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Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor Inhibitors as Anti-Angiogenic Agents in Cancer Therapy
Anand Veeravagu1, Andrew R. Hsu1, Weibo Cai2, Lewis C. Hou1, Victor C.K. Tse1 and Xiaoyuan Chen2,*
1

Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5484, USA; 2Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Stanford University School of Medicine, Stanford, CA 94305-5484, USA
Received: June 20, 2006; Accepted: August 15, 2006; Revised: November 16, 2006

Abstract: New blood vessel formation (angiogenesis) is fundamental to the process of tumor growth, invasion, and metastatic dissemination. The vascular endothelial growth factor (VEGF) family of ligands and receptors are well established as key regulators of these processes. VEGF is a glycoprotein with mitogenic activity on vascular endothelial cells. Specifically, VEGF-receptor pathway activation results in signaling cascades that promote endothelial cell growth, migration, differentiation, and survival from pre-existing vasculature. Thus, the role of VEGF has been extensively studied in the pathogenesis and angiogenesis of human cancers. Recent identification of seven VEGF ligand variants (VEGF [A-F], PIGF) and three VEGF tyrosine kinase receptors (VEGFR- [1-3]) has led to the development of several novel inhibitory compounds. Clinical trials have shown inhibitors to this pathway (anti-VEGF therapies) are effective in reducing tumor size, metastasis and blood vessel formation. Clinically, this may result in increased progression free survival, overall patient survival rate and will expand the potential for combinatorial therapies. Having been first described in the 1980s, VEGF patenting activity since then has focused on anti-cancer therapeutics designed to inhibit tumoral vascular formation. This review will focus on patents which target VEGF-[A-F] and/or VEGFR-[1-3] for use in anti-cancer treatment.

Keywords: Tumor angiogenesis, vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR). 1. INTRODUCTION 1.1. VEGF Family Proteins Vascular endothelial growth factors (VEGFs) have been shown to be key molecules implicated in embryonic development, angiogenesis, vascular permeability, tumor progression and cardiovascular disease [1]. VEGF is a homodimeric, basic, 45 kDa glycoprotein specific for vascular endothelial cells [1,2]. VEGF was first described as vascular permeability factor (VPF) by Dvorak and colleagues after it was discovered to increase the permeability of tumor blood vessels [3]. Currently, the VEGF family consists of seven members - VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGFE, VEGF-F and placental growth factor (PlGF). Each isoform is distinct in its composition of 121, 145, 165, 183, 189 and 206 amino acids by monomer (respectively VEGF121, VEGF 145, VEGF 165, VEGF 183, VEGF 189, VEGF 206) and VEGF165 is the predominant protein among the major splice variants [4]. Each isomoer is the result of alternative splicing of messenger RNA (mRNA) from a common gene composed of eight-cysteine residues [5]. 1.2. VEGF Receptors These VEGF isoforms have different physical and biological properties and act through three specific tyrosine kinase receptors - Fms-like tyrosine kinase Flt-1 (VEGFR*Address correspondence to this author at the Molecular Imaging Program at Stanford (MIPS), Department of Radiology & Bio-X Program, Stanford University School of Medicine, 1201 Welch Road, Room P095, Stanford, CA 94305-5484, USA; Tel: (650) 725-0950; Fax: (650) 736-7925; E-mail: shawchen@stanford.edu 1574-8928/07 $100.00+.00

1/Flt-1), the kinase domain region, also referred to as fetal liver kinase (VEGFR-2/KDR/Flk-1), and Flt-4 (VEGFR-3). Each receptor has seven immunoglobulin-like domains in the extracellular domain, a single trans-membrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insert domain [6]. The complete function of each receptor has not been fully determined, however certain VEGFRs have been targeted by cancer therapeutics due to their known roles in angiogenesis. While the distinct implication of VEGFR-1 has yet to be determined since its discovery over a decade ago [7], VEGFR-2 and more recently VEGFR-3 have been labeled as the receptors responsible for the angiogenic consequence of VEGF signaling. The role of VEGFR-1 in blood vessel development and vascular permeability remains unclear. VEGFR-1 has been shown to be weaker in kinase activity and is thus incapable of provoking endothelial cell proliferation when stimulated with VEGF [8]. However, recent studies suggest a temporal component to VEGFR-1 effector function. VEGFR-1 has been shown to modulate endothelial cell proliferation during early stages of vascular development preceding the formation of primitive blood vessels and vascular networks. The role of VEGFR-1 in post-fetal blood vessel formation has yet to be proven and pre-clinical experiments have continued to reveal VEGFR-2 as a more potent mediator of post-embryonic vascular formation. It is currently understood that the major mediator of endothelial cell proliferation, angiogenesis, and heightened vessel permeability as caused by VEGF signaling is VEGFR-2. The key role of this receptor in developmental
2007 Bentham Science Publishers Ltd.

