letters, and the mean central thickness had decreased by 178 µm. The average number of injections over the first year was 5.6. Visual acuity improved ≥15 letters in 35% of patients, and once macular fluid resorbed completely, the mean interval before another injection was 4.5 months. During the second year, the retreatment criteria were amended to include retreatment if any qualitative increase in the amount of fluid was detected using OCT.¹⁷³ Forty patients were enrolled and 37 completed the 2-year study. At month 24, the mean visual acuity improved by 11.1 letters (P < .001) and the OCT-CRT decreased by 212 µm (P < 0.001). Visual acuity improved by 15 letters or more in 43% of patients. These visual acuity and OCT outcomes were achieved with an average of 9.9 injections over 24 months. The PrONTO Study data suggested that OCT-guided variable-dosing regimens with intravitreal ranibizumab were capable of achieving visual acuity outcomes comparable to those from the phase III clinical studies, but with fewer intravitreal injections.

Although the risk of arterial thromboembolic events was not increased to a statistically significant degree among ranibizumab-treated patients in these phase III studies (overall risk ~2.1% in ranibizumab-treated patients during year 1 vs ~1.1% among controls), some concerns were expressed about relative safety compared with alternative therapies, efficacy aside, as the studies were not powered for statistical significance between the two different ranibizumab doses. Year 2 data from the MARINA study indicate the overall rate of antiplatelet trialists’ collaboration (APTC)-defined arterial thromboembolic events, which includes nonfatal myocardial infarction, nonfatal stroke, and death from a vascular or unknown cause,¹⁷⁴ was 4.6%, 4.6%, and 3.8% among the 0.5 mg ranibizumab, 0.3 mg ranibizumab, and control cohorts, respectively. Year 2 data from the ANCHOR study indicate the overall rate of APTC arterial thromboembolic events was 5%, 4.4%, and 4.2% in the 0.5 mg, 0.3 mg, and verteporfin-PDT cohorts, respectively. The PIER^168,169 and EXCITE¹⁷⁰ studies had similar results in this regard.

The SAILOR study was a phase IIIb study whose objectives are to evaluate the safety of 0.3 mg and 0.5 mg Lucentis in patients with AMD-associated subfoveal CNV. In cohort 1 of this study, the dose was randomly assigned and administered once a month for 3 months and thereafter as needed based on retreatment criteria. In SAILOR, there was a numerically higher rate of cerebrovascular stroke with 0.5 mg ranibizumab compared with 0.3 mg ranibizumab (1.2 vs 0.7%), which was not statistically significant in patients with a history of stroke.¹⁷⁵ In the SUSTAIN study, a total of 249 patients (48.5%) reported ocular adverse events, 5 patients (1.2%) with ocular serious adverse events involving the study eye (retinal hemorrhage, cataract, retinal pigment epithelial tear, reduced visual acuity, vitreous hemorrhage), 19 patients (3.7%) with arteriothromboembolic events were observed, with 8 deaths (1.5%).¹⁷¹ The most frequent adverse events in the study eye were reduced visual acuity (18.5%), retinal hemorrhage (7.2%), increased intraocular pressure (7.0%), and conjunctival hemorrhage (5.5%). Overall, the ocular and systemic risks associated with ranibizumab use appear to be low and within a reasonable range when taking into account the associated likelihood of visual improvement in properly selected patients (Table 67.2).¹⁶⁵

Table 67.2 Summary of key ocular and non-ocular adverse events in ranibizumab clinical trials

Comparison of Age-related Macular Degeneration Treatments Trial (CATT)

Despite a relative lack of level 1 evidence prior to 2011, bevacizumab had become the most widely used agent for AMD in the world due to a number of factors, including its low cost compared to ranibizumab, similarity in therapeutic mechanism, and its availability to physicians prior to the FDA approval of ranibizumab. A large amount of data from retrospective reviews, interventional case series, and anecdotal reports have provided physicians with evidence to support the continued off-label use of bevacizumab. However, in the absence of a large, randomized clinical controlled trial comparing bevacizumab to ranibizumab, the current gold standard, several questions have arisen:

1. Does one treatment offer superior visual outcomes to another?

2. What is the optimal treatment regimen and interval?

3. Is the safety profile of bevacizumab comparable to that of ranibizumab, which has been subjected to rigorous phase I – phase IV evaluation?

The CATT was designed to address these questions.¹⁵⁰

The CATT is a multicenter, randomized clinical trial, that enrolled 1208 patients with previously untreated subfoveal CNV due to AMD, as determined by leakage on fluorescein angiogram and fluid on time domain OCT. The primary outcome measure in the study was visual acuity change. The study designers also elected to examine the proportion of patients with a 15-letter difference in vision, number of injections, fluid and thickness on OCT, lesion size on fluorescein angiography, incidence of ocular and systemic adverse events, and annual treatment cost as secondary treatment endpoints. Patients were randomized to one of four groups: (1) ranibizumab 0.5 mg monthly (every 28 days); (2) bevacizumab 1.25 mg monthly; (3) ranibizumab as needed (when signs of active neovascularization were present); (4) bevacizumab as needed (with similar indications for retreatment as group 3). The study was designed to determine noninferiority of one treatment group with respect to each of the remaining three groups, using a statistically robust 99.2% confidence interval and a noninferiority limit of 5 letters on the ETDRS chart.

Visual acuity data were available for 1105 patients at one year. The six pairwise comparisons showed the following:

1. Bevacizumab monthly injections (8.0 letters gained) and ranibizumab monthly injections (+8.5 letters) yielded equivalent visual outcomes.

2. Bevacizumab given as needed (+5.9 letters) was also equivalent to ranibizumab given as needed (+6.8 letters).

3. Ranibizumab given as needed was equivalent to ranibizumab given monthly.

4. Ranibizumab given as needed was also equivalent to bevacizumab given monthly.

5. Bevacizumab given as needed was neither inferior nor not-inferior (inconclusive) to bevacizumab given monthly.

6. Bevacizumab given as needed was neither inferior nor not-inferior (inconclusive) to ranibizumab given monthly.

Detailed visual and anatomic outcome data are shown in Fig. 67.14 and Table 67.3.

