Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

3.5.5Squalamine: an Aminosterol

Squalamine (EvizonTM, Genaera Co. (www.genaera.com), Plymouth Meeting, PA) is a naturally occurring antibiotic aminosterol that has antiangiogenic properties and was originally isolated from the dogfish shark.404 It is structurally different from steroids and has no mineralocorticoid or glucocorticoid function.405 Its anti-angiogenic effects have been demonstrated in various tumor models406-408 and in numerous ocular models. For example, systemic administration of squalamine inhibited and partially

regressed iris NV in primate models,409 inhibited preretinal NV in the mouse OIR model,410,411 and prevented laser-induced CNV in the rat.412

Regarding squalamine’s mechanism of action, it has been shown to inhibit endothelial cell proliferation and migration induced by a wide range of growth factors, including VEGF.406 Its broad anti-angiogenic activity may result, in part, from its inhibition of surface Na+/H+ exchangers and other downstream signaling pathways in endothelial cells.413 Another proposed mechanism involves its selective entry into endothelial cells and activity as a calmodulin chaperone.414 All of these actions may lead to disruption of growth factor signaling including VEGF and integrin expression and cytoskeletal formation, thereby resulting in endothelial cell inactivation and apoptosis.

In early 2007, Genaera announced that it was halting its development of squalamine for exudative AMD. The company cited that the “introduction by competitors of new and off-label products that improve vision in wet AMD” had slowed their trial enrollment significantly and that preliminary information from investigators suggested that squalamine was “unlikely to produce vision improvement with the speed or frequency necessary to compete.”415 Prior to the announcement, a completed phase I/II trial of 40 patients with all types of CNV lesions revealed that once-weekly intravenous injections of squalmine for 4 weeks preserved vision in 100% of subjects and improved visual acuity by three lines or more in 26% of subjects at 4 months.416 Preliminary data from a phase II randomized, controlled, masked study of the effects of squalamine in combination with Visudyne® showed no evidence of any adverse drug-drug interaction.417

3.6Naturally-derived Inhibitors

3.6.1Interferon

Naturally occurring inhibitors of angiogenesis were described in the early years of angiogenesis research, and included molecules such as thrombospondin-1, platelet factor-4, angiopoietin 2, angiostatin and

endostatin, and interferon α.418-420 Interferon α-2a was the first pharmacological anti-angiogenic therapy to be clinically evaluated for the treatment of PSNV in large, randomized, placebo-controlled trials. Unfortunately, it also was subsequently the first pharmacological therapy to fail during posterior segment trials. Interferon α-2a inhibits the proliferation and migration of endothelial cells and was systemically efficacious against nonocular, angiogenesis-dependent diseases, e.g., Kaposi’s sarcoma.421-423 An early positive report found that subcutaneous interferon α-2a administered over 4 months in patients with proliferative DR provided stabilization of both vision and retinopathy scores.424 Conversely, subcutaneous interferon α-2a (1.5, 3, and 6 MIU) administered 3 times per week for one year failed to demonstrate efficacy versus placebo against CNV in patients with exudative AMD.425 Not only did the AMD results show no trend toward a positive dose-response in this 481-patient trial, patients treated with 1.5 and 6 MIU for one year appeared to have statistically significant worsening of visual acuity when compared to placebo-treated controls. Then, in a separate empirical study of 2 patients with exudative AMD, intravitreal injection of interferon α-2a resulted in a marked generalized reduction in the amplitude of the bright-flash darkadapted electroretinographic response one month post-injection as compared to baseline retinal function, for which the study was discontinued.426 Based on the above findings, all clinical trials using interferon α-2a as an inhibitor of ocular angiogenesis were halted, but the era of exploring a pharmacological means for treating angiogenesis-dependent ocular diseases was born.

