vegg as angiogenic recepts

8/3/2019 VEGG as Angiogenic Recepts

http://slidepdf.com/reader/full/vegg-as-angiogenic-recepts 1/13

Recent Patents on Anti-Cancer Drug Discovery, 2007, 2, 59-71 59

1574-8928/07 $100.00+.00 © 2007 Bentham Science Publishers Ltd.

Vascular Endothelial Growth Factor and Vascular Endothelial GrowthFactor Receptor Inhibitors as Anti-Angiogenic Agents in Cancer Therapy

Anand Veeravagu1, Andrew R. Hsu1, Weibo Cai2, Lewis C. Hou1, Victor C.K. Tse1 and XiaoyuanChen2,*

1 Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA 94305-5484, USA;

2 Molecular

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 beenshown to be key molecules implicated in embryonic deve-lopment, angiogenesis, vascular permeability, tumor progres-sion and cardiovascular disease [1]. VEGF is a homodimeric,basic, 45 kDa glycoprotein specific for vascular endothelialcells [1,2]. VEGF was first described as vascular perme-

ability factor (VPF) by Dvorak and colleagues after it wasdiscovered to increase the permeability of tumor bloodvessels [3]. Currently, the VEGF family consists of sevenmembers - VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F and placental growth factor (PlGF). Eachisoform is distinct in its composition of 121, 145, 165, 183,189 and 206 amino acids by monomer (respectivelyVEGF121, VEGF145, VEGF165, VEGF183, VEGF189, VEGF206)and VEGF165 is the predominant protein among the majorsplice variants [4]. Each isomoer is the result of alternativesplicing of messenger RNA (mRNA) from a common genecomposed of eight-cysteine residues [5].

1.2. VEGF Receptors

These VEGF isoforms have different physical andbiological properties and act through three specific tyrosinekinase receptors - Fms-like tyrosine kinase Flt-1 (VEGFR-

*Address correspondence to this author at the Molecular Imaging Programat Stanford (MIPS), Department of Radiology & Bio-X Program, StanfordUniversity School of Medicine, 1201 Welch Road, Room P095, Stanford,CA 94305-5484, USA; Tel: (650) 725-0950; Fax: (650) 736-7925; E-mail:[email protected]

1/Flt-1), the kinase domain region, also referred to as fetalliver kinase (VEGFR-2/KDR/Flk-1), and Flt-4 (VEGFR-3).Each receptor has seven immunoglobulin-like domains in theextracellular domain, a single trans-membrane region, and aconsensus tyrosine kinase sequence interrupted by a kinaseinsert domain [6]. The complete function of each receptorhas not been fully determined, however certain VEGFRshave been targeted by cancer therapeutics due to their knownroles in angiogenesis. While the distinct implication of

VEGFR-1 has yet to be determined since its discovery over adecade ago [7], VEGFR-2 and more recently VEGFR-3 havebeen labeled as the receptors responsible for the angiogenicconsequence of VEGF signaling.

The role of VEGFR-1 in blood vessel development andvascular permeability remains unclear. VEGFR-1 has beenshown to be weaker in kinase activity and is thus incapableof provoking endothelial cell proliferation when stimulatedwith VEGF [8]. However, recent studies suggest a temporalcomponent to VEGFR-1 effector function. VEGFR-1 hasbeen shown to modulate endothelial cell proliferation duringearly stages of vascular development preceding theformation 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 havecontinued 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 heightenedvessel permeability as caused by VEGF signaling isVEGFR-2. The key role of this receptor in developmental



60 Recent Patents on Anti-Cancer Drug Discovery, 2007 , Vol. 2, No. 1 Chen et al.

vasculogenesis and blood island formation is evidenced bythe failure of VEGFR-2 knockout mice to develop organizedblood vessels and typical vasculature resulting in death in-utero [9]. While VEGF binds to VEGFR-2 with loweraffinity when compared to VEGFR-1 (K d = 75-250 pM vs.25 pM) [8,10-12], the mediation of mitogenesis andangiogenesis through Flk-1 has been clearly established inboth in vitro and in vivo models [13,14]. Upon bindingVEGF, VEGFR-2 undergoes dimerization and ligand-

dependent tyrosine phosphorylation. The major phosphory-lation site, Y1175, is known to be responsible for endothelialcell proliferation via Raf-Mek-Erk pathway [15,16] andendothelial survival via PI3 kinase/Akt pathway [17].Specific activation of VEGFR-2 with VEGF-E has againdemonstrated potent endothelial cell activity in vitro and invivo, supporting the notion that activation of VEGFR-2 alonecan efficiently stimulate angiogenesis [18]. Further studiesdemonstrate that VEGFR-2 is exclusively expressed onendothelial cells, making it a prime target for anti-angiogenictherapeutics [10,12].

The third VEGF receptor, VEGFR-3, displays slightlydifferent signaling characteristics. Contrary to the previouslydescribed mechanisms, VEGFR-3 undergoes proteolytic

cleavage in the extracellular domain into two disulfide-linked peptides. While this receptor is capable of stimulatingcell migration, differentiation, and mitogenesis, VEGFR-3 ispredominantly localized to the surface of lymphaticendothelial cells [19, 20]. Thus, VEGF-C, VEGF-D, andtheir receptor, VEGFR-3, present a strong molecularsignaling system for tumor lymphangiogenesis and anotherpossible avenue for anti-angiogenic and anti-metastatictherapeutics [21].