Fig. 67.14 (A) Mean change in the visual acuity score during the first year of follow-up. (B) Differences between pairs of study groups in the mean change from baseline to 1 year in the visual acuity score. The red vertical lines indicate means, and the gray bars 99.2% confidence intervals. Negative values reflect a greater mean increase in group 2. Confidence intervals within −5 and +5 letters (dashed vertical lines) indicate that the two groups are equivalent. Confidence intervals extending beyond the noninferiority limit of −5 letters indicate that the comparison of the two groups is inconclusive with respect to noninferiority. (C) Proportions of patients in each group with a decrease of 15 letters or more, a change within 14 letters, or an increase of 15 letters or more from baseline values during the first study year.

(Reproduced with permission from Martin DF, Maguire MG, Ying GS, et al. Ranibizumab and bevacizumab for neovascular age-related macular degeneration. N Engl J Med 2011;364:1897–908.)

Table 67.3 CATT one-year outcomes: primary outcome (visual acuity) and additional outcomes

Secondary visual outcomes were similarly equivalent, with 91.5% (bevacizumab as needed) to 95.4% (ranibizumab as needed) of patients not having a decrease in vision of 15 letters or more from baseline, and 24.9% (ranibizumab as needed) to 34.2% (ranibizumab monthly) experiencing a gain of at least 15 letters. Anatomic outcomes showed that all groups had substantial decreases in retinal thickness as measured by OCT at 1 year, although the monthly ranibizumab group experienced a greater decrease in thickness (196 ± 176 µm) than those patients receiving bevacizumab as needed (152 ± 178 µm, P = 0.03). The mean number of injections given in the as-needed groups was 7.7 ± 3.5 (bevacizumab) and 6.9 ± 3.0 (ranibizumab) out of 13 possible, translating to an average yearly cost of $385 vs $13 800, respectively. The average cost to patients given monthly injections was $595 for bevacizumab and $23 400 for ranibizumab (Table 67.3).

Deaths occurred in 2.0% of the entire study group over the one-year period, with no significant difference among any of the groups. The occurrence of arteriothrombotic events was 2–3% for each group, also without significant differences. Venous thrombotic events occurred in 4 out of 286 patients receiving bevacizumab monthly (P = 0.08 difference between all groups) but occurred infrequently in all groups. When serious systemic adverse events were pooled among drug-specific groups, they occurred in 24.1% of patients receiving bevacizumab, vs 19.0% of those receiving ranibizumab (P = 0.04). Endophthalmitis occurred in 0.05% of the total number of injections, without a significant difference between medication groups. All other serious ocular adverse events occurred with rare frequency (Table 67.4).

Table 67.4 CATT first-year serious adverse events*

The impact of the one-year CATT study results was significant, establishing the equivalent visual outcomes of ranibizumab and bevacizumab when given monthly. Although bevacizumab as needed, when compared to ranibizumab or bevacizumab monthly, yielded inconclusive results, all other groups showed similar efficacy. Rosenfeld proposed in explanation that the treatment effect for bevacizumab may be less durable in some patients; thus, giving more frequent injections might improve visual outcomes in that subgroup.¹⁷⁶ Importantly, the first-year study results also appeared to establish that as-needed ranibizumab (as well as monthly bevacizumab) was as effective as monthly ranibizumab, which not only has significant implications for treatment cost, but also for the cumulative risk of injection-associated adverse events as well as patient anxiety surrounding treatment administration. Interestingly, although the effect of the medication on retinal thickness was more prominent for ranibizumab, this did not translate to better vision as compared to bevacizumab. Finally, the study authors recognized that the CATT was insufficiently powered to determine a difference in infrequently occurring systemic and ocular adverse events, and although bevacizumab was associated with a higher overall rate of systemic adverse events, these events involved organ systems not typically associated with systemic anti-VEGF therapy. In particular, the rate of arteriothrombotic and venous thrombotic events was no different between the two medications, and was low in all groups. Additionally, the risk of adverse events was not noted to correlate with increased exposure to medication, with a higher rate of occurrence in patients receiving injections on an as-needed basis.

Interested readers might also refer to the one-year interim report from the IVAN study. The efficacy and safety of ranibizumab and bevacizumab intravitreal injections to treat neovascular age-related macular degeneration were compared, and participants were randomize to four groups: ranibizumab or bevacizumab, given either every month (continuous) or as needed (discontinuous), with monthly review. The authors concluded: “The comparison of visual acuity at 1 year between bevacizumab and ranibizumab was inconclusive. Visual acuities with continuous and discontinuous treatment were equivalent. Other outcomes are consistent with the drugs and treatment regimens having similar efficacy and safety.”^176a

The second year results of CATT, published in mid-2012, described the visual impact of switching from monthly to as-needed treatment after the first year, as well as any differences in disease activity or progression, as demonstrated by fluid on OCT, leakage on fluorescein angiography, and lesion size. In addition, the use of high-resolution spectral-domain OCT in year two allowed for comparison between standard and high-resolution imaging in the detection of disease activity.¹⁷⁷

At week 52 of the study, the study groups were modified such that monthly treatment groups were reassigned at random to either maintain monthly dosing or switch to as-needed dosing of the same medication. The existing as-needed groups maintained the same dosing regimen. Comparisons in the second year were not made between each drug regimen group, as the reassignment resulted in a larger number of groups, and consequently reduced group size and statistical power. Rather, the analysis compared bevacizumab with ranibizumab, and monthly with as-needed dosing groups overall.

In patients maintaining the same dosing regimen (either monthly or as needed) through the two-year study period, vision change remained similar, with monthly ranibizumab showing the greatest visual improvement (+8.8 letters), followed by monthly bevacizumab (+7.8), as-needed ranibizumab (+6.7), and as-needed bevacizumab (+5.0). There was no significant difference when comparing all ranibizumab groups with all bevacizumab groups (which showed a 1.4-letter smaller gain relative to ranibizumab), but as-needed therapy showed less vision improvement by 2.4 letters relative to monthly therapy (P = 0.046). Secondary visual outcomes were also similar, including the final mean acuity (Snellen equivalent about 20/40 in all groups), the proportion gaining or losing 15 letters, and the proportion with 20/20 or better or 20/200 or worse final vision. Of note, those receiving bevacizumab as needed required more injections over the course of the study (14.1 total injections) than those receiving ranibizumab as needed (12.6), a significant difference consistent with the higher proportion of visits with detectable fluid.

