Fig. 14.4 Strategic points of inhibition in the VEGF cascade

Sequestration

Kinase

Inhibition

Biologic

Effects

plays a role in inßammatory processes, immunity and wound healing, and acts as a survival factor for endothelial cells and as a neuroprotectant for neurons in the central nervous system and the retina. The essential role relevant for its clinical inhibition, however, is the angiogenic cascade and vascular permeability. The blockade of these principal mechanisms has been shown to be effective in the treatment of ocular neovascularization such as neovascular AMD.

Prove of the binding afÞnity and efÞcacy of pegaptanib were supplied by in vitro studies [4] demonstrating that in human plasma pegaptanib remains stable for 18 h. Several animal studies were performed, some of them with primates. In rhesus monkeys a single intravenous or subcutaneous injection (1 mg/kg body weight) of pegaptanib proved to have a half-life of 9.3 and 12 h, respectively [5].

After a single intravitreal injection of 0.5 mg, the biologically active pegaptanib molecules remained at least 28 days in the vitreous [6]. Pegaptanib clearance

The recently approved medications primarily target and sequester the released factor. Further targets are the synthesis of the factor and its receptors, the receptor binding and the intracellular pathway (Fig. 14.4).

14.3.1 Sequestration of Released VEGF

Pegaptanib sodium (Macugen¨; OSI-Eyetech/PÞzer) was the Þrst FDA- (2004) and EMEA-approved antiVEGF medication for the treatment of exudative AMD. The molecule is a pegylated oligonucleotide aptamer with high binding speciÞcity for the VEGF-165, -189 and −201 splice isoforms that exhibit a heparin-binding domain (Fig. 14.2). The name aptamer derives from the greek words aptein (binding) and meroz (part, site). An aptamer is a nucleotide sequence with the ability to bind to a certain site of the target molecule. A particularly high binding afÞnity is achieved through the 3D structure of the oligonucleotide enabling it to fold around the binding site. The oligonucleotide, which consists of 28 ribonucleic acids, is pegylated, which means that two 20-kDa polyethylene glycol molecules are bound to each end of the aptamer. This protects the molecule from quick degradation [3].

Therefore, an elevated plasma concentration has to be anticipated in patients with renal insufÞciency. In a monkey model no signs of toxicity after intravenous or intravitreal application could be detected.

An important base for the development of Macugen¨ were the results of studies showing that VEGF165 plays an important role in neovascularization processes [7], without impairing physiological vessels, which is in contrast to complete VEGF blockage [8]. The selective inhibition of certain VEGF-A isoforms was supposed to cause fewer side effects and still has the same efÞcacy. However, the distribution and function of the different isoforms in the human eye and in AMD seem to differ from the animal model.

Ranibizumab (Lucentis¨, Genentech/Novartis) was approved as a second VEGF inhibitor for the treatment of wet AMD in June 2006 by the FDA in the US and in 2007 by the EMEA in Europe. Ranibizumab is a humanized antigen-binding antibody fragment (Fab), binding to all isoforms of VEGF-A. This antibody fragment was developed for easier penetration through the retina [9]. The antibody fragment originates from a mouse monoclonal full antibody against human VEGF-A. The binding domains within the light chains (Fab fragment) were modiÞed several times to achieve a 50-fold increased binding afÞnity compared with the primary molecule.

The Fab fragment is supposed to confer additional beneÞts as the Fc-part with its pro-inßammatory characteristics is missing.

The half-life of ranibizumab is signiÞcantly shorter than that of its full antibody. This is a disadvantage in terms of the long-term effect in the eye, but may be an advantage in terms of the systemic circulation. The systemic half-life of the Fab fragment is only a few hours, whereas the full antibody remains in the circulation for several weeks.

