Ординатура / Офтальмология / Английские материалы / Retinal and Choroidal Angiogenesis_Penn_2008

thereafter (check www.clinicaltrials.gov for a full listing of active trials). One year results from the PIER study suggested that although quarterly dosing did provide a visual acuity benefit, the magnitude of effect observed was less than that observed with monthly dosing in the other phase III trials.121 Finally, Lucentis® is also being pursued for other indications, such as DME. In an investigator-sponsored study, intravitreal injections of 0.3 and 0.5 mg ranibizumab were well tolerated, reduced retinal thickness, and maintained or improved visual acuity in patients with DME.110

3.1.3Avastin®

Prior to the publication of this book, a unique phenomenon occurred that will affect the treatment of patients with exudative AMD and may have substantial long-term consequences for pharmaceutical companies pursuing retinal indications. Initially, a group of retina specialists published their empirical findings from an open-label, uncontrolled study involving nine patients treated with systemic administration of the full-length monoclonal antibody, Avastin®, for subfoveal CNV associated with exudative AMD.122 Patients received intravenous infusions of 5 mg/kg Avastin® (as per its oncology label) followed by 1–2 repeat doses at 2 week intervals. Systemic Avastin® was considered to be effective, with improvements in visual acuity by one week post-treatment, reductions in retinal thickness via optical coherence tomography (OCT), and decreased fluorescein leakage from CNV lesions reported. The only adverse event identified during the 12-week follow-up period was elevation of systolic blood pressure (7 of 9 patients), which was considered on average to be mild and controllable with antihypertensive medications. (In large cancer trials, significant adverse events associated with systemic Avastin® treatment have been potentially fatal thromboembolisms, hypertension, epistaxis, hemoptysis, and proteinuria.123-125) Subsequently, these physicians reported similar preliminary efficacy in exudative AMD patients following intravitreal injection of Avastin®.126 During 2005 and 2006, a variety of preclinical and clinical reports were published describing the safety and empirical efficacy

of intravitreal Avastin® in indications spanning from exudative AMD to DME and rubeosis iridis.127-148 The use of Avastin® for an ophthalmic

indication or when delivered using local administration is considered “offlabel,” since the drug is only approved for intravenous infusion in patients with metastatic colorectal cancer. Because Avastin® is sold in a larger volume for intravenous use, an intravitreal injection volume can be prepared by compounding pharmacies and results in a reduced cost per dose more similar to generic products rather than a prescription therapy. Because of the empirical results and the low cost, intravitreal Avastin® has become a widely

accepted practice, particularly in those areas where Lucentis® is still unavailable or not affordable. What impact off-label Avastin® will have on the market acceptance of intravitreal Lucentis®, as well as other retinal therapies in clinical trials for exudative AMD, is yet to be fully realized.

3.1.4VEGF Trap

The VEGF TrapR1R2 is a recombinant chimeric protein (approximately 110 kDa) comprising portions of the extracellular, ligand-binding domains of the human VEGFR1 (Flt-1, Ig domain 2) and VEGFR2 (KDR, Ig domain 3) expressed in sequence with the Fc portion of human IgG.149 The VEGF TrapR1R2 binds all isoforms of VEGF and placental growth factor (PlGF), where the affinity for VEGF is Kd = 1–5 pM. Preclinically, subcutaneous or intravitreal delivery suppressed laser-induced CNV, as well as VEGFinduced retinal vascular permeability, in the mouse.94 Intravitreal administration has been preliminarily reported to inhibit preretinal NV in the mouse OIR model, diabetes-induced retinal vascular permeability in the rat, and laser-induced CNV in the monkey. Pharmacokinetic data following intravitreal injection of 500 μg VEGF TrapR1R2 in the adult rabbit suggests a

vitreous half-life of 4.5 days and a potential duration of activity of 6

weeks.150-154

Regeneron has evaluated systemic delivery of the VEGF TrapR1R2 in both human oncology and ophthalmology trials. In a phase I randomized, double-

masked, dose-escalation, placebo-controlled trial using intravenous infusion

of 0.3, 1.0, or 3.0 mg/kg, the VEGF TrapR1R2 was used to treat 25 patients with exudative AMD.155 The results showed a statistically significant