1.3. VEGF and Cancer

Tumor development and growth greatly depends onaccess to oxygen, nutrients, growth factors, hormones, andhemostatic factors carried by blood vessels [22]. To this

regard, it has been shown that a primary tumor’s ability torecruit and create a network of blood vessels will oftendetermine and contribute to its growth and clinical severity[23-25]. The pathology of tumorigenesis indicates a markedtransition from a prevascular to vascular phase. In theprevascular state, the tumor does not induce angiogenesis, islimited in size, and rarely metastasizes. However, thevascularized tumor induces host microvessels to undergoangiogenesis, has the potential to rapidly expand its cellpopulation, and has a propensity to metastasize [26]. Eventsincluded in this process are the proliferation, migration, andinvasion of endothelial cells, organization of endothelialcells into functional tubular structures, maturation of vessels,and vessel regression [27]. To date, the most influentialmolecular signaling pathway involved in such angiogenicactivity is VEGF. It is one of the most potent inducers of vascular permeability known-50,000-fold more potent thanhistamine [28]. Being responsible for the hyper-permeabilityof tumor vessels, VEGF has been shown to allow for theleakage of several plasma proteins, including fibrinogen andother clotting proteins to transform the stroma of normaltissues into a pro-angiogenic environment [25,27,28]. Theexpression of VEGFR-1, 2, and 3 has been show to be up-regulated on vascular and lymphatic endothelial cells during

tumor angiogenesis in particular [29-32]. Thus anti-angio-genic treatments specifically targeting VEGF or VEGFRspresent a diverse pathway towards tumor control andtreatment [33].

The VEGF family of proteins is widely studied for itscritical role in neovascularization during wound healing,tumor growth, and embryological development. Whilecurrent clinical trials insinuate a positive outlook for VEGF

antagonists, continued understanding of the biological role of VEGFs in angiogenesis will remain essential for sheddinglight on promising advancements. Here, we review thepublished patents and patent applications, and relevantliterature 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 beenthe target of anti-angiogenic therapies for cancer. Pre-clinicalresults of VEGF/VEGFR related antagonists show strongefficacy in reducing tumor size, blood vessel density, andmetastatic potential. Companies have developed a widerange of strategies for VEGF-mediated tumor growthinhibition 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 initialexperiments predicted a cytostatic effect, VEGF therapieshave also shown cytotoxic anti-vascular effects, possiblyextending their use to late stage carcinomas [47,48]. Whilemany of these compounds may result in similar effectorfunction, optimized cytochrome P450 (CYP) enzymeprofiles, solubility, selectivity, and toxicity are areas targetedfor refinement.

2.1. Monoclonal Antibodies and Antibody FragmentsAgainst VEGF and VEGFR

Monoclonal antibodies (mAb) are pure antibodiesdesigned to bind to a specific antigen target. The initialdevelopment of mAbs by Milstein and Köhler in 1975 [49]has allowed for the large-scale production of mAbs for useas anti-cancer therapeutics. Recently, mAbs developed totarget various isoforms of VEGF have shown bothpreclinical and clinical efficacy.

Bevacizumab (Avastin) is a humanized monoclonalantibody developed by Genentech Inc. Bevacizumab binds toall VEGF isoforms as well as all bioactive proteolyticfragments and thus attempts to block the biological activityof this growth factor by inhibiting the interaction of VEGFwith its corresponding receptor [50]. Bevacizumab washumanized from a previously developed mouse anti-VEGF

antibody (muMAb), with retention of high affinity binding(K d = 1.8 nM) [51]. In January 1997, Genentech filed anInvestigational New Drug Application (IND) forbevacizumab, and phase I clinical trials were initiated inApril 1997. These phase I studies showed that bevacizumabas a single agent was relatively non-toxic and that addingbevacizumab to standard chemotherapy regimens did notsignificantly exacerbate chemotherapy associated toxicities[52,53]. In a Phase III trial involving 813 patients withmetastatic colorectal cancer, those patients who received



VEGF and VEGFR Inhibitors for Cancer Treatment Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 61

bevacizumab with irinotecan, 5-fluorouracil (5-FU), andleucovorin (IFL) showed an increase in progression freesurvival of 10.6 months vs. those given placebo plus IFL of 6.2 months. Median duration of survival was also increasedfrom 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 otherneoplasms including pancreatic adenocarcinoma, advancedrenal cell carcinoma, and metastatic colorectral carcinoma.

