Putting the Brakes on AMD

A primer for the general ophthalmologist.

By Louis K. Chang, MD, PhD

Though intravitreal injections for the treatment of neovascular age-related macular degeneration are almost exclusively the province of retina specialists, the huge advances made in the treatment of so-called "wet AMD" should be of interest to all ophthalmologists.

In some remote areas of the United States, where retina specialists are not immediately available nearby, general ophthalmologists have sometimes responded to the needs of primarily elderly patients by performing intravitreal injections, almost always in consultation with a retina specialist who makes the initial diagnosis and produces a treatment plan. Though we in the retina community believe that the diagnosis and treatment of retina disease should always involve the participation of a retina specialist, it is important that relevant knowledge be shared with the entire ophthalmic community.

Given the increasing incidence of both the wet and dry forms of macular degeneration, and an abundance of new therapies being studied, an update on current and potential treatments for AMD should be of value to all practitioners.

Advances in Anti-VEGF

The identification of a central role for vascular endothelial growth factor (VEGF) in the development of choroidal neovascularization (CNV) began the molecular era in the treatment of neovascular age-related macular degeneration. Pegaptanib sodium (Macugen, Eyetech), a selective blocker of VEGF-165, was approved for the treatment of wet AMD in December 2004 by the FDA.¹ It demonstrated the ability to slow the rate of vision loss, but did not show significant improvement in vision in a majority of patients.

The introduction of ranibizumab (Lucentis, Genentech) 18 months later dramatically changed the treatment paradigm for AMD-related CNV. Randomized controlled clinical trials showed that monthly ranibizumab treatment resulted in stabilization of vision in more than 90% of patients, and a three-line improvement in vision in about a third of all patients treated.^2,3 Bevacizumab (Avastin, Genentech) is a VEGF-neutralizing antibody with properties very similar to Lucentis that is used for the treatment of several systemic cancers. Off-label use of Avastin for the treatment of CNV has become widespread in the United States and worldwide.⁴ The Comparisons of Age-related macular degeneration Treatment Trial (or CATT), being conducted under the direction of the National Eye Institute, is a large-scale comparative clinical trial that is currently underway to evaluate the relative benefits of these two molecules in the management of CNV. Initial data are expected to be available in 2011.

Emerging Therapies

■ Other strategies to directly inhibit VEGF. Goals for new drugs designed to neutralize VEGF include higher binding affinity, longer duration of action (thereby decreasing the lifetime risks associated with repeated intravitreal injections), and safer delivery systems.

VEGF Trap-Eye (Regeneron Pharmaceuticals, Bayer Healthcare) is a fusion protein that combines features of two different VEGF receptor sites, thus allowing a higher binding affinity than the anti-VEGF drugs currently in clinical use (pegaptanib, ranibizumab and bevacizumab).¹⁰ This molecule has demonstrated effectiveness in improving visual acuity and reducing CNV size and OCT thickness in a phase 2 clinical trial and is currently in phase 3 testing in a direct head-to-head comparison with Lucentis.¹¹ With a longer intraocular half-life than Lucentis, the VEGF Trap-Eye may have a favorable lifetime risk profile.

Another approach to VEGF neutralization involves a soluble form of the high-affinity VEGF receptor VEGFR1/Flt-1. This soluble receptor sequesters VEGF in the extracellular space preventing the activation of downstream signalling pathways that result from VEGF binding to the native receptors on the cell surface. Preclinical trials have shown that adeno-associated virus serotype 2 (AAV2)-mediated intravitreal gene delivery of sFLT01 efficiently inhibits angiogenesis in murine and primate models.^12,13 With prolonged gene expression, viral vector-based therapy could greatly reduce the number of treatments required to maintain the biological effects.