3.6.2Thalidomide

Thalidomide was identified by the Folkman laboratory as an existing drug that possessed previously unappreciated anti-angiogenic activity.427 Thalidomide was originally used in the 1950’s as an oral sedative and was later banned due to its ability to cause severe birth defects. These specific side effects were clues that eventually propelled the scientists to evaluate thalidomide for anti-angiogenic properties in preclinical models.428 A small prospective clinical trial in patients with exudative AMD, however, was halted due to the inability of patients to tolerate the compound. The most common systemic side effects observed following oral delivery were drowsiness, constipation, and peripheral neuropathy.429

3.6.3adPEDF

Pigment epithelium derived factor (PEDF) was originally discovered in the late 1980’s from cultured RPE cells,430 where ensuing research revealed

expression in ocular and extraocular tissues, including blood plasma. PEDF is a 50-kDa protein and a member of the large superfamily of SERPINs (serine protease inhibitors).431 PEDF has multifactorial effects in both physiology and disease of the eye, where it exhibits neurotrophic, neuroprotective, anti-inflammatory, and anti-angiogenic properties.432-436,309

PEDF may be the major endogenous inhibitor of angiogenesis in the

cornea and posterior segment, and its expression is downregulated, in part, by hypoxia.434,435,437 Various reports indicate that, in both DR and exudative

AMD, the relationship between PEDF (anti-angiogenic) and VEGF (proangiogenic) is unbalanced and favors a pro-angiogenic environment. For

example, human vitreous levels of PEDF decrease during active DR, while VEGF levels increase,438,439 and low PEDF levels in the aqueous humor are a

predictor for the progression of DR.440 Similarly, vitreous levels of PEDF are decreased in patients with exudative AMD.441 Preclinically, a role for PEDF in PSNV is supported by results showing it inhibits endothelial cell proliferation and migration, downregulates hypoxia-induced TNFα and ICAM-1 expression, protects retinal pericytes from advanced glycation

injury, and exhibits decreased production associated with hypoxia in the rat

OIR model.434,442,437

The anti-angiogenic potential of PEDF has been demonstrated in a wide variety of preclinical models. Initially, systemic daily dosing of PEDF in a mouse OIR model achieved nearly complete inhibition of preretinal NV.435 Later, intravitreal injection of human recombinant PEDF was shown to

significantly reduce preretinal NV in a similar mouse OIR model, as well as retinal vascular permeability in rat models of diabetes and OIR.443,437 Based

on these scientific findings, GenVec (www.genvec.com, Gaithersburg, MD) obtained an exclusive license for ocular PEDF gene therapy (2000), an exclusive license for gene therapy technology (2001), and a license for all ocular indications for PEDF (2004). Subsequently, GenVec engineered the full length, open reading frame of PEDF into a viral vector, based on Adenovirus type 5 (adPEDF), for delivery into the eye.444 Intravitreal and periocular delivery of adPEDF was then shown to prevent preretinal NV and laser-induced CNV in preclinical models.444-448 In toxicology studies, adPEDF administered locally to the eye was well tolerated at potential therapeutic doses, and repeat dosing did not increase side effects.449

A multicenter, dose-escalating, phase I trial in up to 51 patients with severe exudative AMD has focused primarily on the safety and tolerability of intravitreal injections of adPEDF. Preliminary findings indicate that adPEDF was well tolerated with no severe adverse events or dose-limiting toxicities. No cases of significant ocular inflammation or endophthalmitis have been reported,450-452 and the results are expected to establish the dose levels for phase II trials.453

3.6.4Angiostatin and Endostatin

Angiostatin and endostatin are naturally occurring, anti-angiogenic protein

fragments that were originally isolated from cell culture media and are known to be generated by primary tumors and inhibit metastasis.419,420

Endostatin is the C-terminal fragment of collagen XVIII, is roughly 20 kDa, and has local and circulating forms in man.454,455 Its mechanism of action in

choroidal angiogenesis, at least in part, appears to be related to inhibition of MMP-2 production and cell migration.456 Endostatin has been identified in CNV lesions from patients with exudative AMD, as well as Bruch’s membrane, RPE, and choroidal vessels in normal and diseased eyes.457 The authors of these findings suggest that production of endostatin is a counter feedback mechanism to the CNV lesion and that therapeutic upregulation of the protein fragment may be beneficial. Indeed, in a previous study assessing vitreous levels of VEGF and endostatin from patients with PDR undergoing vitreous surgery, it was found that patients with high VEGF and low endostatin levels had a significantly greater risk of postoperative progression of PDR.458 As seen with PEDF therapy, therapeutic production of an endogenous protein is a significant challenge pharmaceutically, when compared to delivery of a small, organic inhibitor. In preclinical attempts to address this delivery challenge, intravenous and intravitreal delivery of

endostatin using viral vectors has been shown to inhibit PSNV in animal models.459,460