Anatomic outcomes showed improved retinal thickness in patients treated monthly as compared to those treated as needed (29 µm difference, P = 0.005). Additionally, the proportion of patients without fluid as detected by OCT was lower in both the ranibizumab group as well as the monthly group (up to 45.5% in the monthly ranibizumab group); similarly, the proportion of patients with no fluorescein leakage was higher in patients treated monthly, and lesion area remained stable in these patients while growing in the as-needed group. However, the study also found that the proportion of monthly patients developing geographic atrophy was significantly higher in patients receiving monthly treatment as compared to the as-needed group, with the highest rate in those receiving ranibizumab monthly. (Vision and anatomic outcomes are detailed in Fig. 67.15 and Table 67.5).

Fig. 67.15 (A) The mean change in visual acuity from enrollment over time by dosing regimen within drug group ranibizumab (left) and bevacizumab (right). (B) Differences in mean change in visual acuity at 2 years and 95% confidence intervals in patients treated with the same dosing regimen for 2 years. (C) The 3-line change in visual acuity from enrollment by treatment group and follow-up time.

(Reproduced with permission from Martin DF, Maguire MG, Fine SL, et al. Ranibizumab and bevacizumab for treatment of neovascular age-related macular degeneration: two-year results. Ophthalmology 2012. Corrected proof published online 2012 May 2. http//:www.ophsource.org/periodicals/ophtha/article/S0161-6420(12)00321-1/ [cited 2012 June 8].)

Table 67.5 CATT 2-year outcomes: patients treated with the same dosing regimen for 2 years

In contrast to patients maintaining monthly therapy through two years, those switching to as-needed therapy at week 52 lost from 1.8 (ranibizumab) to 3.6 (bevacizumab) letters after year one (P = 0.03 when comparing monthly to as-needed therapy), resulting in very similar visual outcomes to patients maintained on as-needed therapy since study enrollment for both medications. Final visual acuity was similar for all groups, without significant differences between bevacizumab and ranibizumab, or between monthly and as-needed therapy. Anatomically, retinal thickness increased significantly, with a range of +19 µm (ranibizumab) to +31 µm (bevacizumab) in patients who were reassigned to as-needed therapy. The proportion of patients without fluid on OCT was significantly higher in patients who switched regimens, as well as in patients receiving bevacizumab. As in patients receiving the same regimen through year two, the proportion demonstrating dye leakage was higher in switched patients. Although the lesion area was not significantly different in either group, the rate of development of geographic atrophy was higher in ranibizumab patients as well as those receiving monthly dosing (Table 67.6).

Table 67.6 CATT 2-year outcomes: patients whose dosing regimen was reassigned at 1 year

In contrast to the first year of the study, when all study patients underwent testing with time domain OCT only, in year two 22.6% of scans were performed with high-resolution spectral domain OCT. Treatment decisions in the second year were consistent with reading center interpretation of OCT fluid in 68.5% of the ranibizumab group and 69.6% of the bevacizumab group; 95% of these cases resulted in undertreatment (the reading center interpreted fluid, yet the patient did not receive injection). The use of spectral domain OCT did not appear to increase the consistency between treating physicians and the reading center, with 70.1% agreement in the spectral domain group versus 68.7% in the time domain group.

As reported in the first year of results, the rate of systemic adverse events was significantly higher in the bevacizumab group than in the ranibizumab group, at 39.9% as compared to 31.7% overall, and 24.4% vs. 18.0% in the second year alone. Again, this difference persisted after excluding vascular events previously associated with systemic anti-VEGF drugs (P = 0.02), while the difference in events previously associated with anti-VEGF therapy approached statistical significance (P = 0.07). In addition, as noted in the first year, over the course of two years, patients treated as needed had a higher rate of systemic serious adverse events than those treated monthly (risk ratio of 1.30, P = 0.009), although this difference in the second year alone was not significant. There was no difference between drugs in the proportion of patients who died (6.1% vs 5.3%) or in the number of arteriothrombotic (5.0% vs 4.7%) or venous thrombotic events (1.7% vs 0.5%). Endophthalmitis occurred in 7 patients receiving bevacizumab and 4 patients receiving ranibizumab (P = 0.38), with 10 of these 11 cases occurring in patients receiving monthly therapy (Table 67.7).

Table 67.7 CATT adverse events within 2 years of enrollment

The second year of the CATT was not designed to determine the relative efficacy of each dosing regimen for each medication. However, one may conclude the following:

1. In contrast to the first-year results, longer-term follow-up suggests that as-needed therapy is somewhat less effective than monthly therapy overall in terms of visual gain.

2. Visual acuity and visual gain are associated with, but do not closely mirror retinal thickness or the presence of fluid.

3. One year of monthly therapy does not appear to confer lesion stability when subsequently changing to as-needed therapy.

4. When given as needed, bevacizumab is required more frequently, on average 1.5 more injections over the two-year period.

5. Geographic atrophy occurs with greater frequency in patients receiving monthly therapy.

6. Spectral domain OCT does not appear to increase the accuracy of identifying retinal fluid by the treating physician.

7. Endophthalmitis occurs at similar rates in ranibizumab- and bevacizumab-treated patients, although (as one may expect) it is appears to be associated with an increased rate of injections, occurring most often in patients treated monthly.

8. The increased rate of systemic serious adverse events in bevacizumab persisted in the second year. The authors remained unsure of the reason for the increased risk ratio of systemic adverse events. As in the first year, the rate of vascular events previously associated with anti-VEGF therapy was similar in both groups.

The two-year CATT results certainly support the continued off-label use of bevacizumab for exudative AMD. In addition, it appears that monthly dosing results in better and more sustained visual outcomes than as-needed dosing. However, any treatment regimen should still be made with careful consideration, given the cost of monthly therapy (roughly 60–80% higher than that of as-needed therapy for each medication), as well as the increased risk of geographic atrophy, which over a treatment course of many years may result in significant visual loss.

