AMD Research: Current Needs and Future Directions
Muge R. Kesen, MD
Duke Eye Center
Scott Cousins, MD
Professor of Ophthalmology
Director of the Duke Center for Macular Diseases
Duke University Eye Center
Attempts to better understand the pathophysiology of angiogenesis in neovascular age-related macular degeneration (AMD) with hopes to improve the quality of life in patients with this disease resulted in the emergence of revolutionary discoveries and led us into the era of more targeted treatment approaches that include administration of monoclonal antibodies targeting vascular endothelial growth factor (VEGF). Although the treatment success for AMD has increased dramatically with these new agents, there is still much room to improve the overall care for these patients.
Identifying additional VEGF pathway antagonists with improved characteristics, such as prolonged duration of action and a less invasive route of delivery, is an important goal. Targeting multiple components of the angiogenesis cascade may also be more effective in AMD treatment. Lower costs, reduced risks and treatment burden, better safety profiles and consistent management algorithms are some of the current needs and important objectives of ongoing AMD research.
In the drug discovery and development pipeline, there are many research studies. Here is a summary of some of these, bringing us closer to an optimal treatment for AMD.
VEGF Trap (Regeneron Pharmaceuticals, Tarrytown, NY, USA) is a 110kDa soluble recombinant protein with the binding portions of VEGF receptor 1 and 2 fused to the Fc region of human IgG that binds all VEGF isoforms with a very high affinity (about 140 times that of ranibizumab). In a phase I, randomized, placebo-controlled trial of VEGF Trap administered intravenously for treatment of choroidal neovascularization, the Clinical Evaluation of Antiangiogenesis in the Retina (CLEAR)-AMD 1 group found a dose-dependent increase in systemic blood pressure with a maximum tolerated dose of 1mg/kg. This dose resulted in elimination of about 60% of excess retinal thickness after either single or multiple administrations.1 CLEAR IT-1 was a phase I dose escalation study of a single intravitreal injection of various doses of VEGF Trap (0.05, 0.15, 0.5, 1, 2, and 4mg). At 6 weeks, mean gain in visual acuity was 4.8 letters and mean OCT central retinal thickness decreased from 298μm to 208μm across all groups.2 Higher doses resulted in gaining more letters. The potential benefit of VEGF Trap is its longer duration of action compared with single injections of other VEGF-binding agents because of its high affinity and presumed longer intravitreal half-life based on its molecular mass. Based on the data, the biologic activity of ranibizumab (Lucentis) 0.5mg lasts for 30 days after intravitreal injection.3 Compared with the intravitreal binding affinity of ranibizumab using a time and dose-dependent mathematical model, the activity of a single injection of VEGF Trap (1.15mg) at 79 days was predicted to be comparable to the activity of ranibizumab (0.5mg) at 30 days.4 If this theoretical model is indeed correct, VEGF Trap would offer less frequent dosing resulting in fewer injections, lower cost and reduced risk of complications. The phase III trial with VEGF Trap will assess its efficacy and safety in patients with neovascular AMD. Approximately 1200 patients will be randomized in Europe, Asia, Japan, Australia and South America.