Angiostatin is a 38-kDa internal fragment (kringle 1-4) of plasminogen that was originally shown to block angiogenesis and primary tumor growth,

and accounted for the ability of metastases to self-inhibit their growth following removal of the primary tumor.461,462 This endogenous inhibitor has

the ability to inhibit endothelial cell proliferation and induce apoptosis.463 Direct intravitreal injection decreases VEGF expression and subsequent retinal vascular permeability in rat OIR and diabetic models.464 Similar to

endostatin, intravitreal injection of angiostatin in a viral vector inhibits retinal and choroidal NV in several preclinical studies.465,445,466 To date,

however, no human trials have been initiated with either of the native proteins or by using a viral vector modality.

3.6.5Estradiol Derivatives

Endogenous 2-methoxyestradiol (2ME2, Panzem®, EntreMed (www.entremed. com), Rockville, MD) is synthesized in vivo by catechol-O- methyltransferase (COMT) from catechol estrogens (2- or 4- hydroxyestradiol and 2- or 4-hydroxyestrone).467 COMT is an enzyme present in many tissues such as the liver, kidney, brain, placenta, uterus, and

choroid and RPE to induce a low increase in temperature (<10 °C, i.e., not enough heat to induce photocoagulation), which is maintained for 60 seconds to induce vascular occlusion. TTT with a higher power setting has

been successfully used as an adjunctive treatment for choroidal melanoma.480,481

Several pilot studies showed encouraging data indicating that TTT may have a high closure rate and resolution of the CNV complex without apparent retinal complications.482-484 The multicenter prospective randomized clinical trial of patients with occult subfoveal CNV (TTT4CNV study, a physician-initiated clinical study supported by IRIDEX) has been completed, and final results were presented in May 2005.485 Overall, the visual outcome data showed that TTT did not result in a significantly beneficial effect relative to sham. However, subgroup analysis of eyes with poorer visual acuity at baseline (20/100 or less) indicated a statistically significant treatment benefit, specifically, less mean visual acuity loss and a greater percentage of patients showing improvement.

External-beam, low-dose radiation is a technique that targets low levels of radiation to the macular region using a linear accelerator.486,487 This type

of radiation displays a relative selectivity for damaging proliferating cell types. Although some positive trends have been reported in uncontrolled clinical studies, the true utility of this technology is not known, nor has it gained widespread clinical use.488

4.2Rheopheresis

Rheopheresis (RHEO™, OccuLogix (www.occulogix.com), Mississauga, ON, Canada) is a treatment modality under clinical investigation in patients with microcirculatory disorders. For patients with AMD, the therapy involves application of double filtration plasmapheresis and removal of high molecular weight proteins, such as fibrinogen, LDL-cholesterol, alpha2macroglobulin, fibronectin, and von Willebrand factor.489 It has been suggested that removal of these macromolecules may reduce plasma viscosity and erythrocyte and platelet aggregation, that could then modify the diffusion characteristics of Bruch’s membrane, the rheology of the choriocapillaris, and impact sites of inflammation.490 Interim data from two multi-center, randomized, controlled, double-masked clinical studies (MIRA-1) and an open-label trial (PERC) in patients with dry AMD have been reported.491-494 The objectives of these studies are to modify the natural

history of the disease and to reduce the risk of progression into late-stage AMD and subsequent vision loss. To date, the treatment appears to be relatively safe, but the level of efficacy is yet to be determined.