Soluble receptor: aflibercept (VEGF-TRAP EYE)

Aflibercept (VEGF-TRAP EYE, EYLEA™) is a product of Regeneron Pharmaceuticals. It is soluble fusion protein which combines ligand-binding elements taken from the extracellular components of VEGF receptors 1 and 2 fused to the Fc portion of IgG1,¹⁷⁸ It has at least a 200-fold higher affinity for VEGF than ranibizumab,¹⁷⁹ and aflibercept is thought to penetrate all layers of the retina.¹⁸⁰ Unlike bevacizumab and ranibizumab that inhibit just VEGF-A, aflibercept also binds VEGF-B and PlGF.^179,181 Aflibercept has undergone phase I and II clinical trials in wet AMD, and recently concluded phase III clinical testing. The phase I study, known as CLEAR-IT 1 (CLinical Evaluation of Anti-angiogenesis in the Retina Intravitreal Trial), showed safety and a functional and anatomical improvement with a dose of 0.5 and 2 mg.¹⁸² The phase II trial, known as Clear-IT 2, evaluated the biologic effects and safety of aflibercept during a 12-week fixed-dosing period in patients with exudative AMD followed by PRN dosing out to 1 year.^183,184 Patients were randomized 1 : 1 : 1 : 1 : 1 to aflibercept during the fixed-dosing phase (day 1 to week 12): 0.5 or 2 mg every 4 weeks on day 1 and at weeks 4, 8, and 12; or 0.5, 2, or 4 mg every 12 weeks on day 1 and at week 12. At week 12, treatment with aflibercept resulted in a significant mean decrease in central retinal thickness from baseline in all groups combined (P < 0.0001). The reduction with the 2 mg every 4 weeks and 0.5 mg every 4 weeks regimens was significantly greater than each of the quarterly dosing regimens. Visual acuity increased significantly by a mean of 5.7 letters at 12 weeks in the combined group (P < 0.0001), with the greatest mean gain of >8 letters in the monthly dosing groups.

In the PRN dosing phase of the study out to week 52, the decrease in central retinal thickness observed at week 12 versus baseline remained significant at weeks 12–52 (−130 µm from baseline at week 52) and CNV size regressed from baseline. After achieving a significant improvement in visual acuity during the 12 weeks, PRN dosing for 40 weeks maintained improvements in visual acuity out to 52 weeks (5.3-letter gain; P < 0.0001). The most robust improvements and consistent maintenance of visual acuity generally occurred in patients initially dosed with 2 mg every 4 weeks for 12 weeks. These patients demonstrated a gain of 9 letters at 52 weeks. Overall, a mean of 2 injections was administered after the 12-week fixed-dosing phase, and the mean time to first reinjection was 129 days. Nineteen percent of patients received no injections and 45% received 1 or 2 injections. During the year-long study, PRN dosing with VEGF-TRAP EYE at weeks 16–52 maintained the significant anatomic and vision improvements established during the 12-week fixed-dosing phase with a low frequency of reinjection, and the drug was generally safe and well tolerated.

Based on the phase II results, aflibercept appeared to be a new anti-VEGF treatment requiring fewer intravitreal injections compared with ranibizumab and bevacizumab. This possibility is being tested in the recently completed phase III VIEW 1 and VIEW 2 studies (VEGF Trap-Eye: Investigation of Efficacy and Safety in Wet AMD) in which aflibercept is being compared with the standard monthly dosing regimen of 0.5 mg ranibizumab (NCT00509795). In these two parallel phase III studies, patients with exudative AMD are randomized 1 : 1 : 1 : 1 to aflibercept 0.5 mg monthly, aflibercept 2 mg monthly, aflibercept 2 mg every 2 months (following three monthly loading doses), or ranibizumab administered 0.5 mg every month during the first year of the studies. As-needed (PRN) dosing with both agents, with a dose administered at least every 3 months (but not more often than monthly), is being evaluated during the second year of each study. The primary endpoint was statistical noninferiority in the proportion of patients who maintained (or improved) vision over 52 weeks compared to ranibizumab.

After 1 year, there was no difference in the outcomes when the three aflibercept groups were compared with the ranibizumab group.^185,186 This was true with respect to visual acuity, OCT central retinal thickness, and safety outcomes. The most important outcome for patients and clinicians was the observation that aflibercept 2 mg dosed every 2 months was equivalent to ranibizumab dosed monthly suggesting that aflibercept could be dosed less frequently. The phase III main results manuscript is expected to be published in 2012.

KH902

KH902 (Chengdu Kanghong Biotechnology) is also a fusion protein that combines ligand-binding elements taken from the extracellular domains of VEGF receptors 1(Flt-1) and 2 (KDR) to the Fc portion of IgG1.¹⁸⁷ However, compared with aflibercept (VEGF TRAP-EYE), KH902 also includes the extracellular domain 4 of VEGF receptor 2(KDRd4) which may stabilize the three-dimensional structure and increase the efficiency of dimerization.¹⁸⁸ KH902 has a much higher affinity for VEGF due to this additional extracellular domain and may have a longer half-life in the vitreous.^187,189 In vivo experiments have shown that intravitreal KH902 was able to inhibit leakage and growth of CNV in rhesus monkeys without signs of toxicity at doses of 300 and 500 µg.¹⁸⁷ In a phase I, dose escalation study, intravitreal injections of KH902 up to a dose of 3.0 mg were well tolerated in 28 patients. On day 42 after a single injection, the mean change in visual acuity from baseline was +19.6 letters with no subjects losing 1 letter or more and 57% of patients gaining 15 letters or more from baseline. The mean change in center-point retinal thickness from baseline was −77.2 µm and the mean decrease in CNV area was 12.6%. No safety concerns were detected after a single, intravitreal injection. A phase III trial is currently underway in China.

Adeno-associated viral vector (AAV) gene transduction

As previously discussed, the angiogenic effects of VEGF are mediated through the endothelial receptor tyrosine kinases, VEGFR-1 (also called Flt-1) and VEGFR-2 (Flk-1/KDR),⁷³ with Flt-1 having a tenfold-higher affinity for VEGF.¹⁹⁰ A naturally existing soluble form of Flt-1 (sFlt-1) has no transmembrane domains, and thus has no signal transduction properties, instead forming a heterodimer with Flt-1 and Flk-1 extracellular domains.¹⁹¹ This molecule has provided a unique target for gene therapy using an adeno-associated viral (AAV) vector.