decrease in enhanced retinal thickness via OCT, which was the predetermined primary efficacy outcome measure. However, systemic

delivery of VEGF TrapR1R2 did induce an adverse, dose-dependent increase in blood pressure. Intravenous delivery of 0.3 mg/kg VEGF TrapR1R2 also

has been preliminarily reported in six patients with DME and was found to reduce the mean excess foveal thickness by 42% in treated patients.156 Moreover, initial findings in a phase I, dose-escalation study using

intravitreal injection of VEGF TrapR1R2 in patients with exudative AMD have been published.157 Data at day 29 post-injection from 3 patients treated in

each of 4 dose groups (0.05, 0.15, 0.5, and 1.0 mg) showed a reduction of excess foveal thickness by <70% and stable or improved visual acuity in 75% of treated patients.

3.1.5RTKi’s

Receptor tyrosine kinases (RTKs) possess both extracellular and intracellular domains and function as membrane-spanning cell surface receptors.158,159 They

represent a critical signaling network that transmits extracellular stimuli into the cell, regulating critical cellular functions such as proliferation, migration, and survival.160,161 Consequently, RTK dysregulation is associated with a variety of human disorders, including ocular diseases and various forms of cancer. Over the last several years, numerous pharmaceutical companies have initiated clinical trials using systemic administration of RTK inhibitors (RTKi’s) for the treatment of various cancers, with multiple candidates now in phase III studies. Recently, Alcon®, Allergan, Merck, Novartis, and Pfizer have all publicly claimed to be actively targeting the RTKi class for the treatment of exudative AMD and DR using local and/or systemic delivery platforms. Because of their ability to simultaneously block multiple signaling pathways, the RTKi’s are anticipated to provide advantages in efficacy over current therapies directed at a solitary growth factor.

VEGF Receptor Family

The VEGF receptors mediate the biological functions of the VEGF family and consist of three RTKs: VEGFR1 or Flt1 (Fms-like tyrosine kinase), VEGFR2 or KDR (kinase insert domain-containing receptor), and VEGFR3 or Flt4.74 The VEGFRs are each composed of an extracellular domain containing 7 immunoglobulin-like motifs (the growth factor binding site), a

single transmembrane domain, and an intracellular split kinase domain that confers the tyrosine kinase activity.162,163 Similar to many RTKs, the binding

of VEGF to the extracellular domain induces receptor dimerization and autophosphorylation of specific intracellular tyrosine residues, which serve as docking sites for other proteins that induce downstream signaling.164 VEGFR2 is primarily expressed by vascular endothelial cells and is the

major mediator of the pathological vascular permeability and angiogenic effects of VEGF.163,165,166,74 Its role in developmental angiogenesis is

consistent with the embryonic lethality and abnormal blood vessel formation observed in VEGFR2-/- mice.167,168 In contrast, VEGFR1 binds not only

VEGF, but also VEGF-B and PlGF, with high affinity, and it is expressed on

endothelial cells, smooth muscle cells, monocytes, and hematopoietic stem cells.169,170 Notably, VEGFR1 signaling results in the mobilization of

marrow-derived endothelial progenitor cells that are recruited to tumors, and potentially the diseased retina/choroid, where they contribute to new blood vessel formation.171-174 Interestingly, a locally delivered siRNA against VEGFR1 has been shown to inhibit experimental CNV,175 and soluble VEGFR1 appears to play a major role in the avascularity of the cornea.176

VEGFR3 binds VEGF-C and VEGF-D, but not VEGF, and mediates lymphangiogenesis and aspects of metastasis in animal models.177 Its role in ocular diseases remains undetermined.

PDGF Receptors

The α and β isoforms of the platelet-derived growth factor (PDGF) receptors occur as homodimers or α/β heterodimers and are found most commonly on

the surface of fibroblasts, smooth muscle cells, pericytes, and vascular endothelial cells.178,179 Blood vessel differentiation and remodeling appears

to be defined by pericyte coverage of the endothelium, which is regulated by PDGF-β and VEGF.179 The relationship between endothelial cells and pericytes is particularly significant in the human retina, where the endothelial cell/pericyte ratio is 1:1. Tumor-associated fibroblasts are a source of numerous growth factors, and consequently paracrine PDGF signaling is thought to contribute to disease progression in these cancers.180182 PDGFR-β contributes to tumor angiogenesis through the proliferation and migration of pericytes, the peri-endothelial cells that associate with and stabilize immature blood vessels.183-187 Inhibition of PDGF receptor signaling in fibroblasts and pericytes has been shown to enhance the antitumor effects of chemotherapy by regulating tumor interstitial fluid pressure.188 Similarly, PDGF and PDGFRs may be important in the retinal neurons and microvasculature and modulate angiogenesis in the eye.189,190