Protein Design Labs, Inc./Toagosei Co. Ltd developedHuMV833, a humanized anti-VEGF monoclonal IgG4antibody that similarly binds VEGF121 and VEGF165 (K d =0.1 nM) [55,56]. In a phase I trial of HuMV833, pharmaco-kinetic, pharmacodynamic and toxicity data revealed dosesof 1 and 3 mg/kg as having possible clinical efficacy [57].Preliminary positron emission tomography (PET) imagingresults have shown that different tumor deposits within thesame patient may display distinct pharmacokinetic profilesof HuMV833 [58]. Recent phase I trials isolated two distinctdoses which display promising clinical activity, 1 and3mg/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/humanchimeric IgG1 derived from a single chain Fv isolated from aphage display library [59-61]. Preclinical testing showed thatantibody concentration required to inhibit 50% of VEGF-induced mitogenesis of human umbilical vein endothelialcells (HUVECs) is 0.8 nM [62]. ImClone continued withPhase I clinical trials in May 2002 in patients with metastaticcolorectal carcinoma. Results were negative for grade-3 or -4IMC-1C11-related toxicities and a 5 µg/mL dose of IMC-1C11 prevented KDR phosphorylation in vitro . At a dose of 4 mg/kg, a half-life of 67 h was obtained. Anti-tumor effectswere noted in 11 patients; specifically, dynamic contrastenhanced magnetic resonance imaging (DCE-MRI) was used

to assess drug-induced vascular regression. After 4 weeks of therapy, patients showed a marked reduction in tumorenhancement factor (EF) and tumor influx rate constant Kin(min-1), both of which are proportional to the perfusioncapacity of the tumoral vascular network [63].

In addition to IMC-1C11, ImClone recently screened alarge naïve human antibody phage display library andproduced several fully human anti-VEGFR-2 Fab fragments.Affinity maturation of one of these Fab clones led to thedevelopment of 1121B Fab [64]. The affinity of 1121B Fabfor VEGFR-2 was evaluated using ELISA on immobilizedreceptor 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 wasalso demonstrated that 1121B Fab binds to VEGFR-2 in adose-dependent manner (ED50 = 0.15 nM). Furtherpreclinical testing showed that cell proliferation induced byVEGF stimulation was significantly inhibited by 1121B Fab(IC50 = 20 nM) [65]. Imclone Systems initiated phase Iclinical trials of IMC-1121B in January 2005 [66].

2.2. Soluble VEGF Receptors

Soluble VEGF receptors are designed to irreversibly bindsuspended VEGF ligand in hopes of preventing receptor(VEGFR-1, 2, and 3) activation. Currently being developedcollaboratively by Regeneron Pharmaceuticals Inc. andSanofi-Aventis, VEGF-Trap is a high affinity soluble VEGFreceptor 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 VEGF-Trap for VEGF is significantly higher than that of monoclonal antibody bevacizumab (1-5 pM) [46,51]. ThisVEGF inhibitor has shown both anti-angiogenic [48] andanti-tumor [46] activity as well as efficacy against xenograftmodels of Wilm’s tumor [69], ovarian [70] and pancreaticcancer [71]. It has been hypothesized that high doses of VEGF-Trap efficiently inhibits VEGF signaling by blockingstatic low levels of VEGF required to support the long-termintegrity of co-opted vasculature in addition to the VEGFexpression required for neovascularization [72]. Cytotoxicstudies revealed that VEGF-Trap is capable of inducingregression of co-opted vascular development thus expandingits treatment capability to larger tumors [72].

2.3. Small Molecule VEGF Receptor InhibitorsTo date, the number of VEGFR-2 inhibitors undergoing

advanced preclinical and clinical evaluation is steadilyrising. Investigators now take advantage of combinatorialchemistry and high throughput screening to optimize thesolubility, bioavailability, binding efficiency, productioncost, and therapeutic efficacy of various small molecule RTKinhibitors. Recent patenting activity has been grounded inclaims of basic structural features with countless numbers of substitutions and variations, opening the door to a widerange of potent inhibitors capable of pharmacokineticrefinement.

VEGF receptor tyrosine kinases (RTK’s) have typicallybeen classified into families based on structural features of their respective extracellular domains. While the extracel-lular portion of each receptor expresses unique ligandbinding, the intracellular portion of RTK’s are generallyarchitecturally very similar. Furthermore, favorable pharma-cokinetic profiles of many of these compounds aids in theability to deliver these inhibitors orally, making them mostattractive for further clinical development.

2.3.1. Anilinoquinazolines

AstraZeneca redesigned the scaffold of a previouslydeveloped EGFR inhibitor by rearranging the halogensubstitution pattern around the aniline ring to reveal acompound 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 (IC50’s range from 1 - 40 nM),particularly ZD4190 (1) [73,74]. Murine testing revealed thatZD4190 maintained the highest plasma levels following oraldosing. Characteristics of this compound in particular areneutral C7 heteroaromatic side chains [74]. However, furthertesting revealed low aqueous solubility and variable




pharmacokinetic (PK) properties that made ZD4190unfavorable for clinical evaluation. AstraZeneca refinedZD4190 by manipulating the C7 side chain. One resultingcompound, ZD6474 ( N -(4-bromo-2-fluorophenyl)-6-methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-amine), was formed by incorporating a basic nitrogen in theC7 side chain (2) [75]. Hennequin et al. showed that anilino-quinazolines modified with basic C-7 side chains displayedincreased aqueous solubility over anilinoquinazolines with

neutral C-7 side chains (30 - 80 fold) and had PK charac-teristics that were more clinically favorable [76]. Preclinicaltesting 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 ZD6474resulted in a 5.4-fold decrease in vascular volume comparedwith 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-stateconcentrations exceeding the IC50 for VEGF inhibition inpreclinical models. The study concluded that ZD6474monotherapy had limited therapeutic activity in patients withrefractory metastatic breast cancer [80]. Phase III trials arecurrently ongoing [81].