AAV vectors are particular suited for gene therapy in part because of their low immunogenicity and pathogenicity, and ability to induce long-term gene expression in the eye. They have been used with success for gene transduction of PEDF for animal models of CNV,¹⁹² and have been tested in human trials for RPE65-associated Leber congenital amaurosis.^193–195Moreover, for a soluble gene product such as sFlt-1, gene therapy may be especially effective, as transduction need not be cell-specific.¹⁸⁸

AAV-mediated sFlt-1 (and related fusion protein) gene therapy has been successful in a number of animal models, including rodent and primate.^{190,196–199} Lai et al. tested gene transfer using an injected AAV vector with full-length sFlt-1 on both mice and monkeys.¹⁹⁰ In the mouse, they found a marked decrease in hyperfluorescence on fluorescein angiography in sFlt-1 transduced eyes when compared to noninjected and control AAV-Green Fluorescent Protein (GFP)-injected eyes. Morphologically, they found that more extensive CNV-induced photoreceptor loss occurred in the noninjected and control eyes when compared to sFlt-1 eyes. Electroretinographic studies showed no significant loss of function as a result of sFlt-1 transduction. Primate studies showed similarly encouraging results, including prevention of CNV induced by laser, without evidence of toxicity on histology. Importantly, there was long-term expression of transduced gene products, with detection of sFlt-1 mRNA at up to 17 months after injection. A similar recent primate study tested an AAV vector tied to a gene for a novel fusion protein of a single domain of sFlt-1 and a human IgG1 heavy chain Fc fragment (termed sFLT01), finding both long-term expression over 5 months, as well as inhibition of CNV in a dose-dependent fashion.¹⁹⁸ A phase I clinical trial is currently recruiting for AAV2-sFLT01 gene therapy for neovascular AMD (NCT01024998).

Oligonucleotide aptamer (pegaptanib – Macugen)

The first VEGF inhibitor approved for use in macular degeneration was pegaptanib. Although it has largely been replaced by the monoclonal anti-VEGF antibodies bevacizumab and ranibizumab, it represented the first targeted molecular therapy for macular degeneration, and therefore is of particular historical interest. Pegaptanib is an aptamer, or a short single-stranded nucleic acid sequence of specified strand shape and length, which has a high degree of specificity and affinity for a target polypeptide.²⁰⁰ In the case of pegaptanib, a large library of RNA sequences ranging from 10¹⁴ to 10¹⁵ sequences are incubated with the target molecule, VEGF isoform 165. The molecule is capable of selectively binding only VEGF₁₆₅, and not the other isoforms or other receptors and providing sustained inhibition for in excess of 20 days in vitro corresponding to a desired intraocular concentration in excess of the minimal inhibitory concentration necessary to inhibit VEGF₁₆₅ for a period of approximately 6 weeks.²⁰¹

Phase II/III double-masked, multicenter clinical trials were conducted in humans in the United States and in Europe concurrently.²⁰² A total of 1186 patients were randomized to one of three treatment doses or sham. Results showed lower risk of moderate and severe vision loss with pegaptanib vs control, with a favorable safety profile. As stated previously, pegaptanib is currently used infrequently for the treatment of exudative AMD, except in the context of combination therapy, or in some cases, maintenance therapy following induction with ranibizumab or bevacizumab.²⁰³

Small interfering RNA (siRNA)

RNA interference is the process of sequence-specific post-transcriptional gene silencing in animals and plants, which is initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silent gene. Confronted with double-stranded RNA eukaryotic cells respond by destroying their own messenger RNA (mRNA) that shares these sequences with the double-strand.²⁰⁴ The process appears to be mediated by 21 and 22 nucleotide siRNAs that are generated by ribonuclease 3 cleavage from longer dsRNAs. siRNA differs from an aptamer in being double- rather than single-stranded RNA; mechanistically, the presence of siRNA results in the inhibition of protein synthesis. In this context, neovascularization is theoretically prevented because VEGF mRNA is destroyed prior to translation, in contrast to the inhibition through the binding of free VEGF₁₆₅ by an aptamer such as pegaptanib. In testing this hypothesis, siRNA specific for human vascular endothelial growth factor (VEGF) and an enhanced green fluorescent protein (EGFP) were designed and tested as active molecule and negative control in both in vitro cell lines chemically induced to simulate hypoxia as well as in a laser-induced model of CNV in mice. siRNA was delivered by coinjection with recombinant viruses carrying small hVEGF cDNA to induce expression of the appropriate siRNA in the subretinal space. Production of siRNA was confirmed by the expression of EGFP in the subretinal space with positive controls, and as evidence of VEGF transgene expression, which resulted in significant inhibition of CNV compared with negative controls after laser photocoagulation.²⁰⁴ Conventionally injected siRNA at dosages of 70, 150 or 350µg in a 0.05 ml volume with a vehicle serving as a negative control was successful in reducing CNV in a primate model following standardized laser injury between 15 and 36 days after treatment in conjunction with reduced permeability, angiographically and reduced lesion sizes measured by planimetric methods in a masked fashion⁸¹ (Fig. 67.16).

Fig. 67.16 Effects of siRNA treatment in murine model of choroidal neovascularization.

(Reproduced with permission from Reich SJ, Fosnot J, Kuroki A, et al. Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model. Mol Vis 2003;9:210–6.)

A recent phase I study of Sirna-027, a siRNA molecule targeting a conserved region of VEGFR-1, has been conducted. Twenty-six patients were divided into cohorts of 3–6, each receiving sequentially escalating doses of 200–1600 µg in order to determine the maximally tolerated dose. The study authors reported no dose-limiting toxicity, and few medication-specific adverse events. Additionally, visual acuity improved by a mean of +5.5 letters, and stabilized or improved in the vast majority of study participants. Foveal thickness as measured by OCT showed improvement of up to 20.3% in one cohort. The study illustrated the therapeutic principle, as well as showing a favorable safety profile of siRNAs for the treatment of neovascular AMD.²⁰⁵ Two other molecules have also undergone clinical trials. RTP801i-14 (PF-04523655, Quark/Pfizer), a small inhibitory RNA (siRNA) that targets VEGF production via the REDD1/mTOR/HIF1-alpha pathway, is currently in a phase II study (NCT00713518) as adjunctive therapy to ranibizumab for treatment of AMD-associated CNV.

Interestingly, recent evidence suggests that the effect of siRNAs on CNV may in fact be a class effect of the molecule, and not contingent upon sequence specificity of the dsRNA.^206–208 The effect is instead postulated to occur through a toll-like receptor, TLR3, expressed on the endothelial cell surface, and mediated by the interferon-gamma and IL-12 cytokines.²⁰⁸ How this may influence further study of siRNA pharmacotherapy is unclear.