Angiopoietin Receptors

Angiopoietins (Ang1-4) are ligands for the Tie receptors (Tie1 and Tie2), which are a family of RTKs selectively expressed by vascular endothelial cells and some hematopoietic cells.73 Tie2-/- is embryonic lethal in mice, where a phenotype of immature and disorganized vasculature is exhibited.191 Both Ang1 and Ang2 are integrally involved in vasculogenesis and angiogenesis by acting through the Tie2 receptor. Regarding the eye, Ang2 and VEGF appear to be co-upregulated, and Tie2 is expressed on a variety of cell types in choroidal neovascular membranes surgically excised from patients with exudative AMD.192 Ang2 is upregulated in retinal endothelial cells by exposure to VEGF and hypoxia, and its expression is induced during physiological and pathological ocular angiogenesis.193,194 Moreover, signaling through Tie2 may regulate retinal angiogenesis in concert with VEGF signaling and be a critical pathway in NPDR.195-198

As previously stated, numerous pharmaceutical companies have developed medicinal chemistry efforts to design both selective and multitargeted RTKi’s for a variety of human conditions, including cancer and posterior segment disease.199-203 Related to ophthalmic indications, Campochiaro et al. demonstrated that oral administration of PKC412

(Novartis), a compound selective for PKC as well as RTKs such as VEGFRs and PDGFR, inhibited both preretinal and choroidal NV in rodents.204 Using oral RTKi’s including PKC412 and PTK787 (a selective VEGFR inhibitor) from Novartis, Campochiaro et al. subsequently showed that blockade of VEGFR-2 was sufficient to completely prevent retinal NV in rodents and produced no adverse effects on adult quiescent retinal capillaries.205 More recently, intravitreal injection of a nonproprietary RTKi selective for VEGFR-2, IGF-1R, FGFR-1, and EGFR provided a modest reduction (25%) in the median retinopathy score in a mouse OIR model.83 Merck & Co. have published results using an unspecified, novel KDR inhibitor in the rat OIR and laser-induced CNV models.206 When delivered at 30 mg/kg daily per os, the KDR inhibitor provided significant inhibition of preretinal NV (80%) and laser-induced CNV (70%) as compared to vehicle-treated animals. Following these preclinical results, Novartis assessed PKC412 using oral administration in human patients with existing DME.207 Although pilot results suggested a reduction in macular edema and an improvement in visual acuity, increased liver enzymes were reported in treated patients. In 2005, Novartis then began recruiting patients with CNV from exudative AMD to test the safety of oral vatalanib (PTK787) in combination with verteporfin (www.clinicaltrials.gov).

3.1.6Ruboxistaurin

Protein kinase C (PKC) is a family of serine-threonine kinases with 13 members208 that plays a key role in intracellular signaling for hormones and cytokines. Increased activation of certain PKC isoforms is caused by increased synthesis of diacyl glycerol (DAG) following hyperglycemia.209 The β-isoform of PKC is most closely linked to the development of diabetic microvascular complications and abnormalities in retinal hemodynamics.210212 In preclinical models, PKC activation can mediate abnormal changes in retinal capillary blood flow, leukostasis, basement membrane thickening, increases in vascular permeability, and preretinal NV.213-218 Importantly,

PKC activation appears to exhibit a critical interplay with aspects of VEGF signaling.219-221