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vasculogenesis and blood island formation is evidenced by the failure of VEGFR-2 knockout mice to develop organized blood vessels and typical vasculature resulting in death inutero [9]. While VEGF binds to VEGFR-2 with lower affinity when compared to VEGFR-1 (Kd = 75-250 pM vs. 25 pM) [8,10-12], the mediation of mitogenesis and angiogenesis through Flk-1 has been clearly established in both in vitro and in vivo models [13,14]. Upon binding VEGF, VEGFR-2 undergoes dimerization and liganddependent tyrosine phosphorylation. The major phosphorylation site, Y1175, is known to be responsible for endothelial cell proliferation via Raf-Mek-Erk pathway [15,16] and endothelial survival via PI3 kinase/Akt pathway [17]. Specific activation of VEGFR-2 with VEGF-E has again demonstrated potent endothelial cell activity in vitro and in vivo, supporting the notion that activation of VEGFR-2 alone can efficiently stimulate angiogenesis [18]. Further studies demonstrate that VEGFR-2 is exclusively expressed on endothelial cells, making it a prime target for anti-angiogenic therapeutics [10,12]. The third VEGF receptor, VEGFR-3, displays slightly different signaling characteristics. Contrary to the previously described mechanisms, VEGFR-3 undergoes proteolytic cleavage in the extracellular domain into two disulfidelinked peptides. While this receptor is capable of stimulating cell migration, differentiation, and mitogenesis, VEGFR-3 is predominantly localized to the surface of lymphatic endothelial cells [19, 20]. Thus, VEGF-C, VEGF-D, and their receptor, VEGFR-3, present a strong molecular signaling system for tumor lymphangiogenesis and another possible avenue for anti-angiogenic and anti-metastatic therapeutics [21]. 1.3. VEGF and Cancer Tumor development and growth greatly depends on access to oxygen, nutrients, growth factors, hormones, and hemostatic factors carried by blood vessels [22]. To this regard, it has been shown that a primary tumors ability to recruit and create a network of blood vessels will often determine and contribute to its growth and clinical severity [23-25]. The pathology of tumorigenesis indicates a marked transition from a prevascular to vascular phase. In the prevascular state, the tumor does not induce angiogenesis, is limited in size, and rarely metastasizes. However, the vascularized tumor induces host microvessels to undergo angiogenesis, has the potential to rapidly expand its cell population, and has a propensity to metastasize [26]. Events included in this process are the proliferation, migration, and invasion of endothelial cells, organization of endothelial cells into functional tubular structures, maturation of vessels, and vessel regression [27]. To date, the most influential molecular signaling pathway involved in such angiogenic activity is VEGF. It is one of the most potent inducers of vascular permeability known-50,000-fold more potent than histamine [28]. Being responsible for the hyper-permeability of tumor vessels, VEGF has been shown to allow for the leakage of several plasma proteins, including fibrinogen and other clotting proteins to transform the stroma of normal tissues into a pro-angiogenic environment [25,27,28]. The expression of VEGFR-1, 2, and 3 has been show to be upregulated on vascular and lymphatic endothelial cells during

tumor angiogenesis in particular [29-32]. Thus anti-angiogenic treatments specifically targeting VEGF or VEGFRs present a diverse pathway towards tumor control and treatment [33]. The VEGF family of proteins is widely studied for its critical role in neovascularization during wound healing, tumor growth, and embryological development. While current clinical trials insinuate a positive outlook for VEGF antagonists, continued understanding of the biological role of VEGFs in angiogenesis will remain essential for shedding light on promising advancements. Here, we review the published patents and patent applications, and relevant literature reports up to May 2006 which concern the use of VEGF antagonists as a potential form of anti-cancer therapy. 2. VEGF AND VEGFR ANTAGONISTS Certain VEGF receptors and ligands have recently been the target of anti-angiogenic therapies for cancer. Pre-clinical results of VEGF/VEGFR related antagonists show strong efficacy in reducing tumor size, blood vessel density, and metastatic potential. Companies have developed a wide range of strategies for VEGF-mediated tumor growth inhibition including neutralizing monoclonal antibodies [34], a retrovirus-delivered dominant negative Flk-1 mutant [35], small molecule inhibitors of VEGFR-2 signaling [36-39], antisense oligonucleotides [40,41], anti-VEGFR-2 antibodies [42], and soluble VEGF receptors [43-46]. Although initial experiments predicted a cytostatic effect, VEGF therapies have also shown cytotoxic anti-vascular effects, possibly extending their use to late stage carcinomas [47,48]. While many of these compounds may result in similar effector function, optimized cytochrome P450 (CYP) enzyme profiles, solubility, selectivity, and toxicity are areas targeted for refinement. 2.1. Monoclonal Antibodies and Antibody Fragments Against VEGF and VEGFR Monoclonal antibodies (mAb) are pure antibodies designed to bind to a specific antigen target. The initial development of mAbs by Milstein and Khler in 1975 [49] has allowed for the large-scale production of mAbs for use as anti-cancer therapeutics. Recently, mAbs developed to target various isoforms of VEGF have shown both preclinical and clinical efficacy. Bevacizumab (Avastin) is a humanized monoclonal antibody developed by Genentech Inc. Bevacizumab binds to all VEGF isoforms as well as all bioactive proteolytic fragments and thus attempts to block the biological activity of this growth factor by inhibiting the interaction of VEGF with its corresponding receptor [50]. Bevacizumab was humanized from a previously developed mouse anti-VEGF antibody (muMAb), with retention of high affinity binding (Kd = 1.8 nM) [51]. In January 1997, Genentech filed an Investigational New Drug Application (IND) for bevacizumab, and phase I clinical trials were initiated in April 1997. These phase I studies showed that bevacizumab as a single agent was relatively non-toxic and that adding bevacizumab to standard chemotherapy regimens did not significantly exacerbate chemotherapy associated toxicities [52,53]. In a Phase III trial involving 813 patients with metastatic colorectal cancer, those patients who received

