The early 1980s, a group of researchers revealed that the abnormal accumulation of fluids in malignant ascites is attributed to a potent protein-base factor termed tumor vascular permeability factor (VPF) that is released from the tumor cells and rapidly increase the microvasculature permeability 1. A few years later, in an independent work, Ferrara and Henzel from Genentech’s South San Francisco, California succeeded in identifying a mitogenic growth factor isolated from pituitary follicular cell culture that stimulates the proliferation of microvascular endothelial cells. Based on its target selectivity, it was called Vascular Endothelial Growth Factor (VEGF) 2. Soon, it became clear that both VPF and VEGF were one and the same protein 3.

VEGF (also, referred to as VEGF-A) is a secreted disulfide-linked homodimeric glycoprotein of 45 kDa, occurs in four main different isoforms that derived through the alternative exon splicing events for the common mRNA. They vary with the different degrees in their affinity for heparin and therefore, their relative abundance within a given tissue as matrix-bound or/and free diffusible forms 4. VEGF exerts their biological effects by binding to specific receptors on cell surfaces and triggers a complex sequence of events that stimulates endothelial cells to proliferate, migrate, and produce matrix-degrading enzymes which contribute to the formation of new vessels in a process termed ‘angiogenesis’ 5. VEGF signaling often represents a critical rate-limiting step in physiological angiogenesis which is essential for tissue repair and reproductive functions in the adult 5. However, angiogenesis is also implicated in the pathogenesis of a variety of disorders, of these: malignant tumor and inflammations such as rheumatoid arthritis (RA), and psoriasis 6, diabetic proliferative retinopathies 7 and age-related macular degeneration (AMD)8. n this context, a research team proved that intravitreous injections of physiologically relevant amounts of human recombinant VEGF165 into the eyes of healthy cynomolgus monkeys are sufficient to produce retinal vascular abnormalities that are similar to the human diabetic retinopathy and other ischemic retinopathies 9. Taken together, these documented pieces of pieces of evidence provided a strong rationale for the targeting of VEGF in many human disorders that manifest such as ocular neovascularization and increased vascular permeability. Importantly, VEGF165 which represents the most prominent isoform and has greater biological potency, than VEGF121 10.

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Therefore, it was considered as a promising therapeutic target for the treatment of many ocular disorders such as AMD. Indeed, the complete inhibition of intracellular VEGF signaling by blocking all VEGF isoforms resulted in substantial suppression of normal vascular development. For these reasons, ophthalmologists are looking for a promising therapy that can selectively target pathological neovascularization, while not affecting the physiological compensatory revascularization 11.   Moreover, It is interesting to note that VEGF also bind to heparin, a polyanion like nucleic acids, which rivals that the developing aptamer technology could provide the anticipated new drug therapy that can selectively inhibit growth factor signaling from its beginning, by interfering with the binding of potent VEGF 165 with its receptor 12.                 First FDA Approved Therapeutic Aptamer: VEGF-Targeting Macugen   SELEX with VEGF-165 and the Composition of NX1838   The development of Anit-VEGF 165 aptamer began in early 1994, were Jellinek and co-researcher from NeXstar Pharmaceuticals, Inc. isolated six families of unmodified RNA ligands against VEGF 165 from a random pool of 1014 RNA molecules using Tuerek and Gold SELEX procedure 13.   The representative RNA aptamers from each family were further truncated to obtain the minimal sequences necessary for high-affinity binding to VEGF 165.

The results showed that the truncated RNA ligands had high binding affinities and specificity for VEGF 165. In addition, they all competitively and particularly bound to the heparin-binding domain in a way that antagonizes the binding of VEGF to its cell surface receptor with an IC-50 range from 20-40 nM 13.   The selection protocol for anti-VEGF aptamers was further developed in 1995 by using 2′-aminopyrimidine RNA random library, in an attempt to increase the stability of selected ligands in vivo.

Moreover, Further post SELEX modifications had been performed to the truncated candidate ligands by substituting ribopurine nucleotides at particular positions with 2′-O-methyl purines and adding phosphorothionate caps at both ends 14.   It was clear that such chemical modifications enhanced the therapeutic potential of selected aptamers through increasing their resistance to nucleases as was shown in the tested urine samples.  Interestingly, the binding affinity to VEGF was also improved up to 17 folds as demonstrated in the NX-213 ligand. Finally, they observed that 2′-O-methyl substitutions also improve both the specificity and thermal stability of the given aptamers 14.   In a complementary fashion, strenuous efforts had been employed to obtain outweighed anti-VEGF 165  aptamers which have higher binding affinities than the previously selected 2′-aminopyrimidine-based aptamers, more thermodynamic stability and being more economical. To achieve these purposes Ruckman et al used 2′-fluoropyrimidine RNA library based on the assumption that 2′-F-pyrimidine groups will allow aptamers to adopt more rigid conformations than the more flexible conformation conferred by 2′-NH2 groups which may exhibit higher binding affinities for their targets.