AZD2171, 4-[(4-fluoro-2-methyl-1 H -indol-5-yl)oxy]-6-methoxy-7-[3- (pyrrolidin-1-yl)propoxy]quinazoline (3), isanother VEGFR-2 inhibitor developed by AstraZenecaPharma [WO0047212]. AZD2171 displayed activity againstVEGF-stimulated KDR autophosphorylation in HUVECproliferation assays with IC50 = 40 + 20 pM. Murine tumormodels showed that AZD2171 (1.5 and 6 mg/kg/day)abolished VEGF-dependent blood vessel formation.AZD2171 is advantageous in that it elicits anti-angiogenicresponses at significantly lower doses than those required byother 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 thecatalytic domain suggests that oxindoles bind in the ATPpocket with the indolin-2-one core participating in key H-bond donor/acceptor capacities with the carbonyl of Glu915and the NH of Cys917 [75]. This was further supported by adrop in VEGFR-2 inhibitory activity seen after N -methy-lation of the oxyindole. Sugen (Pharmacia, Pfizer) developeda number of compounds designed to take advantage of thisATP binging binding pocket and further refined theirpharmacokinetic profiles for possible clinical development.Allergan Inc., recently began patenting various 3-(aryl-amino) methylene-1, 3-dihydro-2H-indol-2-ones as kinaseinhibitors as well [85].

Developed by Sugen (Pharmacia, Pfizer) is SU5416(semaxanib) (4), an oxindole VEGFR-1, 2, 3 inhibitor (IC 50 =43 + 11 nM, 220 + 34 nM, 50 nM, respectively) [86,87].Anti-tumor activity has been demonstrated in both humansand rodents [88-91], however the limited solubility of SU5416 required a cremophor formulation to allow forintravenous administration [92]. In February of 2002, Phar-macia announced that they would end clinical developmentof SU5416 because of pharmacokinetic-related problems

[93]. Sugen (Pharmacia, Pfizer) continued in their develop-ment of more soluble versions of this compound.

Sugen (Pharmacia, Pfizer) then developed SU6668 (5)after determining that appending carboxylic acid residuesonto the pyrrole ring of the scaffold would allow foradditional binding interaction in the sugar-binding region of the ATP pocket [94]. The resulting propionic acid analog of SU5416 displayed an increased PDGFR-β inhibition profile

(IC50 = 39 + 1 nM, vs. 2220 + 1500 nM) and an oralbioavailability that coincided with a longer half-life [90,95,96]. Further preclinical testing using biochemical assaysrevealed potent VEGFR-2 inhibition (IC50 = 2.1µM) and invivo xenograft experiments showed that SU6668 is effectiveagainst large established epidermoid (A431), colon (Colo205and 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/m 2

when given orally, thrice daily under fed conditions. Becauseof the low plasma levels reached at this dose level, theefficacy of SU6668 is still undetermined and further clinicaldevelopment has not been encouraged [98].

Another variant of SU5416, SU11248 (SUTENT) (6),

was developed by appending a carboxy-diethylamino-ethylamido group onto the pyrrole ring and attachingfluoride at C5 [99,100]. in vitro Testing of SU11248 showedcompetitive inhibition against Flk-1 and PDGF-dependentPDGFR-β phosphorylation with IC50 = 10 nM for bothRTKs. It was also shown that SU11248 inhibited VEGF-induced proliferation of HUVECs (IC50 = 40 nM) [99].Preclinical tumor models also showed efficacy in tumorregression and apoptotic activity in response to SU11248administration. Phase I clinical trials have shown a dailydose 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 thehalf-life of SU11248 [102].

2.3.3. PhthalazinesPTK787 (1-[4-chloroanilino]-4-[pyridylmethyl]-phthala-

zine succinate), also known as vatalanib, CGP-79787D,ZK222584 or PTK/ZK (7), was first reported by Novartisand Schering AG in 1998 and is currently recognized as oneof the most promising VEGFR inhibitors currently in clinicaldevelopment [103]. Biochemical assays reveal PTK787inhibits both VEGFR-1 and VEGFR-2 (IC50 < 100 nM).Studies conducted on HUVECs show that PTK787 displayspotent activity in a VEGF-driven cellular autophosphory-lation assay (IC 50 = 17 nM). The Phase I clinical trial of thiscompound introduced the use of DCE-MR imaging for thevalidation of its end effect. This functional component usedto 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 humancarcinoma cell lines and is currently being investigated forits potential when combined with irradiation.

Patents surrounding PTK787 have developed in twomajor areas, combinatorial therapies and unique methods of delivery. Novartis, Inex Pharmaceuticals, Beth IsraelDeaconess Medical Center, Schering AG, Eisai, andPharmacia have each submitted patents proposing the use of




PTK787 in combination with various other therapeuticsincluding tie-2 inhibitors [106], EGFR inhibitors [107], andhistone deacetylase inhibitors [108]. Other patents relatePTK787 to a specific method of treatment. Novartis, DanaFarber, and Academisch Zeikenhuis Groningen maintainpatents that propose the use of PTK787 in the treatment of myeloma, ocular neovascular disease [109], angiogenicmyeloid metaplasia [110], and mesothelioma [111].