Intracellular signaling blockers

Complement inhibitors*

Complement inhibitors are thought to be a promising class of agents for the treatment of both atrophic and exudative AMD. POT-4 (AL-78898A, Alcon/Potentia Pharmaceuticals), a cyclic peptide derivative of Compstatin 13 amino acids in length, is a C3 inhibitor administered by intravitreal injection. Gel-like deposits form in the vitreous when POT-4 is injected at high concentrations (>0.45 mg dose), which may last as long as 6 months, thus providing a sustained-release delivery system. A phase I study of POT-4 in AMD eyes with CNV was completed successfully without any safety concerns at doses up to 1.05 mg (NCT00473928), although the systemic effects of intravitreal administration are unknown. A phase II study is currently underway (NCT01157065). C3 inhibition should block complement activation arising from many of the currently described complement pathway mutations, as the classical, lectin, fibrinolytic, and alternative pathways all generate the bioactive fragments C3a and C5a and the membrane attack complex (C5b,6,7,8,9) via C3 cleavage (see Fig. 67.3). This targets a relatively large population of AMD patients; however, the broad degree of complement inhibition may theoretically increase risk of intravitreal injection-associated endophthalmitis. In a murine model, it seems that C3 deficiency does not increase the risk of Staphylococcus aureus endophthalmitis.²¹⁷ In contrast, in a guinea-pig model, complement depletion with cobra venom factor does seem to increase the risk of S. aureus, S. epidermidis, and Pseudomonas aeruginosa endophthalmitis.^209,218,219 Other treatments related to complement factors are being examined in the context of nonexudative AMD and have been discussed previously.

Pigment epithelial-derived factor

Pigment epithelial-derived factor (PEDF) was first purified in the conditioned media of human retinal pigment epithelial cells that induced neuronal differentiation as well as inhibiting microglial growth. The protein shares sequence and structural homology with the serum proteinase inhibitor family (Serpen). It acts in counterpoint to VEGF, inhibiting signaling pathways of the VEGF receptors.^101,102 One of its most striking abilities is the capacity to inhibit endothelial cell migration in a dose-dependent manner with a greater degree of potency than either angiostatin, thrombospondin-1 or endostatin. It has been possible to use adenovirus vector-mediated gene transfer of cDNA constructs to iris pigment epithelium (IPE) resulting in autologous IPE cells gaining the capacity to support the choriocapillaris and outer retina in vivo.²²¹ Iris pigment epithelial cells overexpressing PEDF have been found to be capable of rescuing photoreceptors in RCS rats, reducing laser-induced CNV, and partially reversing the effects of ischemic retinopathy in various animal models.⁸⁷ An open-label phase I study of AdPEDF.11 in patients with CNV was designed to test both the safety and feasibility of adenoviral gene transfer for retinal diseases.²²² In the 28 patients administered the gene vector, there were no serious adverse events, and only transient intraocular pressure rise and intraocular inflammation without evidence of adenoviral infection. Additionally, although the study was not designed to determine efficacy, there appeared to be a trend toward visual stability in patients receiving a 10⁸ particle units (PU) or higher dose of the vector, as well as stability in lesion size in patients receiving ≥10⁸ PU, compared with increase in size in those receiving 10⁶–10^7.5 PU. The study authors concluded that gene transformation using an adenoviral vector was both safe and viable for future study.

Pathologically expressed circulating molecules

Integrin antagonists

Examination of tissues from eyes with pathologic neovascularization indicate that at least two cytokine-dependent pathways of angiogenesis exist and both may be important in pathologic neovascularization. In in vivo models of angiogenesis induced by basic fibroblast growth factor (bFGF) or tumor necrosis factor-alpha (TNF-α), the cell surface-expressed integrin alpha ν beta 3 predominates, whereas in models initiated by VEGF transforming growth factor-alpha (TGF-α), or phorbol ester the pathway is characterized by dependence on and expression of the related cytokine alpha ν beta 5.^69,70 These findings are further confirmed by evidence that inhibition of protein kinase C (using the specific inhibitor calphostin C) blocked angiogenesis induced by VEGF and TGF-α, but had only a small effect on bFGF or TNF-α-mediated angiogenesis. This suggests the specificity of the two pathways: (1) that dependent on alpha ν beta 3 and relatively independent of PKC; and (2) the second potentiated by alpha ν beta 5, and dependent upon both VEGF stimulation and PKC activation.^69,70

Therapeutically, several alternative methods for the inhibition of alpha ν beta 3 and alpha ν beta 5 expression exist. In immunohistochemical examination of tissues removed from eyes with pathologic neovascularization, it appears that for the purposes of inhibiting choroidal rather than retinal neovascularization, selective inhibition of the alpha ν beta 3 pathway may be preferred. When neovascular membranes removed from patients with CNV secondary to age-related macular degeneration were examined, only the expression of alpha ν beta 3 was observed whereas both alpha ν beta 3 and alpha ν beta 5 were present on vascular cells and tissues from patients with proliferative diabetic retinopathy.

A cyclic peptide antagonist (cyclo RGDv) of alpha ν beta 3 and alpha ν beta 5 was capable of dramatically reducing retinal vessel blood growth compared with a control peptide in a neonatal mouse model of retinopathy of prematurity. Other molecules in preclinical studies, including XJ735 (a selective alpha ν beta 3 antagonist), and XK002, a combined antagonist of alpha ν beta 3 and alpha ν beta 5, also inhibit retinal neovascularization in a murine model.²²⁷ Cyclic RGDfV peptide was found to represent an antagonist of vitronectin receptor type integrins by other groups.²

Another approach rather than specific receptor binding with polypeptide molecules is the use of selective monoclonal antibodies directed against cell surface integrins. Specific mouse monoclonal antibodies to integrins alpha ν beta 3 (LM609) and alpha ν beta 5 (P1F6) have been prepared, and in addition to demonstrating the presence of expressed integrins on vascular cell surfaces, have also been used therapeutically to inhibit neovascularization. Antibodies directed against alpha ν beta 3 appeared to selectively block bFGF-induced angiogenesis whereas antibodies directed against antibeta 1 and alpha ν beta 5 had dramatically less effect and appeared to spare pre-existing preformed vessels.^69,70 Other approaches have included the conjugation of mitomycin C dextran to a monoclonal anti-integrin alpha ν beta 3 antibody, which was more effective than the use of a mouse monoclonal alone in a rat model of laser-induced CNV.²²⁸ Yet another strategy involves the targeting of an proapoptotic peptide domain to a homing target that localizes to the alpha ν beta 3/beta 5 integrins expressed preferentially in neovascular endothelial cells, thus inducing apoptosis in the newly forming vessels. This strategy takes advantage of the expression pattern of the alpha ν beta 3/beta 5 integrins without specifically targeting their function or downstream effects, and has proven effective in a mouse model of retinopathy of prematurity.²²⁹