Bevacizumab (Avastin¨, Genentech/Roche) is a full antibody against VEGF-A. The drug was developed for intravenous application and was FDA approved in the US for the treatment of metastasized colorectal cancer in February 2004. Regarding the Þrst promising study results with ranibizumab (Lucentis¨), which was not yet approved at that date, the full antibody bevacizumab was used for the treatment of patients with wet AMD, Þrst intravenously and later intravitreally. The growing off-label use was followed by a number of in vitro and in vivo studies in order to test the efÞcacy, toxicity and biocompatibility of bevacizumab. Also the full antibody was shown to be able to penetrate all retinal layers to reach subretinal pathologies [10]. At present, experimental and clinical studies are investigating whether the signiÞcant molecular differences between ranibizumab and bevacizumab lead to signiÞcant biological and clinical differences.

The authors of the so far only randomized comparative clinical trial (CATT-Study) concluded on the basis of 1-year-results that bevacizumab and ranibizumab achieve equivalent results with regard to visual acuity when given in identical therapy regimen [22].

VEGF trap (Aßibercept¨, Regeneron/Bayer) approved in the US by the FDA in November 2011, has a similar mode of action as an antibody. However, it is a fusion protein, consisting of the key domains of the human VEGF1 and VEGF2 receptors, coupled to the Fc part of a human IgG molecule. The molecule consists completely of human amino acids; therefore, inßammatory processes may be reduced. VEGF trap has a signiÞcantly higher afÞnity to the VEGF molecule than the normal VEGF receptor of endothelial cells and also a higher afÞnity than other VEGF antibodies, such as bevacizumab. VEGF trap not only binds to VEGF-A, like bevacizumab and ranibizumab, but also to all other VEGF isoforms (A-D) as well as PIGF. As intravenous application led to hypotony and severe cardiovascular side effects [11], the intravitreal application mode is currently preferred for clinical use. The molecular weight of 110,000 Da enables complete

penetration through the retina. Because of the high afÞnity to VEGF, good efÞcacy at a low dose with a long half-life is expected [12].

Anticalines (Pieris) are another alternative to the antibody strategy. Anticalines belong to a new group of drugs, originating from lipocalines. Lipocalines belong to a family of natural human proteins, serving as transport and storage devices. Through molecular changes at the binding domains, different proteins can be captured [13]. PRS-055 is an anticaline that was speciÞcally developed for the blockage of free VEGF.

Receptor-Chimeras, e.g., sFLT01 (Genzyme Corp.), consists of an immunoglobulin domain and VEGF-R1 (Flt-1). The mixed protein is not directly injected intravitreally, but delivered by recombinant adeno-associ- ated viruses (rAAV). In vitro studies demonstrated a high binding afÞnity for VEGF. In a mouse model the expression of sFlt-1 could be induced by intravitreal injection of the virus vectors, leading to an inhibition of angiogenesis [14]. The advantage of this method is the long-term presence of the inhibitor in the eye, due to its constant synthesis and release from the virus. However, the risk of potentially triggered neoplastic disease by adenoviruses is not yet clear.

14.3.2Inhibition of VEGF and VEGF Receptor Synthesis by Small Interfering RNA (siRNA)

Inhibition of VEGF and VEGF receptor synthesis by small interfering RNA (siRNA) functions by inhibiting the intracellular synthesis of proangiogenic molecules [21]. This mode of action differs from the previously described drugs, which target the extracellularly secreted molecule. Therapies based on RNA interference use small synthetically produced RNA fragments, silencing the effect of a particular target gene by speciÞc binding and degradation of mRNA (messenger RNA). The normal cell uses this mechanism for example against RNA viruses. A single siRNA molecule degrades a large number of mRNA chains coding for an even larger number of protein copies [3]. Therefore, this is a very potent mechanism. The inhibition does not take place at the level of extracellularly circulating VEGF as it does for antibodies and aptamers but the cascade is blocked at an earlier step, inhibiting the intracellular synthesis of VEGF. Another potential target for siRNA would be the degradation of VEGF receptor mRNA.

Bevasiranib¨ (Acuity Pharmaceuticals) silences the VEGF synthesizing gene, whereas Sirna-027¨ (Sirna/ Allergan) silences the gene coding for the VEGF recepetor-1. After intravitreal injection hardly any of the active drug reaches the systemic circulation, thus reducing the risk of cardiovascular side effects. The effect starts after a certain latency, when remaining VEGF is eliminated. It is advantageous that the effect lasts particularly long (at least 12 weeks). This means that the required number of re-injections is lower than for other drugs, reducing the cumulative surgical risk (endophthalmitis) compared to drugs with shorter retreatment intervals.