4.3Submacular Surgery and Macular Translocation

Submacular surgery utilizes advanced microsurgical techniques to manually remove blood and choroidal neovascular lesions from underneath the macula.495-497 Results from prospective clinical trials using submacular surgery to treat patients with exudative AMD did not demonstrate the ability to improve or preserve visual acuity at 24 months when compared with observation.498,499 Some benefit in patients with predominantly hemorrhagic subfoveal CNV was reported with regard to reducing the risk of severe vision loss at 2 years.499 Reported limitations of this procedure are RPE

defects, photoreceptor atrophy, and persistent CNV associated with the inherent surgical trauma and ongoing pathogenesis of the disease.496,498,500

Because loss of the central RPE is likely a primary impediment to the success of submacular surgery, surgical macular translocation was developed to restore contact between the subfoveal photoreceptors and healthy RPE following removal of the CNV lesion. Macular translocation actually denotes a wide variety of described techniques.501-506 For example, free or pedicle grafts of RPE have been placed under the fovea in patients with exudative AMD.506 Long-term follow-up (5-6 years) from the same group of patients that underwent submacular surgery combined with macular translocation of the RPE showed that improved visual function was transient (2-5 years) even though the RPE graft remained viable.507 The general effectiveness of these procedures is still highly debated; however, improved understanding of how surgical manipulations damage retinal cell types as well as the successful use of stem cells may advance this therapeutic area in the future.

5.FUTURE DIRECTIONS

It is a very exciting time to be involved in the research and treatment of pathological ocular angiogenesis. More importantly, it is a time of increasing hope for those afflicted with devastating retinal diseases. Less than a decade ago, very few options were available for someone newly diagnosed with exudative AMD, and none of the possible treatments involved pharmacological intervention. Today, numerous pharmacological therapies

are available, both approved and off-label, along with a variety of other treatment modalities. Anti-angiogenic therapies for the eye will have an even higher demand in the future as the elderly and diabetic percentages of the population substantially expand in developed countries. The individual and societal costs of blindness in this vast number of people warrants continued and aggressive pursuit of effective and safe treatments.

The obvious next phase in the evolution of treatment strategies for ocular angiogenesis is the robust determination of viable combination therapies. Similar to treatment regimens in oncology, retina specialists have already

begun exploring the use of combined treatment modalities in attempt to achieve additive, if not synergistic, effects.346,43,46 Based upon mechanism of

action and route of administration, a theoretical approach could be to intravitreally inject with an anti-VEGF therapy, e.g., ranibizumab, prior to using PDT with verteporfin to close an actively leaking CNV lesion. This combination could then be followed by a posterior juxtascleral depot of anecortave acetate, with the expectation of reducing the frequency of retreatment. Numerous other combination paradigms could be imagined;99 however, empirical success with any combination strategy should be viewed with caution. The retina community should actively pursue well-controlled, randomized prospective trials to substantiate positive data from pilot studies.

Drug delivery will play a vital role in developing next generation treatments that offer substantial advantages over current therapies. The platform technology and route of delivery will eventually be tailored to specific disease processes, and even to stages within a given pathological condition. For example, although intravitreal administration of Lucentis® appears to be very effective in the majority of patients with exudative AMD when administered as a monthly injection, repeated intravitreal injections are not without risk. Extending the time between treatments is clearly an area to explore that could increase the utility and value of the therapy. The drug delivery chapter written by Drs. Weiner and Marsh provides an excellent review of this area and the available options for such strategies. Several new methods for improved delivery to the back of the eye are moving into the horizon and are worth mentioning here, such as targeted delivery systems, viral vectors, cellular delivery, and stem cell therapy. Targeted delivery of anti-angiogenic agents is a concept designed to reduce or eliminate significant systemic drug exposure and its associated side effects. It is theoretically plausible because of the differential expression of various membrane proteins on activated microvascular endothelium. An example of membrane-expressed molecules that may function as homing sites for targeting agents during ocular angiogenesis are growth factor receptors, such

as VEGFR-2. Proof-of-concept studies in animals have provided initially positive results using this target.508-511

With the identification of endogenous anti-angiogenic proteins, such as PEDF, in ocular tissues, and because of the advent of siRNA therapy, viral vector delivery has received increased attention. Although direct intraocular injection of proteins or “naked” siRNA have provided preclinical efficacy,238,175 viral-mediated gene delivery, specifically recombinant adeno-

associated viruses (AAV), provides the opportunity for long-term expression of a desired anti-angiogenic transgene.465,512-514 An excellent review of AAV

vectors for the treatment of retinal diseases is available.514 Similarly, delivery platforms for injection of live cells that secrete a treatment protein have been explored for retinal neurodegeneration indications, and could be used for the intraocular delivery of anti-angiogenic molecules as well. Perhaps the most futuristic of the potential new therapies is the use of stem cells for retinal disease. Bone marrow-derived hematopoietic progenitor cells