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bevacizumab with irinotecan, 5-fluorouracil (5-FU), and leucovorin (IFL) showed an increase in progression free survival of 10.6 months vs. those given placebo plus IFL of 6.2 months. Median duration of survival was also increased from 15.6 to 20.3 months corresponding to a reduction of 34% in the risk of death associated with bevacizumab [54]. Currently, phase II trials are being conducted for other neoplasms including pancreatic adenocarcinoma, advanced renal cell carcinoma, and metastatic colorectral carcinoma. Protein Design Labs, Inc./Toagosei Co. Ltd developed HuMV833, a humanized anti-VEGF monoclonal IgG4 antibody that similarly binds VEGF121 and VEGF165 (Kd = 0.1 nM) [55,56]. In a phase I trial of HuMV833, pharmacokinetic, pharmacodynamic and toxicity data revealed doses of 1 and 3 mg/kg as having possible clinical efficacy [57]. Preliminary positron emission tomography (PET) imaging results have shown that different tumor deposits within the same patient may display distinct pharmacokinetic profiles of HuMV833 [58]. Recent phase I trials isolated two distinct doses which display promising clinical activity, 1 and 3mg/kg. Although the maximum tolerated dose (MTD) of HuMV833 was not defined, the long term application of HuMV833 was demonstrated as patients who received 59 cycles of therapy maintained an excellent quality of life [57]. ImClone Systems developed IMC-IC11, a mouse/human chimeric IgG1 derived from a single chain Fv isolated from a phage display library [59-61]. Preclinical testing showed that antibody concentration required to inhibit 50% of VEGFinduced mitogenesis of human umbilical vein endothelial cells (HUVECs) is 0.8 nM [62]. ImClone continued with Phase I clinical trials in May 2002 in patients with metastatic colorectal carcinoma. Results were negative for grade-3 or -4 IMC-1C11-related toxicities and a 5 g/mL dose of IMC1C11 prevented KDR phosphorylation in vitro. At a dose of 4 mg/kg, a half-life of 67 h was obtained. Anti-tumor effects were noted in 11 patients; specifically, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) was used to assess drug-induced vascular regression. After 4 weeks of therapy, patients showed a marked reduction in tumor enhancement factor (EF) and tumor influx rate constant Kin (min-1), both of which are proportional to the perfusion capacity of the tumoral vascular network [63]. In addition to IMC-1C11, ImClone recently screened a large nave human antibody phage display library and produced several fully human anti-VEGFR-2 Fab fragments. Affinity maturation of one of these Fab clones led to the development of 1121B Fab [64]. The affinity of 1121B Fab for VEGFR-2 was evaluated using ELISA on immobilized receptor and BIAcore analysis. The binding affinity of 1121B Fab to VEGFR-2, as determined by BIAcore analysis, was shown to be approximately 8-9 fold higher (0.1nM) than that of VEGF natural ligand for VEGFR-2 (0.88nM). It was also demonstrated that 1121B Fab binds to VEGFR-2 in a dose-dependent manner (ED50 = 0.15 nM). Further preclinical testing showed that cell proliferation induced by VEGF stimulation was significantly inhibited by 1121B Fab (IC50 = 20 nM) [65]. Imclone Systems initiated phase I clinical trials of IMC-1121B in January 2005 [66].

2.2. Soluble VEGF Receptors Soluble VEGF receptors are designed to irreversibly bind suspended VEGF ligand in hopes of preventing receptor (VEGFR-1, 2, and 3) activation. Currently being developed collaboratively by Regeneron Pharmaceuticals Inc. and Sanofi-Aventis, VEGF-Trap is a high affinity soluble VEGF receptor created by fusing the extracellular domains of VEGFR-1 and VEGFR-2 to the Fc portion of human IgG1 [67,68]. in vitro Studies have shown the affinity of VEGFTrap for VEGF is significantly higher than that of monoclonal antibody bevacizumab (1-5 pM) [46,51]. This VEGF inhibitor has shown both anti-angiogenic [48] and anti-tumor [46] activity as well as efficacy against xenograft models of Wilms tumor [69], ovarian [70] and pancreatic cancer [71]. It has been hypothesized that high doses of VEGF-Trap efficiently inhibits VEGF signaling by blocking static low levels of VEGF required to support the long-term integrity of co-opted vasculature in addition to the VEGF expression required for neovascularization [72]. Cytotoxic studies revealed that VEGF-Trap is capable of inducing regression of co-opted vascular development thus expanding its treatment capability to larger tumors [72]. 2.3. Small Molecule VEGF Receptor Inhibitors To date, the number of VEGFR-2 inhibitors undergoing advanced preclinical and clinical evaluation is steadily rising. Investigators now take advantage of combinatorial chemistry and high throughput screening to optimize the solubility, bioavailability, binding efficiency, production cost, and therapeutic efficacy of various small molecule RTK inhibitors. Recent patenting activity has been grounded in claims of basic structural features with countless numbers of substitutions and variations, opening the door to a wide range of potent inhibitors capable of pharmacokinetic refinement. VEGF receptor tyrosine kinases (RTKs) have typically been classified into families based on structural features of their respective extracellular domains. While the extracellular portion of each receptor expresses unique ligand binding, the intracellular portion of RTKs are generally architecturally very similar. Furthermore, favorable pharmacokinetic profiles of many of these compounds aids in the ability to deliver these inhibitors orally, making them most attractive for further clinical development. 2.3.1. Anilinoquinazolines AstraZeneca redesigned the scaffold of a previously developed EGFR inhibitor by rearranging the halogen substitution pattern around the aniline ring to reveal a compound that displays strong VEGFR-2 inhibitory efficacy. Replacing the R7 substituent with alkyl linked ((CH2)1-4) neutral and basic heterocycles (e.g. morpholine, thiophene, pyridine, imidazole, and triazole) led to the identification of several VEGFR-2 inhibitors (IC50s range from 1 - 40 nM), particularly ZD4190 (1) [73,74]. Murine testing revealed that ZD4190 maintained the highest plasma levels following oral dosing. Characteristics of this compound in particular are neutral C7 heteroaromatic side chains [74]. However, further testing revealed low aqueous solubility and variable