2.3.4. Anthranilamides

Novartis researchers used conformational analysis,computational modeling, and database searching, to trans-form PTK787 into a new class of anthranilamide VEGFRinhibitors [112]. Novartis first identified anthranilamides asinhibitors of VEGFR and further developed AAL993 (8)[113] and an unnamed compound (9) [114]. While these twocompounds display similar potencies against VEGFR-1 andVEGFR-2 compared with PTK787, the key interactions of AAL993 with VEGFR-2 are two hydrogen bonds of theamid NH and carbonyl groups with a glutamate residue of the αC helix, and the backbone of the aspartate of the Asp-Phe-Gly (DFG) motif [104]. AAL993 has been shown toinhibit VEGF-induced proliferation of HUVECs (IC 50 = 0.84nM).

Novartis continued to refine their new class of VEGFRinhibitors and disclosed several new derivatives. Theyreported the use of alkyl and cycloalkyl anthranylic amides[115], pyridine and pyrimidines as the central core [116-118], limited substitution on the aryl amide moiety witheither a pyridyl group or a benzyl-substituted amide as thehinge-binding element. This led to the development of theirsecond generation VEGFR inhibitors ABP309 (10) whichcombines a pyridone as the hinge-binding element with apyridine core and several other pyridine derivatives [119].ABP309 displays a 10-fold increase in aqueous solubility atpH 4, an improved CYP inhibition profile as compared toPTK787, and is reported to have a selective kinase profilespecific to only VEGFR-2 [104]. Novartis’ most recentpatent activity involves the disclosure of a line of N-aryl

(thio) anthranilic acid amide derivatives [120].

Similarly, Schering AG maintains several patents thatimplicated anthranilic acid amide derivatives. In 2001,Schering AG’s patents included pyridylethyl analogues,pyridylethylene and pyridylethyne derivatives with aryl orhetercyclic amide substitutions. Schering’s discovery thatcyclic ethers or cyclic amines can be used as the hinge-binding element expanded their VEGFR-2 inhibitor profile[121,122]. In a second set of patents, Schering AG addressed

Fig. (1). Anilinoquinazolines & Oxindoles.

N

N

HN

FMeO

O

Cl

NNN N

N

HN

FMeO

O

Cl

N

N

N

O

N

FMeO

ON

HN

HN

CO2H

O

HN

HN

O

HN

HN

HN

O

F

O

NEt2

1

ZD41902

ZD6474

3 AZD2171

4

SU5416

5

SU66686

SU11248




the growing concern of CYP liability associated with initialanthranilamide compounds. Changes included N-oxidederivatives [123] (11), N -benzyl-anthranilic acid (hetero)arylamide derivatives [124], pyridyl substitutions (12), andpyridones with anthranilic acid amides [125] (13).

Amgen began disclosing anthranylic acid amidederivatives in 2002. Their derivatives include heteroaryl corereplacements with broad heterocyclic substitutions at the

amide moiety [126], nicotinamide derivatives [127], and theuse of heterocylics as the hinge-binding element [128].Amgen also patented the use of substituted five- or six-membered heterocycles as the hinge-binding moiety andsubmitted several other patents further defining the centralcore of each compound. The disclosure of anthranilamidepyridinureas by another group has led to development of anew set of VEGFR inhibitors. However, due to its recentdiscovery, 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 inhibi-tion. CP-547632 is an ATP-competitive inhibitor that blocksVEGFR-2 kinase autophosphorylation (IC50 = 11 nM) andVEGF-induced VEGFR-2 phosphorylation (IC50 = 6 nM)[130-132]. It features a pendant pyrrole attached via a urealinkage that is hypothesized to be responsible for an increase

in aqueous solubility [133]. Preclinical experiments showthat CP-547632 inhibits tumor-associated VEGFR-2 phos-phorylation resulting in decreased vascular density andtumor growth [130]. Phase I clinical studies revealed effica-cious dose of 160 mg/kg/day and a resulting half life of 29hours [134].

2.3.6. Pyrroloindolocarbazoles

Cephalon has described a series of compounds whichreplace one indole nitrogen with a carbon. Further optimi-

Fig. (2). Phthalazines & Anthranilamides.

N

N

HN

N

Cl

O

O

HO

OH

NH

HN

N

CF3

ONH

HN

N

N

O

NH

HN

NH

O

O

CF3

NH

HN

N

NH

O

O CF3

O

O-

NH

HN

N

N

O

N

O

OH

ONH

HN

N

NH

7

PTK787

8

AAL993

9

10

ABP309

1211

13




zation led to the development of indenocarbazole KDRinhibitors. Specifically, CEP-5214 (15) was formed byreplacing the bridging heterocycle with a flexible propylchain appended off the indole nitrogen [75,135]. Indeno-pyrrolocarbazole CEP-5214 is an efficient inhibitor of VEGFR-2 (IC50 = 8 nM) and demonstrates significant in vivoanti-tumor activity in murine tumor models [136,137].Studies conducted on HUVECs showed strong efficacy inblocking VEGFR-2 autophosphorylation (IC50 = 10 nM).