Small Interfering RNA (siRNA)
Miami-based OPKO Health, Inc. reported that the pioneering gene silencing agent bevasiranib (formerly known as Cand5; Acuity Pharmaceuticals, Philadelphia, PA), OPKO's most advanced therapeutic compound for the treatment of wet AMD, was named one of the top five most promising drugs entering phase III clinical trials. Bevasiranib is a first-in-class small interfering RNA (siRNA) drug that suppresses RNA translation by inactivating mRNA. These drugs are administered as double-stranded RNA molecules that are imported across the cellular membrane and processed by an enzyme, Dicer, which shortens the siRNA. The processed siRNA is incorporated into an RNA-induced silencing complex (RISC), which, when activated, binds complimentary mRNA and digests it. This allows a single molecule of siRNA to degrade multiple copies of mRNA. It is the first therapy based on the Nobel Prize-winning RNA interference (RNAi) technology to advance to phase III clinical trials. The CARE (Cand5 Anti-VEGF RNA Evaluation) study was a phase II trial that included three dose levels of bevasiranib (0.2, 1.5, and 3mg) injected intravitreally 6 weeks apart. Results revealed a trend towards dose dependent efficacy without discernible adverse effects in AMD patients with serious progressive disease. However, it was considered that by targeting a relatively upstream component of the VEGF pathway, bevasiranib may have a delayed effect in influencing disease processes. Therefore, combining bevasiranib with a VEGF-binding agent, such as ranibizumab or bevacizumab, to immediately bind existent VEGF while interfering with the upstream production, might actually offer a more rapid response. The multi-national phase III trial, COBALT (Combining Bevasiranib and Lucentis Therapy), is designed to compare the efficacy of bevasiranib administered every 8 weeks or 12 weeks with that of ranibizumab administered every 4 weeks. The safety profile and its potential for prolonged duration make it a promising drug for treatment of wet AMD. The study is currently enrolling patients at multiple clinical sites and will further examine efficacy parameters, and dose scheduling regimens.
A second siRNA drug, Sirna-027 (Sirna therapeutics, San Francisco, CA), a chemically stabilized siRNA directed against VEGF receptor 1 (VEGFR1) mRNA, has shown to reduce the levels of retinal VEGFR1 in two mouse models. With either route of delivery, intravitreal or periocular, there was sufficient reduction of VEGFR1 mRNA to suppress laser-induced CNV. Also, intravitreal injection of siRNA significantly suppressed ischemia-induced retinal NV, with corresponding reductions in VEGFR1 mRNA and protein.5 Early phase clinical trials are investigating the safety of a single injection at various doses.
VEGF Receptor Tyrosine Kinase Inhibition
VEGF binds two different receptor tyrosine kinases, VEGFR1 and VEGFR2. VEGFR2 is the major mediator of endothelial cell proliferation, migration, survival, and permeability. VEGFR2 is autophosphorylated after VEGF engagement, which leads to activation of a complex array of intracellular signal-transducing pathways. The concept of disrupted signaling appears to be effective in the pharmacological treatment of neovascularization. Oral administration of PTK787, a tyrosine kinase inhibitor that blocks phosphorylation of VEGF and PDGF receptors, provides complete inhibition of retinal neovascularization. This approach can prevent the development of new vessels while it has no effect on mature retinal vessels in murine oxygen-induced ischemic retinopathy.6
Vatalanib (formerly PTK-787; Novartis International, Basel, Switzerland) is a potent inhibitor of all known VEGF receptor tyrosine kinases, VEGFR1, VEGFR2, and VEGFR3. The "Safety and Efficacy of Oral PTK787 in Patients With Subfoveal Choroidal Neovascularization Secondary to Age-Related Macular Degeneration" (ADVANCE) study evaluated the tolerability and safety of 3 months treatment with PTK787 tablets given daily.
Multi-targeted kinase inhibitors have been shown to be effective in oncology. Newly developed small molecule kinase inhibitors (including TG100572, which inhibits VEGF, PDGF, and FGF receptors in addition to Src family of kinases, and TG100801, a prodrug version of TG100572) were designed and synthesized at TargeGen. Topical administration of TG100801 suppressed CNV in mice and reduced the retinal edema induced by retinal vein occlusion in rats, without observable safety issues.7 Data have suggested that the delivery of these agents occur by local penetration through sclera rather than by systemic absorption as neither compound was detectable in the plasma. Therefore, TG100801 may offer equal efficacy to injectable agents, while offering patients the greater convenience and potential safety advantages due to a non-invasive route of delivery and penetration limited to the eye. A multicenter, open-label, randomized, phase II study is evaluating the effects of 30 days of dosing with two dose levels of TG100801, instilled twice a day, on central retinal/lesion thickness, as measured by optical coherence tomography (OCT). The safety of TG100801 in patients with AMD will also be evaluated in this trial.