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pharmacokinetic (PK) properties that made ZD4190 unfavorable for clinical evaluation. AstraZeneca refined ZD4190 by manipulating the C7 side chain. One resulting compound, ZD6474 (N-(4-bromo-2-fluorophenyl)-6methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4amine), was formed by incorporating a basic nitrogen in the C7 side chain (2) [75]. Hennequin et al. showed that anilinoquinazolines modified with basic C-7 side chains displayed increased aqueous solubility over anilinoquinazolines with neutral C-7 side chains (30 - 80 fold) and had PK characteristics that were more clinically favorable [76]. Preclinical testing has shown ZD6474 to possess efficacy in anti-tumor, anti-metastatic and anti-angiogenesis applica-tions [77,78]. Treatment of murine renal cell carcinoma with ZD6474 resulted in a 5.4-fold decrease in vascular volume compared with untreated tumors [79]. A multicenter phase II trial of ZD6474 showed that all patients receiving 300 mg and 90% of patients receiving 100 mg achieved steady-state concentrations exceeding the IC50 for VEGF inhibition in preclinical models. The study concluded that ZD6474 monotherapy had limited therapeutic activity in patients with refractory metastatic breast cancer [80]. Phase III trials are currently ongoing [81]. AZD2171, 4-[(4-fluoro-2-methyl-1H-indol-5-yl)oxy]-6methoxy-7-[3- (pyrrolidin-1-yl)propoxy]quinazoline (3), is another VEGFR-2 inhibitor developed by AstraZeneca Pharma [WO0047212]. AZD2171 displayed activity against VEGF-stimulated KDR autophosphorylation in HUVEC proliferation assays with IC50 = 40 + 20 pM. Murine tumor models showed that AZD2171 (1.5 and 6 mg/kg/day) abolished VEGF-dependent blood vessel formation. AZD2171 is advantageous in that it elicits anti-angiogenic responses at significantly lower doses than those required by other VEGFR RTK inhibitors [82]. 2.3.2. Oxindoles Oxindoles (indolin-2-ones) were first discovered in 1993 by Buzzetti et al. [83] and later developed by Sugen (Pharmacia, Pfizer) [84]. Co-crystal X-ray structures of the catalytic domain suggests that oxindoles bind in the ATP pocket with the indolin-2-one core participating in key Hbond donor/acceptor capacities with the carbonyl of Glu915 and the NH of Cys917 [75]. This was further supported by a drop in VEGFR-2 inhibitory activity seen after N-methylation of the oxyindole. Sugen (Pharmacia, Pfizer) developed a number of compounds designed to take advantage of this ATP binging binding pocket and further refined their pharmacokinetic profiles for possible clinical development. Allergan Inc., recently began patenting various 3-(arylamino) methylene-1, 3-dihydro-2H-indol-2-ones as kinase inhibitors as well [85]. Developed by Sugen (Pharmacia, Pfizer) is SU5416 (semaxanib) (4), an oxindole VEGFR-1, 2, 3 inhibitor (IC50 = 43 + 11 nM, 220 + 34 nM, 50 nM, respectively) [86,87]. Anti-tumor activity has been demonstrated in both humans and rodents [88-91], however the limited solubility of SU5416 required a cremophor formulation to allow for intravenous administration [92]. In February of 2002, Pharmacia announced that they would end clinical development of SU5416 because of pharmacokinetic-related problems