However, because suboptimal plasma levels were obtainedwith oral dosing, the HCl salt of the N , N -dimethylglycineester of CEP-5214 (CEP-7055) (16) was tested. Preclinicaltesting showed chronic administration resulted in 50-90%maximum inhibition in the growth of a several differenthuman subcutaneous (s.c.) tumor xenografts in nude mice,including A375 melanomas, U251MG and U87MGglioblastomas, CALU-6 lung carcinoma, ASPC-1 pancreaticcarcinoma, HT-29 and HCT-116 colon carcinomas, MCF-7breast carcinomas, and SVR angiosarcomas [138]. Furthertesting also revealed a more desirable aqueous solubility (40mg/mL) [139]. CEP-7055 is currently in phase I clinicaltrials undergoing MTD and toxicity studies. Several of Cephalon’s candidate compounds are described over a series

of patents pertaining to fused pyrrolocarbazole andisoindolone derivatives [140,141].

Yamanouchi Pharmaceutical Co. Ltd (YP) recentlydeveloped another VEGFR-2 inhibitor for preclinicaldevelopment, YM-359445 (17) [142]. YP identified (3 Z )-3-[6-[(4-methylpiperazin-1-yl)methyl]quinolin-2(1 H )-ylidene]-2-oxoindoline-6-carbaldehyde O-(1,3-thiazol-4-ylmethyl)oxime mono-L-tartrate, YM-359445, while screening for amore potent anti-tumor VEGFR-2 inhibitor. Preclinicaltesting in an enzyme assay for VEGFR-2 revealed strongVEGFR-2 inhibition (IC50 = 8.5 nM). Against HUVECproliferation induced by VEGF, YM-359445 displayedpotent anti-proliferation activity (IC50 = 1.5 nM). Pharma-cokinetic analysis revealed a single dose at 1 mg/kg in mice

resulted in a bioavailability of 23%, and maximum

plasmaconcentration of 16 nM. A recent study conducted by YPshowed YM-359445 to be more potent than other VEGFR-2tyrosine kinase inhibitors, namely SU11248, ZD6474, andAZD2171 [143].

2.3.7. 2-Amino-(thiazol-2-yl) pyridines

Merck has developed a variety of distinct KDR inhibi-tors, which may be grouped into six major classes: pyrimi-dines [144,145], pyrazolo[1,5-a]pyrimidines [146-149], inda-zoles [150], 1-H-quinolin-2-ones [151-154], acyl-2-amino-thiazoles [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). Byappending the basic tertiary amine group off the C-4-position

of the pyridyl nucleus, the resulting compound showed animproved pharmacokinetic profile over the previouslypatented compounds and IC50 values range from 0.1 - 5 µM[159] (19). It also appears that the 2-aminopyridyl moiety iscritical to the KDR inhibition. Merck subsequentlydeveloped a series of KDR inhibitors based on 2-subsitutedindole 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 enhancedpharmacokinetic properties of previous compounds.Schering AG recently disclosed their version of pyrazolo-pyrimidines that have yet to be evaluated in preclinicalmodels [161].

2.4. RNA Based Strategies

Progress in the field of RNA therapeutics has led to thewidespread attention of RNA-based anti-cancer treatments[162]. Specifically, RNA interference has evolved over thelast decade to include small interfering RNA (siRNA) andribozymes [163]. These therapeutics, although in early stagesof clinical validation, have shown promising results as anti-angiogenic strategies.

2.4.1. siRNA

SiRNA, sometimes known as short interfering RNA, area class of 20-25 nucleotide-long RNA molecules that play avariety of roles [164-166]. For purposes of cancer treatment,the function of siRNA in the RNA interference pathway isexploited. Specifically, mammalian cells mount a nonspe-cific inhibitory response to dsRNA that results in thetranslational inhibition and degradation of the targeted

mRNA. Cancer treatments take advantage of this mechanismby inducing dsRNA of a targeted protein, marking it fordestruction. The inhibition of corneal angiogenesis [167] andchoroidal neovascularization (CNV) by local delivery of siRNA targeting VEGF has been demonstrated in xenograftmodels [168], displaying both its effector tumor reductionfunction and anti-angiogenic potential.

Sirna Therapeutics Inc. maintains a large patent portfoliothat describes the use of siRNA techniques to limit VEGFand VEGFR expression [169-172]. Preclinical studies haveshown that siRNA targeting VEGFR-1 successfully reducedocular neovascularization by up to 66% [173]. In an RKOcolon cancer model, cells treated with siRNA targetingVEGF showed a 94% knockdown in VEGF expression and a

67% decrease in cellular proliferation [174]. While signifi-cant preclinical work has accomplished marked results inanti-cancer therapy, continued clinical research will provideinsight to its functional efficacy in humans.

2.4.2. Ribozyme

While ribozymes have been in existence for more than adecade, their use in VEGF signaling inhibition has recentlyled to the development of new anti-cancer therapeutics.Ribozymes function by cleaving RNA phosphodiester bondsat specific sites and in doing so destroy the ability of targetedmRNA to direct synthesis of an encoded protein. Whilesingle ribozyme molecules can degrade multiple mRNAstrands, they are limited by their susceptibility to nucleasedegradation that results in poor serum stability. Preclinicalstudies of an anti-VEGF hairpin ribozyme compound hasshown efficacy in significantly inhibiting the growth andproliferation of ovarian cancer SKOV3 cells [175, 176].