Ruboxistaurin mesylate (Arxxant®, LY333531, Eli Lilly & Co.) is a

competitive inhibitor of ATP binding to the isozyme PKC-β(1,2) (IC50 4.7 nM).222 Preclinical efficacy with ruboxistaurin mesylate has been shown in various experimental manifestations of DR.223,216,224,214,219 In 2002, healthy

subjects treated for 7 days with oral ruboxistaurin mesylate showed a significant reduction in endothelium-dependent vasodilation casued by hyperglycemia.225 Phase III results from a trial using oral ruboxistaurin mesylate in patients with moderately severe to very severe NPDR

demonstrated that the PKC inhibitor was well tolerated but had no significant effect on the progression of DR.226 In this multicenter, doublemasked, randomized, placebo–controlled study, 252 subjects received placebo or ruboxistaurin mesylate (8, 16, or 32 mg/day) over a period of 32– 46 months. The subjects were evaluated on progression of DR, DME, and visual acuity. Although the primary endpoint of the study was not achieved, ruboxistaurin mesylate was shown to reduce the risk of sustained moderate vision loss in patients with DME at baseline. An independent study published the same year showed that oral ruboxistaurin mesylate (4, 16, or 32 mg/day), when used for 18 months in 41 patients with DME, provided a significant reduction of treatment at any doseage and baseline permeability as determined by vitreous fluorophotometry.227 Moreover, those patients with a markedly elevated retinal vascular permeability (>3 fold) at baseline exhibited a 30% reduction when treated with ruboxistaurin versus placebo. Late in 2006, Eli Lilly & Co. reported that the FDA had provided an approval letter in regard to the use of ruboxistaurin mesylate for DR but that additional efficacy data would be required prior to approval; thus, the company was assessing its options for further development.228

3.1.7siRNA

One of the most exciting new pharmacological modalities with therapeutic potential in ocular and nonocular disease is RNA interference (RNAi). The concept of RNA interference was first reported by Fire and Mello et al. in 1998, when double stranded RNAs injected into C. elegans were converted into short interfering RNA (siRNA) that initiated sequence-selective degradation of host cytoplasmic RNA.229 Purportedly a protective mechanism against viral dsRNA, siRNA functions to silence gene expression in protozoa, plants, invertebrates, and vertebrate species.230-232 The general sequence of RNAi events involves cleavage of any cytoplasmic dsRNA by the Dicer enzyme (RNAase-III-type enzyme) into 21–28 nucleotide siRNAs, where a single siRNA strand then can be incorporated into the RNA-induced silencing complex (RISC). The RISC cleaves complementary mRNA sequences, effecting highly specific gene silencing. siRNAs typically do not elicit an interferon response, since the base pairs are less than 30 bases long. Consequently, siRNAs have become a widely used research tool in functional genomics and, at the same time, are under intense investigation as therapeutic agents, especially against targets once thought of as not treatable by drug therapy.

At least three major issues must be overcome to achieve a successful siRNA therapeutic agent: specificity, delivery, and duration. Specificity is provided by the targeted nucleotide sequence, and siRNAs have been shown

to selectively silence a particular allele that differs from another by as little as a single nucleotide. Nonetheless, siRNAs can induce “off-target” effects by targeting sequences that are closely related, which could elicit a variety of undesirable effects. Because siRNAs rapidly incorporate into RISC and require low concentrations to elicit gene silencing, they have diminished potential for nonspecific binding of proteins. Therefore, selection of the appropriate dose of siRNA will likely be important in developing a successful drug candidate. Devising effective methods of siRNA delivery into cells of interest is a major area of intellectual property competition. Two common delivery forms are chemical modification of synthetic siRNAs and vector delivery systems, such as adenoviral, adeno-associated viral, retroviral, and lentiviral vectors.233 Lastly, the duration of gene silencing will depend on multiple features of the therapeutic siRNA, including delivery method, stability of siRNA (chemical modifications can decrease nuclease susceptibility), and degree of protein binding. Three pharmaceutical companies (Acuity Pharmaceuticals, Alnylam, and SIRNA) have announced siRNA programs that target the VEGF pathway in ocular disease.