14.3.3Inhibition of the Intracellular Signal Cascade

The activation of VEGF receptors leads to an activation of a complex variety of intracellular signal cascades. This signal cascade induces the phosphorylation of proteins, such as PI3, MAPK and PKC.

Anti-human VEGF receptor Flt-1 antibodies or peptides are in competition with VEGF for the VEGFbinding domain of the receptor. In contrast to the growth factor VEGF itself, the binding of these antibodies or peptides does not activate the receptor [15]. The humanized antibody is produced in hybridoma cells.

Receptor tyrosine kinase inhibitors block the intracellular cascade, which normally starts after binding to this receptor. Whereas Vatalanib¨ (Novartis) only inhibits VEGF receptors (VEGFR1-3), there is another tyrosine kinase inhibitor (AG-013958, Allergan) that also blocks the receptor for PDGF (platelet-derived growth factor). Vatalanib¨ is taken orally (e.g., 1,250 mg/day), and therefore is less invasive. Disadvantageous is the higher risk of systemic side effects. In contrast, AG-013958 is injected subtenonally. Both drugs are well tolerated. Further studies need to examine the efÞcacy, dosing and application frequency. In the animal model experimentally induced neovascularization was reduced by 80% [16].

Other tyrosine kinase inhibitors, such as pazopanib (GlaxoSmithKlein), TG100801 and TG101095 (Targegen), are being developed for topical application, whereas AL39324 (Alcon) should be applied by intravitreal injection.

The PKC-b inhibitor (ruboxistaurin mesylate) was developed to treat diabetic macular edema, but may

also be a useful therapeutic strategy for AMD. Similarly, ICo-007 (iCo Therapeutics), a second-generation antisense oligonucleotide targeting C-raf kinase mRNA, was developed to treat neovascular processes [20]. iCo-007 binds to the C-raf kinase mRNA and inhibits its transcription. Though antisense oligonucleotides act like siRNAs, they are less stable and effective. The second generation of this substance class, however, has been improved.

Squalamine (Genaera) is an aminosterol with antiangiogenic characteristics affecting the intracellular signaling cascade (e.g., of VEGF). After promising experimental and phase I/II results the phase III study was discontinued.

Several drugs known to inhibit VEGF receptor kinase and VEGF-induced effects are now being evaluated for the treatment of AMD. These are quinazolin derivatives (Astrazeneca AB), imidazo[1,2-A] pyridine (Lilly Co.), anthranilamide pyridinureas (Bayer Schering Pharma AG), anthranilic acid amides and 2-aminonicotine amide (Novartis AG), thalidomide analoga and N-aryl- sulfoximine-substituted pyrimidines (Schering AG). In addition to the VEGF-related downstream cascade, targeting of the upstream signaling seems to be promising. HIF-a and mTOR are potential targets. mTOR plays a central role regulating both, inducing and inhibiting pathways that act on angiogenesis, protein synthesis, cell proliferation and metabolism. Clinical studies are evaluating everolimus (RAD-001, Novartis) and palomid 529 (Paloma Pharmaceuticals, Inc.).

14.3.4 Natural VEGF Inhibitors

Angiogenesis is inßuenced by proand anti-angiogenic factors. VEGF is physiologically counterbalanced. Neovascularization reßects an imbalance toward proangiogenic factors such as VEGF. Recreating a balanced situation may therefore be achieved with the addition of natural counter players. The most prominent endogenous inhibitors are PEDF and endostatin.

Phase-I-studies showed that an intravitreally injected adenoviral vector encoding pigment epithelium derived factor (PEDF) (AdPEDF¨, GenVec), could halt the progression of choroidal neovascularization [17]. PEDF inßuences physiological angiogenesis and protects photoreceptors. AdPEDF-based therapy could therefore be both, anti-angiogenic and neuroprotective. Additionally, one injection could have a prolonged effect.