The integrin alpha 5 beta 1 has also been identified as a significant player in choroidal neovascularization.^230–232 It has been found to be significantly expressed in laser-induced CNV in animal models, and an inhibitor of alpha 5 beta 1 have been show to reverse corneal neovascularization as well as hypoxia-induced retinal neovascularization.^233,234 Currently, antagonists to alpha 5 beta 1 have shown mixed results in the inhibition or reversal of CNV in animal models. There is currently a phase I trial underway evaluating Volociximab, an anti-alpha 5 beta 1 monoclonal antibody already being tested for cancer therapy, for exudative AMD (NCT00782093).²³⁵

MMP inhibitors

The naturally occurring class of tissue inhibitors of metalloproteinase (TIMP), particularly TIMP-3, are thought to be critically important in the homeostasis of the extracellular matrix and thereby to factors contributing to the development of CNV. As discussed earlier, large quantities of TIMP-3 have been found with increasing age in Bruch’s membrane where it is thought to be a normal structural component and particularly in areas where there is pathologic thickening of Bruch’s membrane.^30,36,98 The carboxy terminus of TIMP-3, unlike the soluble TIMP-1, TIMP-2, and TIMP-4, is exclusively membrane-bound, while the amino terminus appears to inhibit MMPs 2 and 9, which are known to be expressed in choroidal new vessels membranes removed from patients with subfoveal CNV.⁹⁹ TIMP-3 naturally inhibits VEGF and is capable of reducing angiogenesis in vitro and in vivo including CAM assays and also when overexpressed by selective gene therapy in rat eyes undergoing laser photocoagulation for the induction of CNV.^99,100 Pharmacologic therapies directed at replacement or modification of TIMP-3 and potentially other related compounds may have utility as a pharmacologic strategy not only for regulation of the extracellular matrix in response to VEGF stimulation late, but also potentially earlier in regulating the homeostatic mechanisms that lead to Bruch’s membrane thickening and the other inducible factors for CNV preceding VEGF activation (Fig. 67.17).²³⁶

Fig. 67.17 Computerized rendering of TIMP-1 interacting as its active NH site with MMP-3 catalytic domain.

(Reproduced with permission from Gomis-Ruth FX, Maskos K, Betz M, et al. Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1. Nature 1997;389:77–81.)

Matrix metalloproteinase inhibitors (MMPI) have been tested for their ability to inhibit pathologic ocular neovascularization. AG3340, a synthetic MMP2,9 inhibitor was shown to significantly inhibit neovascularization in a mouse model of retinopathy of prematurity leading to a human phase II/III trial.²³⁷ However, the study was unable to confirm any inhibitory effect of the small molecular weight binding inhibitor of MMP-2 MMP-9 in any dose tested either with regards to lesion size, or final visual acuity and efforts were halted. Other MMP inhibitors continue to be tested in preclinical studies.

Squalamine

Squalamine is an antiangiogenic amino sterol derived from shark liver which inhibits iris neovascularization when administered by intravenous injection, although it is ineffective when administered by intravitreal injection.^239,240 Squalamine also induces regression of experimental retinopathy of prematurity in a mouse model²⁴¹ and has been tested in a phase I study of CNV in patients with no safety concerns expressed leading to several phase II trials. Initial visual results were promising, with improvement or stability in those patients receiving higher doses of the drug. Interestingly, the fellow non-study eyes with advanced macular degeneration also showed significant improvement after drug administration. A phase III study was initiated; however, it was terminated.

Steroids

Steroids have long known to be associated with neovascularization reduction by mechanisms that were not clearly understood. Folkman and colleagues were the first to suggest that the antiangiogenic effects of steroids could be separated into two categories: (1) those related to anti-inflammatory effects paralleling convention glucocorticoid and mineralocorticoid activity; and (2) a separate structural configuration of the pregnan nucleus conferring distinct antiangiogenic capability.^83,84 Although preclinical studies suggested benefit in terms of VEGF-stimulated vascular permeability and angiogenesis, and while steroids have long been used with success for diabetic macular edema, clinical studies employing conventional steroids alone for AMD have shown relatively unimpressive effects on the natural history of the disease. In the era of anti-VEGF therapy, steroid therapy is largely an adjunct; however, steroid monotherapy is still being examined in continuing clinical trials. At this time, a sustained release fluocinolone implant (Iluvien, Alimera Pharmaceuticals) is in phase II clinical trials for both choroidal neovascularization (NCT00605423) as well as geographic atrophy in AMD (NCT00695318). Additionally, there has been enthusiasm for potential beneficial effects associated with the use of intravitreal triamcinolone combined with conventional photodynamic therapy (PDT) using verteporfin for subfoveal CNV^242–252 (discussed below in a separate section on combination therapy).

Rapamycin

Rapamycin (sirolimus) is a macrocyclic, naturally occurring lactone that was initially isolated from the species Streptomyces hygroscopicus and given its name from Rapa Nui (Easter Island) during the course of a search for novel antifungal agents. Rapamycin is structurally related to the immunosuppressive agent FK506 (tacrolimus), which has been used extensively for the prevention of organ transplant rejection. Rapamycin requires direct interaction with at least two intracellular proteins to exert its action of arresting cell phase progression. FK binding protein (FKBP12) forms a rapamycin FK complex, which in turn binds to and inhibits the activity of the mammalian target of rapamycin (mTOR) through which it exerts its intracellular affects. Other FKBPs have been identified and interact with heat shock proteins. When bound to mTOR, phosphorylation does not occur, and the cells’ mitotic ability is blocked in addition to other effects involving protein kinase C delta, and IL-2 dependent functions that control the progression of T cells into the S phase.²⁵³

Rapamycin inhibits primary metastatic tumor growth by inhibition with angiogenesis through direct inhibition of vascular endothelial growth factors as well as endothelial cell responses to VEGF.²⁵⁴ Additionally, rapamycin, through its intrinsic immunomodulatory effects, may reduce macrophage chemotaxis and activation with concomitant reduction in release of VEGF and other angiogenic cytokines.

