Transcripción de la entrevista a Carlos Soto (El Círculo Mágico. 13-05-2021)

VEGF is upregulated by hypoxia during the angiogenesis. VEGF activates quiescent endothelial cells and stimulate cell proliferation, migration and vascular permeability [183]. The intravitreal administration of recombinant human VEGF165 produces non- inflammatory iris neovascularization, ectropion uvea and neovascular glaucoma [184]. An alternative strategy was designed to inhibit the VEGF receptor tyrosine kinase (VEGFR-

2): sunitinib and sorafenib. These drugs were approved for the treatment of renal cell carcinoma and gastrointestinal stromal tumor respectively [185]. Avastin (bevacizumab) is a full-length humanized rhuMAb HER2 antibody (148 kD) and Lucentis (ranibizumab) is the humanized rhuMAb vascular endothelial growth factor Fab antibody (48.3 kD) isolated from bevacizumab [186]. But the inhibition of VEGFR-2 is proportionally related with the risk of blood pressure elevation [187] there is also a regulated increase in vascular permeability to both solutes and water [188] [189]. And VEGF regulates the apoptosis of EC, contributes to the maturation of vascular granulation tissue into avascular scar tissue and, to the pruning of retinal vessels during development [190].

The target of treatment has thus far been direct inhibition of VEGF in the eye with VEGF- A antagonists. There are four anti-VEGF drugs on the market for wet AMD.

2.6.1 Pegaptanib

Pegaptanib (Macugen, Pfizer) is a specific 28-base ribonucleic acid aptamer of 50 kDa aprox, covalently linked PEG [191, 192] with a high selectivity and inhibitor of VEGF- A165 isoform [192, 193], which has been implicated in blood-retinal barrier breakdown and

pathological intraocular neovascularization [21]. Pegaptanib was found as the weaker drug to treat this disease [194]. The IVT half-life is 10 days; given at 0.3 mg every six weeks. In vitro and in vivo studies have shown that precursors of this drug inhibit the enhancement of EC proliferation and vascular permeability [195, 196]. Macugen was approved by the FDA in 2004 and by the EMA in 2006. Therapeutic benefit was very favorable in comparison with photodynamic therapy. However, was overturned in efficacy terms displaced by the next anti-VEGF drug, ranibizumab, since pegaptanib does not inhibit other bioactive isoforms of VEGF fragment in vivo. Pegaptanib is no longer recommended for the treatment of AMD [197].

2.6.2 Ranibizumab

Ranibizumab (Lucentis, Genentech), is a 48 kDa recombinant, monoclonal, humanized antibody Fab fragment which inhibits VEGF-A isoforms [192, 198, 199]. The binding site is located at amino acid sites 88-89 [200]. It is designed from the full-length monoclonal antibody bevacizumab (developed for cancer angiogenesis) by removing the Fc fragment from the parent molecule [201, 202]. Ranibizumab binds with high affinity a site present in all isoforms and their bioactive proteolytic fragments, including the soluble VEGF

isoforms 110, 121 and 165 and the tissue-bound isoforms 189 and 206 [203-205]. The half- life of ranibizumab is 2.9 days, in rabbits at a concentration of >0.1 µg/mL, the compound was maintained in vitreous humor for 29 days [206]. The relative molar binding activity to VEGF is 1, so excess drug has a rapid systemic clearance thus systemic safety of this drug is very high [207] [208]. The recommended dose is 0.5 mg/monthly injection and the initial loading dose is of three monthly injections. After this visual acuity is reached and patients have to be monitored monthly for visual acuity to continue the treatment, 1 injection every month if there is a loss of visual acuity. Lucentis was approved by the FDA in July 2006 and by the EMA in January 2007 [21].

2.6.3 Bevacizumab

Bevacizumab (Avastin, Genentech), is a full-length humanized anti-VEGF monoclonal IgG1 anti-VEGF-A antibody that inhibited the action of all isoforms of VEGF [192, 193, 198]. It binds to both VEGF165a and VEGF165b with equal affinity [209]. The target is the

same as ranibizumab, the MW is 149 kDa and the half-life is longer than ranibizumab [199, 210]. Human IVT half-life is estimated at 4.32 days and its relative molar binding activity is 0.05-0.2, showing it has lower binding affinity than current treatments [211]. With a concentration of >10 µg/mL, bevacizumab was maintained in the vitreous humor for 30 days [212]. Bevacizumab was approved by the FDA and EMA for the treatment of different types of solid tumors (colorectal, non-epithelial lung, breast, ovarian and renal) and glioblastomas [213]. In ophthalmology it is used off-label. The recommended dose for intraocular treatment is 1.25 mg/month [214]. This is usually given as a 50 μL injection of 25 mg/mL protein.

2.6.4 Aflibercept

Aflibercept (Eylea, Regeneron), is a 115-kDa recombinant fusion protein which, in addition to inhibiting VEGF-A, also inhibits VEGF-B binding domains of human VEGFR-1 and VEGFR-2 fused to the Fc domain of human immunoglobulin G1. Aflibercept and ranibizumab have the same effect blocking endothelial cell proliferation and both are 10- fold more potent than bevacizumab and aflibercept is non-inferior to ranibizumab in treating wet AMD [192, 215]. Aflibercept binds to placental growth factor (PLGF), a proangiogenic protein present on EC and leukocytes [208]. It acts as a soluble decoy receptor recognizing and neutralizing all VEGF-A isoforms, with the establishment of a

tighter binding than the native receptors. It binds also PLGF-1 and PLGF-2 with a high affinity, greater than bevacizumab or ranibizumab. Therefore, the predicted binding affinity of aflibercept to its ligand is higher than ranibizumab (aflibercept: kDa = 0.49 pmol/L; ranibizumab: kDa = 0.46 pmol/L; ranibizumab: kDa = 0.58 pmol/L) [191]; [193]; [194]. The intravitreal half-life of aflibercept is 7.1 days and the relative molar binding is 140 thus, the activity in the eye of Eylea is 2.5 months [216] [217]. The FDA approval was granted in 2012. The recommended intravitreal dose is 2.0 mg/month for three consecutive treatments, followed by one injection every two months [218].

VEGF-A antagonists utility is limited by several factors. It has been reported that approximately 15-33% of patients with wet AMD are “nonresponders” despite undergoing treatment with anti-VEGF agents and a further 30-40% of patients will experience no improvement in vision and no change in lesion size [10, 219]. These treatments slow disease progression more often than they improve vision due to adaptive resistance of the system. The choroidal microenvironment responds to VEGF antagonists by increasing VEGF expression, and neovascularization can resurge between treatment cycles [220]. In addition, VEGF is not the only proangiogenic factor contributing to retinal neovascularization and its blockade can cause overproduction of other factors (IL-8, bFGF) as was shown for cancer angiogenesis [220]. In addition, the cons of these treatments, especially in the long-term period, are: the high frequently administration, IVT injection every 1-2 months, the price Eylea and Lucentis ranges from 800-1,900 USD (re-formulated Avastin is 50-100 USD) and most of them have potential side effects related to the eye and the circulatory system like, vitreous hemorrhage, retinal detachment, hypertension, etc [221, 222]. So novel compounds could decrease AMD progression with minimal local and systemic effects, with significantly reduced frequency of administration is important to enhance the effective of the treatment.