■ Upstream targets. The process by which VEGF is generated is a complex cascade of events, each of which offers the possibility of therapeutic intervention. A key step in the signaling cascade that leads to the production of VEGF involves a molecule known as mTOR, the mammalian target of rapamycin. mTOR is a protein kinase that regulates cell proliferation, motility, survival and protein synthesis.⁵ It leads to the activation of hypoxia inducible transcription factors, including HIF1α, which in turn activates transcription of a number of genes, including those that produce VEGF.⁶ A number of agents are being developed to target this portion of the cascade.

Sirolimus (rapamycin, MacuSight/Santen), which targets mTOR1, has demonstrated pre-clinical evidence of anti-inflammatory, antiangiogenic and anti-fibrotic activity.⁷ Phase 1 testing has demonstrated evidence of potential effects in AMD through both subconjunctival and intravitreal delivery approaches. Phase 2 trials are currently underway for this drug, which has been given the name Perceiva and on October 21 received fast-track status from the FDA.

Everolimus (RAD001, Novartis) is a derivative of rapamycin and also inhibits mTOR.⁸ It is currently used in the prevention of allograft transplant rejection and for the treatment of systemic cancers, but research into its ophthalmic application is underway with a Phase 2 trial.

REDD1 also promotes VEGF production through the mTOR/HIF1α pathway.⁹ RTP801i-14 (Quark/Pfizer), now known as PF-4523665, is a small interfering RNA (siRNA) that has been developed to inhibit REDD1 and suppress VEGF production as well as inhibit angiogenesis. Results from a phase 1/2 trial showed that PF-4523655, delivered intravitreally, was safe and well tolerated in patients with exudative AMD. Preclinical evaluation of another siRNA drug that is specifically targeted toward HIF1α is also underway, with plans for bringing it to clinical phase 1 level by 2010.

■ Downstream targets. VEGF in the extracellular space binds to VEGF receptors on the surface of the target cell, initiating a signaling cascade that results in endothelial cell activation and angiogenesis. Many potential therapeutic molecules that target this portion of the cascade are currently under investigation.

One such molecule is AGN- 211745 (Allergan/Merck), a siRNA directed against the VEGF receptor, VEGFR1, that has completed initial phase 1 testing for AMD but that has not moved into further studies.¹⁴ By blocking VEGFR1 production, endothelial cells would be less responsive or unresponsive to elevated levels of VEGF.

A different approach employs immunization against VEGF receptors. Immunization against VEGFR2 resulted in cytotoxic T-cell-mediated regression in a murine model of laserinduced CNV.¹⁵ A phase 1 trial is underway examining the safety of weekly subcutaneous immunization with VEGFR1 and VEGFR2 peptides.

Another potential drug target at this level of the cascade is a family of transmembrane proteins known as integrins, which have a role in signaling and modulating downstream activities.¹⁶

Several agents are currently being studied that target the integrins as a means of controlling neovascularization in AMD. One drug, JSM6427 (Jerini), is a potent, highly specific integrin α5β1-antagonist that is currently undergoing phase 1 trials involving single and multiple injections for patients with CNV.¹⁷ One advantage of this molecule is that it may also inhibit the effects of growth factors and cytokines other than VEGF that promote angiogenesis, inflammation, and fibrosis.

Another integrin antagonist is volociximab (Ophthotech), a highaffinity monoclonal antibody that binds to α5β1 integrin and blocks binding of α5β1 integrin to fibronectin, a critical event in angiogenesis. Volociximab administration has resulted in strong inhibition of rabbit and primate retinal neovascularization and laser-induced choroidal neovascularization in monkeys.¹⁸ A phase 1 study of volociximab, in combination with ranibizumab in patients with wet AMD is ongoing.