[93]. Sugen (Pharmacia, Pfizer) continued in their development of more soluble versions of this compound. Sugen (Pharmacia, Pfizer) then developed SU6668 (5) after determining that appending carboxylic acid residues onto the pyrrole ring of the scaffold would allow for additional binding interaction in the sugar-binding region of the ATP pocket [94]. The resulting propionic acid analog of SU5416 displayed an increased PDGFR- inhibition profile (IC50 = 39 + 1 nM, vs. 2220 + 1500 nM) and an oral bioavailability that coincided with a longer half-life [90,95, 96]. Further preclinical testing using biochemical assays revealed potent VEGFR-2 inhibition (IC50 = 2.1M) and in vivo xenograft experiments showed that SU6668 is effective against large established epidermoid (A431), colon (Colo205 and HT-29), prostate (PC-3), lung (H460), and glioma (SF767T and C6) tumors [97]. A Phase I clinical trial of SU6668 resulted in a maximum tolerated dose of 100 mg/m2 when given orally, thrice daily under fed conditions. Because of the low plasma levels reached at this dose level, the efficacy of SU6668 is still undetermined and further clinical development has not been encouraged [98]. Another variant of SU5416, SU11248 (SUTENT) (6), was developed by appending a carboxy-diethylaminoethylamido group onto the pyrrole ring and attaching fluoride at C5 [99,100]. in vitro Testing of SU11248 showed competitive inhibition against Flk-1 and PDGF-dependent PDGFR- phosphorylation with IC50 = 10 nM for both RTKs. It was also shown that SU11248 inhibited VEGFinduced proliferation of HUVECs (IC50 = 40 nM) [99]. Preclinical tumor models also showed efficacy in tumor regression and apoptotic activity in response to SU11248 administration. Phase I clinical trials have shown a daily dose of 50 mg produces inhibitory activity with plasma Cmax = 120 ng/mL [101]. This led to the confirmation of 42 mg/m2 (i.e. 50 mg daily oral dose) as the MTD and 40 hours as the half-life of SU11248 [102]. 2.3.3. Phthalazines PTK787 (1-[4-chloroanilino]-4-[pyridylmethyl]-phthalazine succinate), also known as vatalanib, CGP-79787D, ZK222584 or PTK/ZK (7), was first reported by Novartis and Schering AG in 1998 and is currently recognized as one of the most promising VEGFR inhibitors currently in clinical development [103]. Biochemical assays reveal PTK787 inhibits both VEGFR-1 and VEGFR-2 (IC50 < 100 nM). Studies conducted on HUVECs show that PTK787 displays potent activity in a VEGF-driven cellular autophosphorylation assay (IC 50 = 17 nM). The Phase I clinical trial of this compound introduced the use of DCE-MR imaging for the validation of its end effect. This functional component used to quantify the therapeutic efficacy of the compound is attributed to its success in establishing its proof-of-principle [104,105]. PTK787 has been tested on a variety of human carcinoma cell lines and is currently being investigated for its potential when combined with irradiation. Patents surrounding PTK787 have developed in two major areas, combinatorial therapies and unique methods of delivery. Novartis, Inex Pharmaceuticals, Beth Israel Deaconess Medical Center, Schering AG, Eisai, and Pharmacia have each submitted patents proposing the use of

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Cl

Cl

HN MeO N N N O N N 1 ZD4190 N O M eO N N 4 SU5416 H N O O H N F H N F O N F M eO

HN N N F

2 ZD6474 H N O H N

O 3 AZD2171

H N

O HN CO2H 5 SU6668 6 SU11248 NEt2

Fig. (1). Anilinoquinazolines & Oxindoles.

PTK787 in combination with various other therapeutics including tie-2 inhibitors [106], EGFR inhibitors [107], and histone deacetylase inhibitors [108]. Other patents relate PTK787 to a specific method of treatment. Novartis, Dana Farber, and Academisch Zeikenhuis Groningen maintain patents that propose the use of PTK787 in the treatment of myeloma, ocular neovascular disease [109], angiogenic myeloid metaplasia [110], and mesothelioma [111]. 2.3.4. Anthranilamides Novartis researchers used conformational analysis, computational modeling, and database searching, to transform PTK787 into a new class of anthranilamide VEGFR inhibitors [112]. Novartis first identified anthranilamides as inhibitors of VEGFR and further developed AAL993 (8) [113] and an unnamed compound (9) [114]. While these two compounds display similar potencies against VEGFR-1 and VEGFR-2 compared with PTK787, the key interactions of AAL993 with VEGFR-2 are two hydrogen bonds of the amid NH and carbonyl groups with a glutamate residue of the C helix, and the backbone of the aspartate of the AspPhe-Gly (DFG) motif [104]. AAL993 has been shown to inhibit VEGF-induced proliferation of HUVECs (IC 50 = 0.84 nM).

Novartis continued to refine their new class of VEGFR inhibitors and disclosed several new derivatives. They reported the use of alkyl and cycloalkyl anthranylic amides [115], pyridine and pyrimidines as the central core [116118], limited substitution on the aryl amide moiety with either a pyridyl group or a benzyl-substituted amide as the hinge-binding element. This led to the development of their second generation VEGFR inhibitors ABP309 (10) which combines a pyridone as the hinge-binding element with a pyridine core and several other pyridine derivatives [119]. ABP309 displays a 10-fold increase in aqueous solubility at pH 4, an improved CYP inhibition profile as compared to PTK787, and is reported to have a selective kinase profile specific to only VEGFR-2 [104]. Novartis most recent patent activity involves the disclosure of a line of N-aryl (thio) anthranilic acid amide derivatives [120]. Similarly, Schering AG maintains several patents that implicated anthranilic acid amide derivatives. In 2001, Schering AGs patents included pyridylethyl analogues, pyridylethylene and pyridylethyne derivatives with aryl or hetercyclic amide substitutions. Scherings discovery that cyclic ethers or cyclic amines can be used as the hingebinding element expanded their VEGFR-2 inhibitor profile [121,122]. In a second set of patents, Schering AG addressed