In addition, the differences in the size and hydrogen bonding properties of the 2′-substituents may also improve the affinity parameter 15.   Accordingly, three representative families of 2′-F-pyrimidine anti VEGF165 aptamers had been identified from the random library using 12 rounds of SELEX, which were further modified and truncated to obtain 2′-F-pyrimidine -2′-O-Methyl-purine-substituted RNA aptamers. It was from this set of SELEX Macugen was identified 15.   In this study, it was reported that these selected aptamers (t22.23-OMe, t2.29-OMe, and t44.27-OMe) specifically bound to the basic exon 7-encoded domain of human VEGF165 with high affinities range between 49 and 130 pM. On the other hand, no binding to VEGF121 isoform had been detected.

Furthermore, these aptamers inhibited the binding of VEGF to its receptor with IC50 values ranging between 50 pM to 0.3 nM for the Flt-1 receptor (fms-like tyrosine kinase) and 2 to 60 pM for KDR receptor (kinase insert domain-containing receptor)15.     It is worth to mention, that these observations were further supported in 2005 by a nuclear magnetic resonance (NMR) spectroscopy studies performed by Lee et al with t44-OMe aptamer, which demonstrated that the aptamer predominantly interacts with the heparin binding domain of full-length VEGF165 and blocks the receptor binding through steric interference mechanism 16. In the same context, other study showed that t44-OMe aptamer undergoes an induced fit mechanism to achieve high-affinity binding 17. For in vivo studies, Miles assay (also called, cutaneous vascular permeability assay) had been performed, in which each of candidate aptamer premixed with VEGF and co-injected intradermally in adult guinea pigs.

The result revealed that the vascular permeability response was inhibited by 58% and 37 % for t44-OMe and t22-OMe, respectively at 1µM concentration. On the other hand, the third aptamer, t2-OMe showed no inhibition in this assay relative to the control oligonucleotide 15.   Finally, t44-OMe aptamer was selected for further development in order to enhance its inhibitory activity by conjugating 40-kDa PEG at it’s 5′-end.  It was demonstrated that PEG conjugation truly increases the potency of the aptamer, an effect that may derive from the relatively slower diffusion rate of the higher molecular weight conjugate which would increase the opportunities for rebinding of the aptamers to its target. Additionally, the data confirmed that PEG conjugation also improved the pharmacokinetic properties of the aptamer through reducing it’s in vivo clearance rate by the kidneys and increasing its plasma half-life, which was further improved by adding deoxythymidine at it’s 3′-terminus via a 3′-3′ linkage. Indeed, such modifications dramatically increased the resistance of the t44-OMe to nucleases in body fluids 15.     The resulted PEG-t44-OMe conjugate RNA aptamer later referred to as NX 1838, inhibited the VEGF-induced vascular permeability by 83% at 0.

1 µM concentration 15.   From NX1838 to Macugen : Preclinical tests   Additional study in 1999, reported that NX1838 inhibited the binding of VEGF 165 to its receptors expressed in human umbilical vein endothelial cells (HUVEC) and human dermal microvascular endothelial cells (HMVEC-ds) with IC50 in the range 0.16-1.3 nM18. Moreover, the binding of NX1838 inhibited both the VEGF-mediated phosphotyrosine signal transduction and VEGF-induced calcium mobilization in vitro.

In addition, inhibited HUVEC proliferation induced by VEGF165 in vitro with an IC50 value of 1.1 nM compare with anti-VEGF-mAb with an IC50 value of 0.1 nM.

In contrast, NX1838 did not inhibit VEGF121-mediated HUVEC proliferation, while inhibited by anti-VEGF-mAb (IC50 value of 0.11 nM) 18. In supportive fashion, the systemic plasma pharmacokinetics was first investigated in a primate model following the administration of a single 1 mg/ Kg dose of NX1838 aptamer 19.

Accordingly, the estimated half-lives of NX1838 in the plasma of Rhesus Monkeys were 9.3 and 12 hours following intravenous and subcutaneous administration, respectively. While the estimated clearance rate was 6.2 ml/(h kg) which was very similar to that previously determined for rats 7.8 ml/(h kg)19.