In experimental models, rapamycin has been shown to inhibit the development of ocular neovascularization in rats in response to subretinal matrigel placement orally at a dose of 2.5 mg per kilograms per day, or as an intraocular injection. Inhibition was also seen in a murine model of retinopathy of prematurity and laser-induced neovascularization when administered by an intraperitoneal route in doses of 2–4 mg per kilogram per day.²⁵⁵

A recent phase I/II pilot study has been conducted, comparing the effectiveness of adjunctive systemic administration of one of three immunosuppressive agents (including rapamycin) when combined with anti-VEGF treatment (either ranibizumab or bevacizumab), against anti-VEGF therapy alone.²⁵⁶ The prospective randomized, unmasked trial assigned patients 1 : 1 : 1 : 1 to receive either intravenous daclizumab, intravenous infliximab, oral rapamycin tablets, or observation for a study duration of 6 months. All patients were administered intravitreous bevacizumab (1.25 mg/0.05 mL or 2.5 mg/1.0 mL) or ranibizumab (0.5 mg) at intervals determined by the treating physician based on the presence of intraretinal or subretinal fluid on OCT imaging. The primary study outcome was the number of injections required in each group over the study period, which was also compared to the number of injections required prior to joining the study. There was a decrease in the number of injections in patients receiving daclizumab as well as rapamycin, although the study was small, and differences were not significant. However, the results do suggest that there may be a role for immunomodulators in the treatment of AMD. There is currently a phase I/II study underway examining subconjunctival administration of sirolimus for the treatment of geographic atrophy (NCT00766649).

Pharmacology of photodynamic sensitizers

Photodynamic therapy actually represents the combination of a pharmacologic therapy and a laser-based therapy in a two-step process capable of successfully treating subfoveal CNV. Photosensitizers have biophysical properties well suited to the thrombotic closure of abnormal neovascularization while preserving normal physiologic vessels. Most agents possess strong absorption properties in the far-red spectral region (660–780 nm), where light has the greatest penetration through blood and tissue. Photosensitizers also selectively bind to abnormal neovascularization through its expression of increased numbers of lipoprotein receptors contrasted with normal mature vessels, thus achieving the desirable feature of selectivity. Most photosensitizing molecules, including verteporfin, which has been approved for human use, as well as a number of agents under clinical evaluation, are structurally related to porphyrins (Fig. 67.18). Porphyrins are fused tetrapyrrolic macromolecules found in nature as pigments such as protoporphyrin IX, the nonprotein portion of hemoglobin.

Fig. 67.18 Structural formulae of photodynamic therapy agents either approved (verteporfin) or under investigation for use in humans.

(Reproduced with permission from Woodburn KW, Engelman CJ, Blumenkranz MS. Photodynamic therapy for choroidal neovascularization: a review. Retina 2002;22:391–405.)

Other molecules undergoing evaluation although not yet approved for use in humans include Tin-ethyl-etiopurpurin (Purlytin), monotexafin lutetium (optrin, Lu-tex), Npe6, and ATX-S10. Their chemical formulas are listed in Fig. 67.18. Purlytin was subjected to a large multicenter phase II/III clinical trial, which demonstrated a trend towards efficacy in selected subgroups, although it failed to meet the preapproved regulatory efficacy endpoint.

Lu-tex underwent a phase I dose escalation trial, in humans, which demonstrated its ability to achieve photodynamic closure, although associated with a greater degree of surrounding choroidal damage than verteporfin and also associated with peripheral paresthesias and occasional periocular pain in selected patients and the trial was discontinued. A phase I dose-ranging study of NPE6 was terminated. ATX-S10 has been the subject of continuing preclinical studies and at the time of this writing no human trials are under way.²⁵⁷

Verteporfin (Visudyne)

The benzoporphyrin derivative monoacid verteporfin (Visudyne) consists of two isomers that differ in the location of the carboxylic acid and methyl ester on the lower pyrrole rings of the chlorine macrocycle. Because verteporfin is hydrophobic, it requires formulation within liposomes, which in addition to improving penetration into cells and delivery by an intravenous route, also may actually further enhance its selectivity. Verteporfin is activated with a monochromatic laser light in the range of approximately 689–691 nm. The molecule is well tolerated following intravenous administration and cutaneous light sensitivity is kept to a minimum compared with other molecules, which either have longer periods of photosensitization, or less favorable therapeutic indices.

Verteporfin photodynamic therapy (PDT) was the first photosensitizer approved for the treatment of exudative AMD. The technique involves infusion of 6 mg/m² verteporfin over a 10 minute period followed by laser irradiation using a 689 nm diode laser (light dose: 50 J/cm²; power density: 600 mW/cm²; duration: 83 seconds) 15 minutes after the start of the infusion. Verteporfin demonstrated safety and efficacy in selectively localizing to choroidal neovascular membranes in preclinical trials in nonhuman primates. The mechanism of action is postulated to occur by activation of verteporfin to a triplet state following laser irradiation, resulting in the creation of singlet and reactive oxygen species that induces endothelial cellular damage and, ultimately, occlusion of the neovascular membranes through activation of the clotting cascade.^258–262 Unfortunately, the generation of free radicals necessary for the photothrombotic effect, may also serve as a proangiogenic stimulus, possibly accounting for the apparent benefit associated with concomitant administration of steroids.

Verteporfin PDT was demonstrated to be safe and effective for treatment of CNV due to AMD in phase I and II testing.^263,264 The Treatment of Age-related macular degeneration with Photodynamic Therapy (TAP) Study demonstrated that patients with CNV due to AMD demonstrated a beneficial effect following verteporfin PDT. The greatest benefit was seen among the subset of patients with predominantly classic subfoveal CNV due to AMD, with 67% of verteporfin-treated eyes versus 39% of placebo-treated eyes prevented from progressing to moderate visual loss – loss of 15 or more letters on the Early Treatment Diabetic Retinopathy Study (ETDRS) scale or approximately 3 lines of vision – P < 0.001. Lesions that were less than 50% classic did not demonstrate a treatment benefit. These results were valid through 2- and 3-year follow-up.^265,266 Further discussion of the role of verteporfin in AMD is covered in Chapter 66 (Neovascular (exudative or “wet”) age-related macular degeneration). Presently, verteporfin PDT is approved for the treatment of AMD lesions that are predominantly classic subfoveal CNV, or for occult or minimally classic subfoveal CNV less than 4 disc diameters in size.^202,265,267