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the growing concern of CYP liability associated with initial anthranilamide compounds. Changes included N-oxide derivatives [123] (11), N-benzyl-anthranilic acid (hetero) arylamide derivatives [124], pyridyl substitutions (12), and pyridones with anthranilic acid amides [125] (13). Amgen began disclosing anthranylic acid amide derivatives in 2002. Their derivatives include heteroaryl core replacements with broad heterocyclic substitutions at the amide moiety [126], nicotinamide derivatives [127], and the use of heterocylics as the hinge-binding element [128]. Amgen also patented the use of substituted five- or sixmembered heterocycles as the hinge-binding moiety and submitted several other patents further defining the central core of each compound. The disclosure of anthranilamide pyridinureas by another group has led to development of a new set of VEGFR inhibitors. However, due to its recent discovery, preclinical data describing the compounds efficacy has not been thoroughly completed [129].

2.3.5. Isothiazoles Pfizer has also developed a compound, CP-547632 (14), that has preliminarily shown efficacy in VEGFR-2 inhibition. CP-547632 is an ATP-competitive inhibitor that blocks VEGFR-2 kinase autophosphorylation (IC50 = 11 nM) and VEGF-induced VEGFR-2 phosphorylation (IC50 = 6 nM) [130-132]. It features a pendant pyrrole attached via a urea linkage that is hypothesized to be responsible for an increase in aqueous solubility [133]. Preclinical experiments show that CP-547632 inhibits tumor-associated VEGFR-2 phosphorylation resulting in decreased vascular density and tumor growth [130]. Phase I clinical studies revealed efficacious dose of 160 mg/kg/day and a resulting half life of 29 hours [134]. 2.3.6. Pyrroloindolocarbazoles Cephalon has described a series of compounds which replace one indole nitrogen with a carbon. Further optimi-

Cl

CF 3 O HN HN OH O NH O 8 AAL993 NH

HN N N HO

N 7 PTK787 N HN O NH NH CF3 HN O NH HN O O N H O
-

N N 9 N

OH

N N H 10 ABP309 O CF3 O 11

N 12

N HN O NH

N H 13

Fig. (2). Phthalazines & Anthranilamides.

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zation led to the development of indenocarbazole KDR inhibitors. Specifically, CEP-5214 (15) was formed by replacing the bridging heterocycle with a flexible propyl chain appended off the indole nitrogen [75,135]. Indenopyrrolocarbazole CEP-5214 is an efficient inhibitor of VEGFR-2 (IC50 = 8 nM) and demonstrates significant in vivo anti-tumor activity in murine tumor models [136,137]. Studies conducted on HUVECs showed strong efficacy in blocking VEGFR-2 autophosphorylation (IC50 = 10 nM). However, because suboptimal plasma levels were obtained with oral dosing, the HCl salt of the N,N-dimethylglycine ester of CEP-5214 (CEP-7055) (16) was tested. Preclinical testing showed chronic administration resulted in 50-90% maximum inhibition in the growth of a several different human subcutaneous (s.c.) tumor xenografts in nude mice, including A375 melanomas, U251MG and U87MG glioblastomas, CALU-6 lung carcinoma, ASPC-1 pancreatic carcinoma, HT-29 and HCT-116 colon carcinomas, MCF-7 breast carcinomas, and SVR angiosarcomas [138]. Further testing also revealed a more desirable aqueous solubility (40 mg/mL) [139]. CEP-7055 is currently in phase I clinical trials undergoing MTD and toxicity studies. Several of Cephalons candidate compounds are described over a series of patents pertaining to fused pyrrolocarbazole and isoindolone derivatives [140,141]. Yamanouchi Pharmaceutical Co. Ltd (YP) recently developed another VEGFR-2 inhibitor for preclinical development, YM-359445 (17) [142]. YP identified (3Z)-3[6-[(4-methylpiperazin-1-yl)methyl]quinolin-2(1H)-ylidene]2-oxoindoline-6-carbaldehyde O-(1,3-thiazol-4-ylmethyl) oxime mono-L-tartrate, YM-359445, while screening for a more potent anti-tumor VEGFR-2 inhibitor. Preclinical testing in an enzyme assay for VEGFR-2 revealed strong VEGFR-2 inhibition (IC50 = 8.5 nM). Against HUVEC proliferation induced by VEGF, YM-359445 displayed potent anti-proliferation activity (IC50 = 1.5 nM). Pharmacokinetic analysis revealed a single dose at 1 mg/kg in mice resulted in a bioavailability of 23%, and maximum plasma concentration of 16 nM. A recent study conducted by YP showed YM-359445 to be more potent than other VEGFR-2 tyrosine kinase inhibitors, namely SU11248, ZD6474, and AZD2171 [143]. 2.3.7. 2-Amino-(thiazol-2-yl) pyridines Merck has developed a variety of distinct KDR inhibitors, which may be grouped into six major classes: pyrimidines [144,145], pyrazolo[1,5-a]pyrimidines [146-149], indazoles [150], 1-H-quinolin-2-ones [151-154], acyl-2-aminothiazoles [155, 156], and 2-amino-(thiazol-2-yl) pyridines [157,158]. In 2003 Merck further developed their 2-amino(5-cyanothiazol-2-yl)pyridine class of molecules (18). By appending the basic tertiary amine group off the C-4-position of the pyridyl nucleus, the resulting compound showed an improved pharmacokinetic profile over the previously patented compounds and IC50 values range from 0.1 - 5 M [159] (19). It also appears that the 2-aminopyridyl moiety is critical to the KDR inhibition. Merck subsequently developed a series of KDR inhibitors based on 2-subsituted indole derivatives. Specifically, in 2004 Merck disclosed 3[5-(4-methanesulfonyl-piperazin-1-ylmethyl)-1H-indol-2yl]1H-quinolin-2-one (20) [160]. The key distinguished feature