Pazopanib (GW786034), by GlaxoSmithKline, is a second-generation multi-targeted tyrosine kinase inhibitor against all VEGF receptors, PDGFRα, PDGFRβ, and c-kit. An early phase trial is evaluating the pharmacodynamics, safety, and pharmacokinetics of Pazopanib eye drops in patients with neovascular AMD.
Pigment Epithelial-Derived Factor (PEDF)
PEDF is one of the most potent antiangiogenic proteins found in humans. This factor also has neuroprotective properties. PEDF was shown to inhibit VEGF-induced proliferation and migration of microvascular endothelial cells in vitro and in vivo.8, 9 PEDF may also reduce VEGF-induced hyperpermeability and cause vessel regression in established neovascularization.10 However, recent studies have also suggested that only low doses of PEDF appear to be antiangiogenic, and that high doses result in increasing neovascularization, when administered intravitreally or subcutaneously, in animal models.11
AdPEDF, by GenVec, Inc., is currently under development for the treatment of wet AMD. AdPEDF uses GenVec's proprietary adenovector, a DNA carrier, to deliver the human PEDF gene, resulting in the local production of AdPEDF in the treated eye. AdPEDF's key differentiation from other therapies is its potential to protect the photoreceptors damaged by abnormal blood vessels and inhibit blood vessel growth.
GenVec has now completed the dose escalation portion of a phase I, multicenter clinical trial in 28 patients with advanced wet AMD. No dose limiting toxicities or drug related severe adverse events were observed and the antiangiogenic effect appeared to last for months.12 The sustained effect and repeatability of this therapy makes it a preferable choice. Additionally, GenVec just completed enrollment of 21 patients with less severe disease to complete this phase I study and the results appear similar to those obtained with the initial group of patients.
Microtubule Inhibitors ("vascular disruptors")
Combretastatins are natural antimitotic agents isolated from the root bark of the South African tree, Combretum caffrum. The most potent of these compounds is Combretastatin A-4 (CA-4), an antitumor drug.13 CA-4 binds to tubulin in endothelial cells at the same site as colchicine leading to strong inhibition of tubulin polymerization, resulting in disruption of the cytoskeletal network, cell-shape changes, and increased monolayer permeability.14 The consequences are vascular resistance, vasoconstriction, increased vascular permeability, platelet thrombi and vascular shutdown. CA4P causes shape changes, cytotoxicity and apoptosis of proliferating endothelial cells, but not of quiescent cells.13 The cytoskeleton of mature cells is not sensitive to CA4P as opposed to newly formed cells, which are particularly sensitive. There is a preferential sensitivity of endothelial cells in tumor vessels to CA4P, which unlike those in normal vessels, become thrombogenic, resulting in hemorrhagic necrosis of tumors.15-17
ZYBRESTAT (CA4P) is OXiGENE's lead vascular disrupting agent product candidate, which is currently being evaluated in multiple clinical trials as a treatment for various solid tumors. The company is currently advancing development of a topical formulation of ZYBRESTAT as a treatment for AMD. Based on the results of preclinical biodistribution studies, OXiGENE believes that it is feasible to deliver ZYBRESTAT topically, in a drop or other formulation, and achieve sufficient drug levels in the posterior segment to have therapeutic effects. This topical formulation could potentially eliminate or decrease the number of intravitreal injections in patients with AMD when administered either as monotherapy or in combination with current products.