Acuity Pharmaceuticals became the first company to attempt a clinical proof-of-concept study with their siRNA, bevasiranib (Cand5), and have now completed a phase II clinical trial using intravitreal injections in patients with exudative AMD, and have begun recruiting for phase III clinical trials.234-236 Bevasiranib is a siRNA directed against VEGF-A that has been shown to provide roughly 50–60% inhibition of laser-induced CNV following a

subretinal injection in the mouse or an intravitreal injection in the nonhuman primate eye.237,238 In the primate study, the authors originally reported a dose-

response effect on fluorescein dye leakage in addition to the inhibition of CNV

lesion growth, but they later retracted the statement due to cited statistical concerns.237,239 In a phase I, open-label, dose-escalation study in 15 patients

with exudative AMD, bevasiranib was found to be safe and well tolerated following repeat intravitreal injections (<3.0 mg) over a 6-week period. Adverse events, such as subconjunctival hemorrhages and ocular pain, were determined to be primarily associated with the administration procedure. During a randomized, double-masked phase II study, 129 patients with serious exudative AMD, classic or active minimally classic AMD, including those patients who had failed previous treatments, received multiple intravitreal injections of 3 doses over 6 months. The company reported that the siRNA was safe and well tolerated and that it was able to inhibit CNV growth and prolong the need for rescue (i.e., treatment with an approved therapy).

Sirna Therapeutics (formerly Ribozyme Pharmaceuticals) has taken their expertise derived from the development of ribozyme therapeutics and converted their core focus to siRNA technology. Sirna was the first to release public data related to a phase I human trial involving siRNA.240 In

May 2005, Sirna reported visual acuity stabilization in 14 patients with exudative AMD following a single intravitreal injection (100–800 μg) of Sirna-027, an anti-VEGFR1 siRNA.241 In a preclinical publication, Sirna-027 was shown to reduce laser-induced CNV by 45–66% following a single intravitreal or periocular injection in the adult mouse.175 Additionally, intravitreal injection of Sirna-027 in the mouse OIR model provided a 32% inhibition of preretinal NV. During late 2005, Sirna entered into a partnership with Allergan Inc. to develop siRNAs for ophthalmic diseases, including continued pursuit of Sirna-027 for exudative AMD.242 At the end of 2006, Merck & Co. bought Sirna.243

Alnylam Pharmaceuticals is another biotechnology company that has reported a substantial siRNA patent portfolio (InterferRx program), including the development of anti-VEGF siRNAs.244 In 2004, Alnylam partnered with ISIS Pharmaceuticals, a leader in the field of medicinal chemistry surrounding oligonucleotides, and in early 2005, they partnered with Merck in the development of RNAi therapeutics for various human diseases, including exudative AMD.245 However, later in 2005, Alnylam announced the discontinuation of their anti-VEGF program for exudative AMD and claimed that the diminished market cap produced by the introduction of off-label, intravitreal Avastin® was a major contributor to the decision. Although many hurdles have yet to be overcome by any company in this field, the anticipated efficacy derived from selectively silencing the pathological expression of an effector gene in blinding ocular diseases likely will continue to drive this fascinating technology.

3.2Other Growth Factor Strategies

3.2.1Somatostatin and somatostatin receptor agonists

Considerable evidence suggests an important role for the Growth Hormone (GH)-Insulin-like Growth Factor 1 (IGF-1) axis in DR. The relationship of GH with DR, specifically PDR, was first noted when a patient with

spontaneous post-partum hypopituitarism exhibited clinical resolution of her retinal disease.246,247 For a time, hypophysectomy was used in retractable

cases of PKD to ameliorate the progression of the blinding condition.248 Subsequent to these findings, a number of reports have demonstrated elevated levels of IGF-1 and its binding proteins in the vitreous of patients with severe DR and/or retinal ischemia.249-253 IGF-1 immunoreactivity also has been found in surgically excised CNV membranes from patients with exudative AMD, where the retinal pigment epithelial (RPE) cells from these membranes were immunopositive for IGF-1R.254 Additional circumstantial

evidence is provided by the finding that GH-deficient dwarfs with diabetes mellitus were free of microvascular abnormalities after being followed for more than 25 years, and a 12-year-old girl with leprechaunism (a rare, congenital syndrome of insulin resistance caused by mutations in the insulin

receptor gene) exhibited progression of her DR to retinal neovascularization following chronic systemic IGF-1 therapy.255,256