of this patent is their use of salt variants that enhanced pharmacokinetic properties of previous compounds. Schering AG recently disclosed their version of pyrazolopyrimidines that have yet to be evaluated in preclinical models [161]. 2.4. RNA Based Strategies Progress in the field of RNA therapeutics has led to the widespread attention of RNA-based anti-cancer treatments [162]. Specifically, RNA interference has evolved over the last decade to include small interfering RNA (siRNA) and ribozymes [163]. These therapeutics, although in early stages of clinical validation, have shown promising results as antiangiogenic strategies. 2.4.1. siRNA SiRNA, sometimes known as short interfering RNA, are a class of 20-25 nucleotide-long RNA molecules that play a variety of roles [164-166]. For purposes of cancer treatment, the function of siRNA in the RNA interference pathway is exploited. Specifically, mammalian cells mount a nonspecific inhibitory response to dsRNA that results in the translational inhibition and degradation of the targeted mRNA. Cancer treatments take advantage of this mechanism by inducing dsRNA of a targeted protein, marking it for destruction. The inhibition of corneal angiogenesis [167] and choroidal neovascularization (CNV) by local delivery of siRNA targeting VEGF has been demonstrated in xenograft models [168], displaying both its effector tumor reduction function and anti-angiogenic potential. Sirna Therapeutics Inc. maintains a large patent portfolio that describes the use of siRNA techniques to limit VEGF and VEGFR expression [169-172]. Preclinical studies have shown that siRNA targeting VEGFR-1 successfully reduced ocular neovascularization by up to 66% [173]. In an RKO colon cancer model, cells treated with siRNA targeting VEGF showed a 94% knockdown in VEGF expression and a 67% decrease in cellular proliferation [174]. While significant preclinical work has accomplished marked results in anti-cancer therapy, continued clinical research will provide insight to its functional efficacy in humans. 2.4.2. Ribozyme While ribozymes have been in existence for more than a decade, their use in VEGF signaling inhibition has recently led to the development of new anti-cancer therapeutics. Ribozymes function by cleaving RNA phosphodiester bonds at specific sites and in doing so destroy the ability of targeted mRNA to direct synthesis of an encoded protein. While single ribozyme molecules can degrade multiple mRNA strands, they are limited by their susceptibility to nuclease degradation that results in poor serum stability. Preclinical studies of an anti-VEGF hairpin ribozyme compound has shown efficacy in significantly inhibiting the growth and proliferation of ovarian cancer SKOV3 cells [175, 176]. Chiron Corporation/Ribozyme Pharmaceuticals Inc. (RPI) holds the rights to more than 100 worldwide patents encompassing ribozyme design, synthesis, chemical modification, delivery, and production [177-181]. Their signature anti-angiogenic product is an anti-Flt-1 ribozyme known as Angiozyme [182]. Preclinical testing revealed strong

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Br

H2N

14 CP-547632 H N O S N N O O N N H O O N N H O 15 CEP-5214

OH

N N

HO2C HO

OH CO2H

16 CEP-7055 N N HN N N R1 R2 N : R3 S HN S N

17

N N R4

HN O N H

N R4

N R2 18 O S O N N O H N NH N R3 19

R5

R1 = alkyl, O-alkyl, halogen, OH R4 = SO2-alkyl, CONH-alkyl R5 = NHCONH-alkyl

20

Fig. (3). Isothiazoles, Pyrroloindolocarbazoles & 2-Amino-(thiazol-2-yl) pyridines.

efficacy in the prevention of tumor growth and metastasis. In a study conducted by Pavco et al, ribozymes targeting either VEGFR-1 or VEGFR-2 significantly inhibited primary tumor growth in a highly metastatic variant of Lewis lung carcinoma and significantly inhibited liver metastasis in a xenograft colorectal cancer model [176]. Chiron/RPI continued with phase I clinical studies to show that Angiozyme was well tolerated with satisfactory pharmacokinetic variables for daily s.c. dosing [183]. Further combinatorial studies have revealed that RPI.4610 (Angiozyme), carboplatin, and paclitaxel can be administered safely in combination without substantial pharmacokinetic interac-tions [184].