The activation of VEGF receptors and many of the downstream signaling molecules is dependent on tyrosine kinases. A number of molecules that inhibit tyrosine kinases, with the expected effect of decreasing blood vessel growth and leakage, are currently under clinical investigation. One such molecule is pazopanib (GlaxoSmithKline), a kinase inhibitor that targets multiple VEGF family members. Pazopanib blocks several receptor tyrosine kinases including VEGF receptors 1, 2 and 3 and PDGFR, c-Kit and fibroblast growth factor receptor 1 and inhibits CNV in a murine model.¹⁹

Many of these other receptor tyrosine kinases activate pericytes and endothelial cells that populate choroidal neovascular membranes. By blocking multiple receptors, it is hoped that multi-kinase inhibitors may not only halt new vessel development but also induce regression of CNV itself. Phase 1 trials are underway to evaluate pazopanib, which is delivered by topical application. Other candidate receptor tyrosine kinase inhibitors include the topical drug TG100801 (TargeGen), the oral drug vatalanib (Novartis), and AG013958 (Pfizer) and AL39324 (Alcon), which are delivered by periocular injection.

VEGF-independent targets

Other molecular pathways are involved in angiogenesis and their role in development and maintenance of CNV are being explored. Fosbretabulin (combretastatin A4 phosphate, OXiGENE) is a novel anti-vascular agent that targets endothelial cells of abnormal vascular structures. The biologically active metabolite CA4 binds to tubulin and inhibits microtubule assembly, leading to occlusion of lumen of actively proliferating blood vessels.²⁰ It is currently undergoing phase 2 testing with intravenous administration in patients with the polypoidal variant of AMD seen commonly in Asia.

Sphingosine-1-phosphate (S1P) is an extracellular signaling and regulatory molecule implicated as one of the earliest responses to stress, promoting cellular proliferation, migration and activating survival pathways. Several lines of evidence suggest that S1P and its receptors play a major regulatory role in the neovascularization, fibrosis and inflammation related to AMD.²¹ Sonepcizumab (LT1009, LPath) is a humanized monoclonal antibody against S1P that binds S1P with high specificity at physiologic concentrations.²² A phase 1 multi-center, dose-escalation study of LT1009 administered as a single intravitreal injection in patients with exudative AMD is underway.

Pigment epithelium derived factor (PEDF) is a naturally occurring inhibitor of angiogenesis.²³ It is normally produced in ocular tissues and regulates normal blood vessel growth. PEDF levels have been found to be significantly decreased in eyes with AMD.²⁴ Ad-PEDF.11D (GenVec) is an intravitreal or peri-ocular injected transgene that uses a viral vector to deliver the PEDF gene, resulting in the local production of PEDF in the treated eye.²⁵ A phase 1, dose-escalation study of this agent in eyes with CNV secondary to AMD showed no significant adverse effects.

Platelet-derived growth factor (PDGF) is responsible for the recruitment, growth and survival of pericytes and serves to regulate vascular maturation. E10030 (Ophthotech) is a pegylated aptamer that binds and inhibits PDGF, and has been shown to inhibit or strip pericytes in preclinical models.²⁶ In a phase 1 clinical study in combination with ranibizumab, E10030 was well-tolerated and caused significant regression of neovascular lesions. As mentioned previously, some receptor tyrosine kinase inhibitors under investigation for the treatment of AMD also inhibit activation of PDGF receptor 1 in addition to VEGF receptors.

Another therapeutic target for neovascular AMD is the complement system, a part of the innate immune system that includes a series of endogenous proteins that act to inhibit excessive activation and protect host cells. Genetic and histopathologic studies support a role for abnormal activation of the complement system in different stages of AMD, including non-exudative AMD.^27-29 Thus, drugs targeting the complement system may find utility in the treatment of both dry and wet AMD. ARC1905 (Ophthotech) is an aptamer directed against complement factor C5 that is currently undergoing phase 1 testing, with intravitreal delivery in combination with ranibizumab in patients with exudative AMD.

Another agent, POT-4 (Potentia/Alcon), a small molecule derivative of Compstatin, is directed against complement factor C3. This drug has completed phase 1 testing in patients with exudative AMD with an excellent safety profile. One unique feature of this molecule is that is persists as a long-lasting gel deposit after injection into the vitreous. The phase 1 study demonstrated that significant levels of drug are maintained in the vitreous cavity for up to six months following a single injection.