Mechanistically, IGF-1 is likely the main effector molecule of this GH- IGF-1 association with DR and potentially exudative AMD. In the 1990’s, IGF-1 was found to be produced by retinal endothelial cells and induce endothelial proliferation in vitro, and intravitreal injection stimulated retinal NV in rabbits and a retinal microangiopathy in domestic pigs.257-260 Several transgenic and knockout studies have been used to confirm the importance of this signaling cascade: (1) mice expressing a GH antagonist gene exhibit inhibition of preretinal NV in the OIR model (that was reversed with IGF-1 treatment), (2) mice with a vascular cell-specific knockout of the IGF-1R or insulin receptor show a significant decrease in preretinal NV in the OIR model, and (3) normoglycemic/normoinsulinemic mice overexpressing IGF- 1 in the retina develop progressive manifestations of both nonproliferative and proliferative DR.261-263 An important aspect of IGF-1’s downstream effects is likely associated with its ability to regulate VEGF expression and

secretion in RPE cells, in part through a HIF-1α-dependent

mechanism.264,254,265

Potential interventions in this important growth factor cascade have

focused upon direct inhibition of IGF-1, or more commonly, antagonism of the GH-IGF-1 axis using synthetic variants of somatostatin.266,267

Somatostatin is a naturally occurring, GH-release inhibiting, tetradecapeptide hormone. It is produced in the central nervous system as well as many peripheral tissues including the retina, and its G-protein coupled membranebound receptors (SSTR1–5) are also expressed in the human retina.268-270 Somatostatin variants are found in the vitreous of patients with or without

DR, and some variants are deficient in patients with PDR; SSTR2 also has been identified in CNV membranes from patients with exudative AMD.271,272

Somatostatin has diverse biological functions such as neurotransmission, anti-secretion and anti-proliferation. Importantly, somatostatin has been shown to inhibit IGF-1-mediated induction of VEGF and stimulate nitric oxide production in human RPE cells through SSTR2, inhibit IGF-1-

stimulated growth of human retinal endothelial cells, and inhibit laserinduced CNV in the mouse following intravitreal injection.258,273,270,274

Stability and duration of action are anticipated disadvantages of many protein therapeutics. Consequently, synthetic analogues of somatostatin have

been developed that demonstrate longer tissue half-lives and varying affinities for its five known receptors.275,276 Octreotide (SMS201-995 or

Sandostatin®, Novartis) was one of the earliest developed somatostatin analogues that had a high affinity for SSTR-2, as well as affinity for SSTR-3 and –5.277 It has been demonstrated to inhibit proliferation and migration of human retinal endothelial cells in vitro in response to bFGF and IGF-1.258 Octreotide also inhibited IGF-1, induced IGF-1 receptor phosphorylation, and decreased subsequent VEGF production in cultured human RPE cells.273 Higgins et al. reported that treatment with octreotide caused a marked decrease in GH mRNA and protein, as well as a decrease in NV in the mouse OIR model.278 Nonetheless, the ability of somatostatin analogues to be effective treatments for human DR remains unproven. Octreotide and its long-acting analogue (Sandostatin® long-acting release or LAR) are delivered subcutaneously and have shown promise as safe and empirically effective treatments for the more severe stages of DR, including cystoid macular edema, in small clinical trials.279-283 However, similar studies in patients with early DR have not demonstrated preventive effects on further progression of the disease.284 Novartis now has apparently discontinued their phase III studies with Sandostatin® in DR.

3.2.2Lisinopril

The ACE-inhibitor, Lisinopril®, has been approved for the systemic treatment of hypertension and chronic heart failure commonly observed in patients with diabetes mellitus.285 In phase III clinical trials, Lisinopril® reduced the risk of developing DR over 2 years in type 1 diabetic patients.286 Those patients with pre-existing DR showed a slowing of disease progression. However, a confounding variable in these results may have been systemic hypertension. The mechanism of action for Lisinopril® in DR and ocular angiogenesis is undetermined; however, it is known that the local, ocular renin-angiotensin system modulates retinal microvascular function, at least in part, through VEGF signal transduction.287

3.3Matrix Metalloproteinase (MMP) Inhibitors

MMPs are a family of more than 20 soluble and membrane-anchored proteolytic enzymes. Details about the classification of MMPs and their substrates can be found in the review by Egeblad and Werb.288 MMPs are known to degrade components of the extracellular matrix (ECM) and facilitate key steps in the angiogenic cascade such as microvascular endothelial cell migration and proliferation and capillary tube formation.289

Inhibition of MMP activity at the initial stages of angiogenesis has the unique potential to prevent NV independent of the cause. The regulation of MMP activity can occur at several levels including: gene transcriptional