3. CURRENT & FUTURE DEVELOPMENTS Anti-cancer therapeutics have been developed over the last decade to include novel strategies based on targeting tumor angiogenesis. The identification of VEGF-related signaling cascades has led to the expansion of possible antiangiogenic compounds, each with a characteristic method of action, binding pattern, bioavailability, toxicity, and clinical efficacy. Preclinical data has revealed strong anti-tumor, anti-metastatic, and anti-angiogenic effects [185]. However, clinical translation of these compounds has been the most challenging. Hurdles faced by poor bioavailability and solubility in human trials have halted the development of several inhibitors that are potent in vitro. Each type of inhibition described in this review offers specific pros and

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cons. Soluble receptors, monoclonal antibodies and antibody fragments offer highly specific VEGF and VEGFR binding, reducing possible toxicity. Whereas small molecule KDR inhibitors offer a much wider range of inhibition, targeting RTKs that do not necessarily involved VEGF [186]. The future of VEGFR inhibitors as therapeutic anti-cancer agents will become clearer as both preclinical and clinical trials further describe angiogenesis signaling and antagonist efficacy. There are several challenges faced by the introduction of anti-angiogenic therapy. Specifically the clinical application of anti-angiogenic compounds is troubled by the temporal dependence of angio-suppressive treatments [187]. The angiogenic switch is a phrase used to describe the level of tumoral angiogenic activity. The pathology of tumor genesis indicates a marked transition from a prevascular to vascular phase. It is hypothesized that VEGF-inhibitive therapy will be most effective against small tumors and should be administrated prior to the development of a well-established vascular network. Phase III clinical trials of Capecitabine + Bevacizumab for women with metastatic breast cancer failed to elicit a positive therapeutic response and may attest to this conclusion [188]. Thus, the use of VEGF antagonists may be better suited for chronic therapy, preventing the recurrence of disease and inhibiting the genesis of new vessels. Furthermore, the development of reliable markers that are able to aid in selecting patients who are more likely to benefit from anti-VEGF therapy will be critical to identifying the treatment regimen of such therapeutics. The recent application of molecular imaging techniques to visualize the expression of receptors and ligands implicated in cancer angiogenesis will aid in such patient selection [189-192]. Molecular imaging probes coupled to therapeutic toxins enable the visualization of therapeutic efficacy and provide a forum for the quantitative assessment of patient response. The future of cancer-related treatment lies in a combinatorial approach that aims to target tumor cells and the corresponding vascular support system. It is unlikely that anti-anngiogenic therapies alone will, without increased toxic risk, sufficiently halt tumor growth within a reasonable time period. To this regard, numerous on-going clinical trials have shown efficacy in combining chemotherapy and antiangiogenic therapy [193,194]. The basis for this specific synergy assumes that cytotoxic agents will reduce the tumor burden of vascularized tumors, while anti-angiogenic agents will prevent neovascularization and growth of small and occult metastatic foci as well as the formation of new metastatic lesions [5]. Early phase clinical trials have suggested that this combinatorial therapy is generally well tolerated. However, instances of thromboembolis and hemorrhage have been reported [98]. It will be critical to develop appropriate treatment regimens that combine the use of anti-angiogenic, chemo-, and radiation-therapy to take full advantage of maximal angiogenic signaling and VEGF blockade. Therapeutics designed to manipulate the VEGF pathway will extend much beyond the treatment of cancer. Conditions currently in preclinical and clinical investigation include asthma, bone lesion, diabetic nephropathy, arthritis, and

psoriasis [195,196]. Currently, several clinical trials are being conducted to explore this possibility, Phase III trials are in progress for patients with age-related macular degeneration. Furthermore, recent studies have determined that VEGFR-1, previously un-implicated in post-fetal angiogenesis, may have a fundamental role in the recruitment of endothelial progenitor cells and hematopoiesis [197,198]. As the role of each VEGF receptor is more clearly elucidated, therapies designed to inhibit distinct VEGFRs will be further refined and demonstrate more targeted effector function. Preclinical experiments have revealed strong treatment efficacy with a variety of VEGF-antagonists. While it has been clearly demonstrated that VEGF related signaling is largely responsible for endothelial cell activity, several other recently discovered signaling systems have also shown similar capabilities. One of these surface proteins, integrinv3, has been shown to be up-regulated on cytokineactivated vascular endothelial cells, smooth muscle cells, and blood vessels in tumor, wound, and granulation tissue [199203]. MEDI-522, a mAb against human v3 developed by MedImmune Inc, has shown promising preclinical evidence and as a result entered phase III clinical trials. Merck GmBH developed a cyclic penta-peptide c(RGDf[NMe]V) targeted to v3 called EMD-121974 or Cilengitide. Phase II clinical trials have revealed positive results for suppressing tumor vascularization and growth [204,205]. Several other candidates are also in various phases of development; these include antagonists to hypoxia-inducible factor-1alpha (HIF1), angiopoietin-1 (ANG-1), and PDGF-. Despite many of the obstacles discussed above, modulating the VEGF signaling cascade presents great opportunity to further understand the process of blood vessel formation. Though validated as a monotherapeutic, the future of comprehensive anti-cancer therapy will require a multifaceted approach, targeting different aspects of tumor development. The exact role of the VEGF-inhibitors will require further clinical investigation, proper patient selection, and acceptable toxicology; many of these tasks have yet to be completed by a potent inhibitor. REFERENCES
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