(HPCs) were first shown to mobilize to sites of tumor angiogenesis172 and subsequently to the retina, choroid, and cornea.173,515-517 The HPC

populations contain endothelial and pericyte precursor cells that differentiate into their respective cell types once they have migrated into the site of NV. Although HPC homing and differentiation processes are still ill-defined,

some investigators have suggested that VEGF, VEGFR1, and R-cadherin likely play a role for endothelial cells.517,174,518 Whether or not HPCs could or

should be targeted for inhibition, actually used as inhibitors,518 used as antiangiogenic gene/drug carriers, or differentiated in such a way as to provide

desired positive vasculotropic and/or neurotropic effects in humans still remains to be determined.170,519,518,520

Further elucidation of the basic pathophysiology of the individual retinal diseases will likely lead to identification of novel therapies. Even though the role of VEGF as a primary mediator of ocular angiogenesis has been clinically validated, new growth factors that regulate microvascular physiology and pathology continue to be discovered. For example, the roles of Ephrins/Eph receptor, stromal-derived factor (SDF-1)/CXCR4, and erythropoietin have recently received increased attention with regard to ocular angiogenesis.521-526 Some of the ligand receptors, e.g., CXCR4, have

known small molecule inhibitors that provide efficacy in animal models of ocular angiogenesis.527,528 Or perhaps more importantly, recent investigations

into the instigators of growth factor production may provide druggable targets upstream of growth factor release.

One of the hottest areas of macular degeneration research that may lead to a novel therapy is the role of inflammation, and more specifically, the role of complement, in the development of dry and exudative AMD.529-539

Targeted and genome-wide scans, initiated because of the known inheritability of certain forms of AMD, have recently identified a single nucleotide polymorphism (rs1061170) in the 1q32 region resulting in a

T→C change at nucleotide position 1277 of exon 9 of the complement factor H (CFH) gene (MIM1223270).533-536,540,541 This single nucleotide change

produces an amino acid change at position 402 from a tyrosine to a histidine

(Y402H), resulting in a 2-10 fold increased risk for AMD for the homozygous CC alleles.542,543 Multiple disease susceptible alleles also are in the region of the CFH variant.544,545 Notably, the effects of the Y402H

variant may be more pronounced in relation to the exudative stage of AMD.543 Regarding a potential mechanism of action, CFH is known to

inhibit the alternative pathway of complement activation, where it and other complement components have been found in drusen.546,536 Activation of the

alternative complement pathway has been shown to play a significant role in the development of laser-induced CNV in mice.538,539 Because the presence

of the Y402H variant produces an increase in risk, it suggests that a reduced inhibitory tone in the alternative pathway may be a key regulator in the underlying pathophysiology of AMD. Similarly, genomic screening very recently has identified a single nucleotide polymorphism in the promoter region of HTRA-1 (a serine protease, also called PRSS11) at 10q26. In a Chinese population, this risk-associated genotype conferred a 10-fold increased risk for exudative AMD.547 Moreover, in a Caucasian cohort from Utah, the HTRA-1 variant conferred a population attributable risk for AMD of 49%, and the protein was labeled in drusen from 3 of the AMD patients with the risk allele.548 Although the function of HTRA-1 in AMD remains unknown, it may prove to be a useful pharmacological target.

Preventing the development of pathological ocular angiogenesis is the ultimate goal for the patient and the treating physician. In AMD, large randomized trials have demonstrated the utility of antioxidants plus zinc for reducing the risk of progression to advanced stages of AMD.549 However, the maximal reduced risk observed was only 25%. Other prophylactic therapies have been tried in AMD, such as laser photocoagulation of soft

drusen, but randomized trials have actually shown a deleterious effect related to the incidence of CNV development in unilateral cases.550,551 Similarly,

tight glycemic control has been shown to reduce the risk of progression into the latter, more severe stages of DR, but it does not completely halt the progression.552 Pharmacological agents that can safely block the production of pro-angiogenic signals may be a viable alternative for risk-reduction therapy. Because of its proven capability to prevent neovascular development in multiple preclinical models and its robust clinical safety package, Alcon® Research Ltd. has begun a 2500 patient, 4-year study to