Nucleic Acid Therapies
A key class of intracellular signaling proteins participating in growth factor and extracellular matrix signal transduction are the raf kinases.18 This is a family of isozymes with various stimulatory factors and substrates. The C-raf-1 kinase is widely distributed in tissues and participates in signal transduction initiated by integrin binding19 and by receptor tyrosine kinases of many growth factors in vascular endothelium, including VEGF.20 The inhibition of C-raf-1 kinase expression has been suggested to decrease the angiogenic response to ischemia produced by branch retinal vein occlusion.21
iCo Therapeutics is developing iCo-007, a second generation antisense inhibitor targeting C-raf kinase messenger ribonucleic acid (mRNA), for the treatment of retinal neovascular diseases, including diabetic macular edema. iCo-007 binds to the mRNA molecule and decreases the production of C-raf kinase. Antisense therapeutics based on second generation antisense chemistry appear to have increased target binding affinity, improved resistance to degradation and decreased toxicities. Due to these improvements, second generation antisense therapeutics degrade more slowly and could potentially offer an effective treatment for treatment of diabetic macular edema and AMD with less frequent dosing. A phase I, open-label, dose escalation study to evaluate the safety, tolerability, and pharmacokinetics of iCo-007 intravitreal injection in subjects with diffuse diabetic macular edema is currently in progress.
The peroxisome proliferator-activated receptor (PPAR) is a class of nuclear receptors (PPARα, PPARβ/δ, PPARγ) from the super family that includes the steroid, thyroid hormone, vitamin D, and retinoid receptors.22 PPARγ was discovered to be the intracellular high affinity receptor for the insulin-sensitizing, antidiabetic thiazolidinediones (TZDs), the activation of which also promotes growth arrest of preadipocytes' differentiation into mature adipocytes.23, 24 Ligand activation of PPARγ also downregulates the transcription of genes encoding inflammatory molecules, cytokines, growth factors, proteolytic enzymes and adhesion molecules.25 The pathogenesis of choroidal neovascularization includes inappropriate production of proinflammatory cytokines, growth factors and proteolytic enzymes. It was shown that the expression of PPARγ1 in human RPE cells and bovine choroidal endothelial cells inhibited VEGF-induced proliferation and choroidal neovascularization.26 These findings suggest that pharmacological activation of PPARγ by TZDs may have a role in treatment of choroidal neovascularization. PPARγ was found mostly in the retinal pigment epithelium, photoreceptor outer segments, choriocapillaries, choroidal endothelial cells, corneal epithelium and, to a lesser extent, in the retinal photoreceptor inner segments and outer plexiform layer, and the iris. The prominent expression of PPARγ in selected parts of the retina makes it an ideal target for treatment of ocular inflammation and proliferative retinopathies. Given the fact that synthetic, non-peptide PPARγ agonists are easy to synthesize and inexpensive to formulate, these agents may provide AMD patients with a much cheaper treatment option.
The complement system is a key component of innate immunity. Studies strongly suggested that the process of drusen formation includes inflammatory and immune mediated events. Complement components, complement activation products (C3a, C5a, MAC) and complement regulatory proteins (CD46, Vitronectin) have been localized in drusen. Further, Complement Factor H (CFH) and FHL-1 (Factor H-like 1) protein have been shown to be present in patients with early AMD.27 A tyrosine to histidine mutation at codon 402 (Y402H) within CFH has been suggested to be a marker for AMD.28 Defective function of CHF and FHL-1 causes unregulated complement activation, induction of inflammatory cascade and drusen formation due to tissue damage.27
Taligen Therapeutics is developing novel compounds, such as TT30 or targeted Factor H, that are intended to selectively deliver complement inhibitors to sites where complement activation is occurring and driving inflammation. Taligen also is developing an intraocular injectable formulation of TA106, a Fab fragment of a monoclonal antibody which inhibits complement factor B that could be positioned for the AMD market. There is currently no approved drug for dry AMD, the less serious though far more prevalent form of the disease; therefore, complement inhibition represents an alternative approach potentially offering synergies or better serving a different subset of patients.
Compstatin is a synthetic 13 amino acid cyclic peptide that binds tightly to complement component C3, preventing its participation in the complement activation cascade. As C3 is a central component of all three known complement activation pathways, its inhibition effectively shuts down all downstream complement activation that could otherwise lead to local inflammation, tissue damage and upregulation of angiogenic factors such as vascular endothelial growth factors. POT-4, a derivative of the cyclic peptide Compstatin developed by Potentia Pharmaceuticals, is a peptide capable of binding to human complement factor C3 (C3) and prevents its activation, resulting in broad and potent complement activation inhibition. The Safety of Intravitreal POT-4 Therapy for Patients With Neovascular Age-Related Macular Degeneration (AMD) (ASaP) study is currently recruiting patients and, when completed, will provide initial safety and tolerability information of intravitreal POT-4 for treatment of patients with AMD.