New Drugs for Dry AMD

Unlike neovascular AMD, non-exudative or dry AMD is associated with slowly-progressive vision loss. Although the spectrum of disease includes asymptomatic patients with a few small drusen, severe vision loss is typically seen in eyes with geographic atrophy affecting the fovea. Currently, dietary supplementation with a combination of high dose vitamins A, C, E and zinc, based on the AREDS study, is the only intervention that has been shown in a large, randomized controlled trial to slow the progression of visual loss in the setting of dry AMD. The development of specific, effective treatments has been hampered by the slow natural progression of the disease and our lack of a clear understanding of the underlying pathophysiology.

Three promising treatments are currently in clinical trials for the treatment of geographic atrophy from AMD.

Fenretinide (Sirion Therapeutics) binds to retinol binding protein and decreases the production and build-up of lipofuscin and A2E, which are thought to exert toxic effects on the retinal pigment epithelium and promote geographic atrophy. A phase 2 double-masked, placebo-controlled trial of oral, once-daily fenretinide is currently underway. Interim results have shown a trend toward slower growth of GA, but the difference did not reach statistical significance.

OT-551 (Othera Pharmaceuticals) is a small molecule that has shown a protective effect in animal models of retinal degeneration. It has anti-oxidant, anti-inflammatory, and antiangiogenic properties and can be delivered topically. Within the eye, esterases convert it to TEMPOL-H, a derivative that cannot penetrate the cornea but has similar biological activities. A phase 2 trial is currently underway. At 12-months, there was a trend toward a reduction in moderate vision loss in treated eyes versus placebo.

NT-501 (Neurotech) is an implant containing human cells genetically modified to secrete Ciliary neurotrophic factor (CNTF). The implant is surgically implanted into the vitreous and continuously secretes CNTF into the vitreous cavity. In a phase 2 trial, eyes with the implant showed increase in the thickness of the outer retina and a trend toward visual stabilization when compared to sham surgery eyes. No serious adverse events were noted. It is also being investigated for the treatment of retinitis pigmentosa.

Summary

Elucidation of the molecular pathways involved in angiogenesis has resulted in the development of a number of promising candidate drugs that may more effectively inhibit the VEGF pathway and/or disrupt VEGF-independent pathways involved in neovascular AMD. Although tremendous gains have already been made, continued efforts on this front will hopefully improve the armamentarium available to ophthalmologists for combating this debilitating condition. OM