Chiron Corporation/Ribozyme Pharmaceuticals Inc.(RPI) holds the rights to more than 100 worldwide patentsencompassing ribozyme design, synthesis, chemical modifi-cation, delivery, and production [177-181]. Their signatureanti-angiogenic product is an anti-Flt-1 ribozyme known asAngiozyme [182]. Preclinical testing revealed strong




efficacy in the prevention of tumor growth and metastasis. Ina study conducted by Pavco et al, ribozymes targeting eitherVEGFR-1 or VEGFR-2 significantly inhibited primarytumor growth in a highly metastatic variant of Lewis lungcarcinoma and significantly inhibited liver metastasis in axenograft colorectal cancer model [176]. Chiron/RPI conti-nued with phase I clinical studies to show that Angiozymewas well tolerated with satisfactory pharmacokineticvariables for daily s.c. dosing [183]. Further combinatorialstudies have revealed that RPI.4610 (Angiozyme), carbop-latin, and paclitaxel can be administered safely in combina-tion without substantial pharmacokinetic interac-tions [184].

3. CURRENT & FUTURE DEVELOPMENTS

Anti-cancer therapeutics have been developed over thelast decade to include novel strategies based on targetingtumor angiogenesis. The identification of VEGF-related

signaling cascades has led to the expansion of possible anti-angiogenic compounds, each with a characteristic method of action, binding pattern, bioavailability, toxicity, and clinicalefficacy. Preclinical data has revealed strong anti-tumor,anti-metastatic, and anti-angiogenic effects [185]. However,clinical translation of these compounds has been the mostchallenging. Hurdles faced by poor bioavailability andsolubility 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

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

F

FBr

N S

H2N

NHN

N

O

O

H

N

HN

OH

O

O

N

HN

O

O

O

N

O

NH

NH

O

N

N

NO

S

N

HO2C

CO2HHO

OH

N

N

HNS

N

R1

N

N

NR2 R3

HN S

N

R3

NR2

N

N

R4

N

R4

NO

HN

NH

R5

HN NH

O

N

NS

O O

14 CP-547632

15

CEP-5214

16CEP-7055

17

18

:

R1 = alkyl, O-alkyl, halogen, OHR4 = SO2-alkyl, CONH-alkyl

R5 = NHCONH-alkyl

19

20




cons. Soluble receptors, monoclonal antibodies and antibodyfragments offer highly specific VEGF and VEGFR binding,reducing possible toxicity. Whereas small molecule KDRinhibitors offer a much wider range of inhibition, targetingRTKs that do not necessarily involved VEGF [186]. Thefuture of VEGFR inhibitors as therapeutic anti-cancer agentswill become clearer as both preclinical and clinical trialsfurther describe angiogenesis signaling and antagonistefficacy.

There are several challenges faced by the introduction of anti-angiogenic therapy. Specifically the clinical applicationof anti-angiogenic compounds is troubled by the temporaldependence of angio-suppressive treatments [187]. The“angiogenic switch” is a phrase used to describe the level of tumoral angiogenic activity. The pathology of tumor genesisindicates a marked transition from a prevascular to vascularphase. It is hypothesized that VEGF-inhibitive therapy willbe most effective against small tumors and should beadministrated prior to the development of a well-establishedvascular network. Phase III clinical trials of Capecitabine +Bevacizumab for women with metastatic breast cancer failedto elicit a positive therapeutic response and may attest to thisconclusion [188]. Thus, the use of VEGF antagonists may be

better suited for chronic therapy, preventing the recurrenceof disease and inhibiting the genesis of new vessels.Furthermore, the development of reliable markers that areable to aid in selecting patients who are more likely tobenefit from anti-VEGF therapy will be critical toidentifying the treatment regimen of such therapeutics. Therecent application of molecular imaging techniques tovisualize the expression of receptors and ligands implicatedin cancer angiogenesis will aid in such patient selection[189-192]. Molecular imaging probes coupled to therapeutictoxins enable the visualization of therapeutic efficacy andprovide a forum for the quantitative assessment of patientresponse.

The future of cancer-related treatment lies in a combina-

torial approach that aims to target tumor cells and thecorresponding vascular support system. It is unlikely thatanti-anngiogenic therapies alone will, without increasedtoxic risk, sufficiently halt tumor growth within a reasonabletime period. To this regard, numerous on-going clinical trialshave shown efficacy in combining chemotherapy and anti-angiogenic therapy [193,194]. The basis for this specificsynergy assumes that cytotoxic agents will reduce the tumorburden of vascularized tumors, while anti-angiogenic agentswill prevent neovascularization and growth of small andoccult metastatic foci as well as the formation of newmetastatic lesions [5]. Early phase clinical trials havesuggested that this combinatorial therapy is generally welltolerated. However, instances of thromboembolis and

hemorrhage have been reported [98]. It will be critical todevelop appropriate treatment regimens that combine the useof anti-angiogenic, chemo-, and radiation-therapy to take fulladvantage of maximal angiogenic signaling and VEGFblockade.