With the increasing evidences that VEGF has an important role in the pathogenesis of choroidal neovascularisation of age-related macular degeneration (ARMD) along with the coincident increasing in its expression in the vitreous and the outer layer of the macula in these patients, Drolet and his colleagues decided to study the pharmacokinetic parameters and toxicological potential of NX1838 in the vitreous humor of model primates 20. The results interestingly demonstrated that NX1838 remained fully active in the vitreous humor up to 28 days following receiving single 0.50 mg /eye dose by injection.In addition, No toxicological effects or antibody responses were evident 20.   Results from these studies allowed for the progression from pre-clinical evaluation in animal models to highly anticipated clinical trials.   Macugen-Clinical Studies   Phase IA clinical study was published in 2002 by the Eyetech Pharmaceuticals Study Group. In this study, 15 patients with subfoveal choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD) were received a single intravitreal dose of NX1838 (referred to as EYE001) varying from 0.25 to 3.

0 mg/eye and were followed up for 3 months. The study demonstrated the safety of NX1838 single dose with no significant ocular or systemic risks. In addition, 80% of patients showed stable or improved vision after 3 months of the treatment, while 26.7% had significant improvement in vision as assessed on the Early Treatment for Diabetic Retinopathy Study (ETDRS) chart at that time 21.

  In phase II clinical safety study, the same pharmaceutical company further tested the therapeutic effect of multiple doses of EYE001 aptamer (now called pegaptanib sodium) injected in the eye of 21 patients with subfoveal CNV secondary to AMD. In this study, patients received three doses, each 3 mg/eye at 28-day intervals with or without photodynamic therapy (PDT). According to this study, the majority of those treated with the aptamer alone showed stable or improved vision at 3 months, while 25% of them had a 3 line improvement of vision on ETDRS chart.

Furthermore, 90% of participants treated with both pegaptanib sodium and PDT showed stabilized or improved vision, and 60% showed a 3 line improvement of vision on the ETDRS chart at 3 months. Also, this study demonstrated that no serious adverse events related to pegaptanib sodium were noted 22. Two concurrent, randomized phase III controlled clinical trials were conducted by V.I.


N. (VEGF Inhibition Study in Ocular Neovascularization) trials to test the short-term safety and effectiveness of pegaptanib (now called, Macugen, Eyetech Pharmaceuticals) with a broad spectrum of patients with neovascular age-related macular degeneration (ARMD)23.   In this study, a total of 1186 patients (divided into two groups ,first trial enrolled 586 patients, while 622 patients for the second trial) spread across 117 test sites around the world were randomly received either nine intravitreous injections of 0.3,1,or 3 mg pegaptanib into one eye or sham injections over a period of 48 weeks 23.

According to this study, there were significant differences between sham injections and all three doses of pegaptanib in term of the prespecified primary efficacy endpoint and the angiographic measurements 23.   Patients received sham injections had twice risks to develop a severe loss of vision as patients administrated 0.3 mg or 1 mg of pegaptanib. Indeed, there was no observed evidence in any of the analyses that the efficiency of pegaptanib was dose-dependent.

These visual results were further supported by the angiographic examinations which observed a regression in the growth of total lesion area, size of choroidal neovascularization, and the severity of leakage in patients received the aptamer 23. In agreement with preclinical observations, no antibodies against pegaptanib were detected and no local or systemic hypersensitivity attributed to pegaptanib was reported 23. These data led to pegaptanib being the first aptamer-based drug to receive approval from the FDA in the December of 2004 for the treatment of wet AMD at the dose of 0.

3 mg, given as an IVT injection every 6 weeks 24.   The safety profile of pegaptanib had been further studied by V.I.S.I.O.

N trials for 1, 2 and 3 years of continuous treatment in patients with neovascular age-related macular degeneration. As in year 1, data from patients received continuous treatment of pegaptanib up to 2 years revealed no evidence of systemic toxicity 25. It was documented, that 45% relative benefit in vision observed in patients randomized to continue 0.

3 mg pegaptanib at the end of 102 weeks compared with those receiving usual care 26.   In addition, the161 elderly patients received continuous treatment for 3 years, showed no new changes in the ocular and systemic safety profile over this additional period. Also, consistent with the previous years, pegaptanib sodium was well tolerated, with no significant intraocular activation of the immune response; adverse events were not unexpected and mainly ocular in nature, mild, transient and none of them were judged to be related with the study drug 27. Since its approval By FDA, Macugen aptamer reached peak sales of $185M in its first full year of sales (2005) in the United States. However, it has been displaced by antibody-based anti-VEGF therapeutics such as Lucentis (ranibizumab), Avastin, and Eylea (aflibercept).

In fact, the most likely explanation is that these mentioned anti-VEGF drugs inhibit all isoforms of VEGF, including VEGF-121 and VEGF-110, whereas Macugen is selective for VEGF-165 26. Here, it is noteworthy to recognize that Macugen represents the first historical success of aptamers as drug therapy, which indicates the high potential of aptamers to be used in the clinical application, and without any doubt, open the door for further ongoing studies for many uncover applications of aptamers in the future.


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