C5a is a potent chemotactic factor for monocytes, macrophages, neutrophils, activated T- and B-lymphocytes and induces a strong pro-inflammatory response. Jerini has developed both a peptidomimetic and a small organic molecule C5a receptor antagonist. These compounds have shown positive preclinical results and therapeutic potential in several indications, including AMD.
An anti-C5 aptamer, ARC1905 (Ophthotech Corp.), which prevents the formation of key terminal fragments (C5a and C5b-9) by inhibiting C5, may also offer therapeutic benefit in both dry and wet AMD.
Genentech is another company that is in the process of developing a potent complement inhibitor for treatment of AMD.
Rapamycin, a macrolide fungicide with potent antimicrobial and immunosuppressive properties, possesses significant anti-tumor and anti-angiogenic features.29 The anti-angiogenic properties are associated with a decrease in VEGF production and a reduction in the response of vascular endothelial cells to stimulation by VEGF. Rapamycin has been shown to reduce significantly the extent of neovascularization in both CNV and ROP models, suggesting that it may provide an effective new treatment for ocular neovascularization.30
Preliminary results from a prospective study of 30 patients with wet AMD demonstrated that MacuSight's proprietary formulation of sirolimus (rapamycin) was safe and well-tolerated in all doses tested with two different routes of administration (subconjunctival injection and intravitreal injection). Based on these positive results from its phase 1 trials, MacuSight is initiating phase 2 studies to enroll AMD patients with treatment-naive sub-foveal choroidal neovascularization.
We have highlighted some of the many AMD research studies in an effort to provide an overview of the current perspective on AMD. These and additional promising investigations will expand our knowledge base regarding the pathogenesis of this disease and will form the foundation for new strategies to improve the prognosis in patients with AMD. Although we have come a long way in understanding and treating AMD, substantial work remains in order to be able to prevent or cure this potentially devastating disease.
- Nguyen QD, Shah SM, Hafiz G, et al. 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.
- Nguyen QD, Hariprasad S, Shah SM. Results of a phase I, dose-escalation, safety, tolerability, and bioactivity study of intravitreal VEGF trap in patients with neovascular age-related macular degeneration: the CLEAR-IT I study. Retina Society/Club Jules Gonin Annual Meeting, 2006.
- Gaudreault J, Fei D, Rusit J, Suboc P, Shiu V. Preclinical pharmacokinetics of Ranibizumab (rhuFabV2) after a single intravitreal administration. Invest Ophthalmol Vis Sci 2005;46:726-33.
- Stewart MW, Rosenfeld PJ. Predicted biological activity of intravitreal VEGF Trap. Br J Ophthalmol 2008;92:667-8.
- Shen J, Samul R, Silva RL, et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1. Gene Ther 2006;13:225-34.
- Ozaki H, Seo MS, Ozaki K, et al. Blockade of vascular endothelial cell growth factor receptor signaling is sufficient to completely prevent retinal neovascularization. Am J Pathol 2000;156:697-707.
- Doukas J, Mahesh S, Umeda N, et al. Topical administration of a multi-targeted kinase inhibitor suppresses choroidal neovascularization and retinal edema. J Cell Physiol 2008;216:29-37.
- Duh EJ, Yang HS, Suzuma I, et al. Pigment epithelium-derived factor suppresses ischemia-induced retinal neovascularization and VEGF-induced migration and growth. Invest Ophthalmol Vis Sci 2002;43:821-9.
- Stellmach V, Crawford SE, Zhou W, Bouck N. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. Proc Natl Acad Sci U S A 2001;98:2593-7.