References

1. Gragoudas ES, Adamis AP, Cunningham ET, et al. Pegaptanib for neovascular age-related macular degeneration. N Engl J Med. 2004;351:2805-2816.
2. Rosenfeld PJ, Brown DM, Heier JS, et al; MARINA Study Group. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med. 2006;355:1419-1431.
3. Brown DM, Kaiser PK, Michels M, et al; ANCHOR Study Group. Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N Engl J Med. 2006;355:1432-1444.
4. Spaide RF, Laud K, Fine HF, et al.Intravitreal bevacizumab treatment of choroidal neovascularization secondary to age-related macular degeneration. Retina. 2006;26:383-390.
5. Ma XM, Blenis J. Molecular mechanisms of mTOR-mediated translational control. Nat Rev Mol Cell Biol. 2009;10:307-318.
6. Shoshani T, Faerman A, Mett I, et al. Identification of a novel hypoxia-inducible factor 1-responsive gene, RTP801, involved in apoptosis. Mol Cell Biol. 2002;22:2283-2293.
7. Dejneki NS, Kuroki AM, Fosnot J, et al. Systemic rapamycin inhibits retinal and choroidal neovascularization in mice. Mol Vis. 2004;10:964-972.
8. Schuler W, Sedrani R, Cottens S, et al. SDZ RAD, a new rapamycin derivative: pharmacological properties in vitro and in vivo. Transplantation, 64: 36-42.
9. Brugarolas J, Lei K, Hurley RL, et al. Regulation of mTOR function in response to hypoxia by REDD1 and the TSC1/TSC2 tumor suppressor complex. Genes Dev. 2004;18:2893-2904.
10. Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent antitumor effects. Proc Natl Acad Sci U S A. 2002;99:11393-11398.
11. Nguyen QD, Shah SM, Hafiz G, et al. CLEAR-AMD 1 Study Group. A phase I trial of an IV-administered vascular endothelial growth factor trap for treatment in patients with choroidal neovascularization due to age-related macular degeneration. Ophthalmology. 2006;113:1522.e1-1522.e14.
12. Rota R, Riccioni T, Zaccarini M, et al. Marked inhibition of retinal neovascularization in rats following soluble-flt-1 gene transfer. J Gene Med. 2004;6:992-1002.
13. Lai CM, Shen WY, Brankov M, et al. Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys. Mol Ther. 2005;12:659-668.
14. Shen J, Samuel R, Silva RL, et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther. 2006;13:225-234.
15. Mochimaru H, Nagai N, Hasegawa G, Kudo-Saito C, Yaguchi T, Usui Y, et al: Suppression of choroidal neovascularization by dendritic cell vaccination targeting VEGFR2. Invest Ophthalmol Vis Sci. 2007;48:4795-4801.
16. Avraamides CJ, Garmy-Susini B, Varner JA. Integrins in angiogenesis and lymphangiogenesis. Nature Rev Cancer. 2008;8:604-617.
17. Umeda N, Shu Kachi S, Akiyama H, et al. Suppression and regression of choroidal neovascularization by systemic administration of an α5β1 integrin antagonist. Mol Pharmacol. 2006;69:1820-1828.
18. Ramakrishnan V, Bhaskar V, Law DA, et al. Preclinical evaluation of an antialpha5beta1 integrin antibody as a novel anti-angiogenic agent. J Exp Ther Oncol. 2006;5:273-286.
19. Takahashi K, Saishin Y, Saishin Y, et al. Suppression and regression of choroidal neovascularization by the multitargeted kinase inhibitor pazopanib. Arch Ophthalmol. 2009;127:494-499.
20. Nambu H, et al. Combretastatin A-4 phosphate suppresses development and induces regression of choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003;44:3650-3655.
21. Maines, LW, et al., Pharmacologic manipulation of sphingosine kinase in retinal endothelial cells: implications for angiogenic ocular diseases. Invest Ophthalmol Vis Sci. 2006;47:5022-5031.
22. Xie B, Shen J, Dong A, et al. Blockade of sphingosine-1-phosphate reduces macrophage influx and retinal and choroidal neovascularization. J Cell Physiol. 2009;218:192-198.
23. Tong JP, Yao YF. Contribution of VEGF and PEDF to choroidal angiogenesis: a need for balanced expressions. Clin Biochem. 2006;39:267-276.
24. Holekamp NM, Bouck N, Volpert O. Pigment epithelium-derived factor is deficient in the vitreous of patients with choroidal neovascularization due to agerelated macular degeneration. Am J Ophthalmol. 2002;134:220-227.
25. Mori K, Gehlbach P, Ando A, et al. Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci. 2002;43:2428-2434.
26. Jo N, Mailhos C, Ju M, et al. Inhibition of platelet-derived growth factor B signaling enhances the efficacy of anti-vascular endothelial growth factor therapy in multiple models of ocular neovascularization. Am J Pathol. 2006;168:2036-2053.
27. Klein RJ, Zeiss C, Chew EY, et al. Complement factor H polymorphism in agerelated macular degeneration. Science. 2005;308:385-389.
28. Haines JL, Hauser MA, Schmidt S, et al. Complement factor H variant increases the risk of age-related macular degeneration. Science. 2005;308:419-421.
29. Edwards AO, Ritter R III, Abel KJ, et al. Complement factor H polymorphism and age-related macular degeneration. Science. 2005;308:421-424.