- Mori K, Gehlbach P, Ando A, McVey D, Wei L, Campochiaro PA. Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor. Invest Ophthalmol Vis Sci 2002;43:2428-34.
- Apte RS, Barreiro RA, Duh E, Volpert O, Ferguson TA. Stimulation of neovascularization by the anti-angiogenic factor PEDF. Invest Ophthalmol Vis Sci 2004;45:4491-7.
- Campochiaro PA, Nguyen QD, Shah SM, et al. Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: results of a phase I clinical trial. Hum Gene Ther 2006;17:167-76.
- Cirla A, Mann J. Combretastatins: from natural products to drug discovery. Nat Prod Rep 2003;20:558-64.
- Tozer GM, Prise VE, Wilson J, et al. Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability. Cancer Res 2001;61:6413-22.
- Campochiaro PA, Hackett SF. Ocular neovascularization: a valuable model system. Oncogene 2003;22:6537-48.
- Nambu H, Nambu R, Melia M, Campochiaro PA. Combretastatin A-4 phosphate suppresses development and induces regression of choroidal neovascularization. Invest Ophthalmol Vis Sci 2003;44:3650-5.
- Eichler W, Yafai Y, Wiedemann P, Fengler D. Antineovascular agents in the treatment of eye diseases. Curr Pharm Des 2006;12:2645-60.
- Daum G, Eisenmann-Tappe I, Fries HW, Troppmair J, Rapp UR. The ins and outs of Raf kinases. Trends Biochem Sci 1994;19:474-80.
- Clark EA, Brugge JS. Integrins and signal transduction pathways: the road taken. Science 1995;268:233-9.
- Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells. Oncogene 1999;18:2221-30.
- Danis R, Criswell M, Orge F, et al. Intravitreous anti-raf-1 kinase antisense oligonucleotide as an angioinhibitory agent in porcine preretinal neovascularization. Curr Eye Res 2003;26:45-54.
- Mangelsdorf DJ, Thummel C, Beato M, et al. The nuclear receptor superfamily: the second decade. Cell 1995;83:835-9.
- Lehmann JM, Moore LB, Smith-Oliver TA, Wilkison WO, Willson TM, Kliewer SA. An antidiabetic thiazolidinedione is a high affinity ligand for peroxisome proliferator-activated receptor gamma (PPAR gamma). J Biol Chem 1995;270:12953-6.
- Altiok S, Xu M, Spiegelman BM. PPARgamma induces cell cycle withdrawal: inhibition of E2F/DP DNA-binding activity via down-regulation of PP2A. Genes Dev 1997;11:1987-98.
- Gelman L, Fruchart JC, Auwerx J. An update on the mechanisms of action of the peroxisome proliferator-activated receptors (PPARs) and their roles in inflammation and cancer. Cell Mol Life Sci 1999;55:932-43.
- Murata T, He S, Hangai M, et al. Peroxisome proliferator-activated receptor-gamma ligands inhibit choroidal neovascularization. Invest Ophthalmol Vis Sci 2000;41:2309-17.
- Skerka C, Lauer N, Weinberger AA, et al. Defective complement control of factor H (Y402H) and FHL-1 in age-related macular degeneration. Mol Immunol 2007;44:3398-406.
- Kaur I, Hussain A, Hussain N, et al. Analysis of CFH, TLR4, and APOE polymorphism in India suggests the Tyr402His variant of CFH to be a global marker for age-related macular degeneration. Invest Ophthalmol Vis Sci 2006;47:3729-35.
- Guba M, von Breitenbuch P, Steinbauer M, et al. Rapamycin inhibits primary and metastatic tumor growth by antiangiogenesis: involvement of vascular endothelial growth factor. Nat Med 2002;8:128-35.
- Dejneka NS, Kuroki AM, Fosnot J, Tang W, Tolentino MJ, Bennett J. Systemic rapamycin inhibits retinal and choroidal neovascularization in mice. Mol Vis 2004;10:964-72.