Therapeutics designed to manipulate the VEGF pathwaywill extend much beyond the treatment of cancer. Conditionscurrently in preclinical and clinical investigation includeasthma, bone lesion, diabetic nephropathy, arthritis, and

psoriasis [195,196]. Currently, several clinical trials arebeing conducted to explore this possibility, Phase III trialsare in progress for patients with age-related maculardegeneration. Furthermore, recent studies have determinedthat VEGFR-1, previously un-implicated in post-fetal angio-genesis, may have a fundamental role in the recruitment of endothelial progenitor cells and hematopoiesis [197,198]. Asthe 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 treatmentefficacy with a variety of VEGF-antagonists. While it hasbeen clearly demonstrated that VEGF related signaling islargely responsible for endothelial cell activity, several otherrecently discovered signaling systems have also shownsimilar capabilities. One of these surface proteins, integrin-αvβ3, has been shown to be up-regulated on cytokine-activated vascular endothelial cells, smooth muscle cells, andblood vessels in tumor, wound, and granulation tissue [199-203]. MEDI-522, a mAb against human αvβ3 developed byMedImmune Inc, has shown promising preclinical evidenceand as a result entered phase III clinical trials. Merck GmBHdeveloped a cyclic penta-peptide c(RGDf[NMe]V) targeted

to αvβ3 called EMD-121974 or Cilengitide. Phase II clinicaltrials have revealed positive results for suppressing tumorvascularization and growth [204,205]. Several other candi-dates are also in various phases of development; theseinclude antagonists to hypoxia-inducible factor-1alpha (HIF-1α), angiopoietin-1 (ANG-1), and PDGF-β.

Despite many of the obstacles discussed above, modula-ting the VEGF signaling cascade presents great opportunityto further understand the process of blood vessel formation.Though validated as a monotherapeutic, the future of comprehensive anti-cancer therapy will require a multiface-ted approach, targeting different aspects of tumor develop-ment. The exact role of the VEGF-inhibitors will requirefurther clinical investigation, proper patient selection, and

acceptable toxicology; many of these tasks have yet to becompleted by a potent inhibitor.

REFERENCES

[1] Roy H, Bhardwaj S, Yla-Herttuala S. Biology of vascularendothelial growth factors. FEBS Lett 2006; 580: 2879-87.

[2] Ferrara N. Vascular endothelial growth factor: basic science andclinical progress. Endocr Rev 2004; 25: 581-611.

[3] Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005; 437: 497-504.

[4] Cui TG, Foster RR, Saleem M, et al. Differentiated humanpodocytes endogenously express an inhibitory isoform of vascular endothelial growth factor (VEGF165b) mRNA andprotein. Am J Physiol Renal Physiol 2004; 286: F767-73.

[5] McMahon G. VEGF receptor signaling in tumor angiogenesis.Oncologist 2000; 5 Suppl 1: 3-10.

[6] Ortega N, Hutchings H, Plouet J. Signal relays in the VEGFsystem. Front Biosci 1999; 4: 141-52.

[7] de Vries C, Escobedo JA, Ueno H, et al . The Fms-like tyrosinekinase, a receptor for vascular endothelial growth factor. Science1992; 255: 989-91.

[8] Terman B, Khandke L, Dougher-Vermazan M, et al. VEGFreceptor subtypes KDR and FLT1 show different sensitivities toheparin and placenta growth factor. Growth Factors 1994; 11:187-95.




[9] Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice.Nature 1995; 376: 62-66.

[10] Quinn TP, Peters KG, De Vries C, et al. Fetal liver kinase 1 is areceptor for vascular endothelial growth factor and is selectivelyexpressed in vascular endothelium. Proc Natl Acad Sci USA1993; 90: 7533-37.

[11] Terman BI, Dougher-Vermazen M, Carrion ME, et al.Identification of the KDR tyrosine kinase as a receptor forvascular endothelial cell growth factor. Biochem Biophys ResCommun 1992; 187: 1579-86.

[12] Millauer B, Wizigmann-Voos S, Schnurch H, et al. High affinityVEGF binding and developmental expression suggest Flk-1 as amajor regulator of vasculogenesis and angiogenesis. Cell 1993;72: 835-46.

[13] Waltenberger J, Claesson-Welsh L, Siegbahn A, et al . Differentsignal transduction properties of KDR and Flt1, two receptors forvascular endothelial growth factor. J Biol Chem 1994; 269:26988-95.

[14] Zachary I. Vascular endothelial growth factor. Int J BiochemCell Biol 1998; 30: 1169-74.

[15] Takahashi T, Ueno H, Shibuya M. VEGF activates protein kinaseC-dependent, but Ras-independent Raf-MEK-MAP kinasepathway for DNA synthesis in primary endothelial cells.Oncogene 1999; 18: 2221-30.

[16] Wu LW, Mayo LD, Dunbar JD, et al. Utilization of distinctsignaling pathways by receptors for vascular endothelial cellgrowth factor and other mitogens in the induction of endothelialcell proliferation. J Biol Chem 2000; 275: 5096-103.

[17] Gerber HP, McMurtrey A, Kowalski J, et al. Vascularendothelial growth factor regulates endothelial cell survivalthrough the phosphatidylinositol 3'-kinase/Akt signaltransduction pathway. Requirement for Flk-1/KDR activation. JBiol Chem 1998; 273: 30336-43.

[18] Ogawa S, Yoshida S, Ono M, et al. Induction of macrophageinflammatory protein-1alpha and vascular endothelial growthfactor during inflammatory neovascularization in the mousecornea. Angiogenesis 1999; 3: 327-34.

[19] Achen MG, Jeltsch M, Kukk E, et al. Vascular endothelialgrowth factor D (VEGF-D) is a ligand for the tyrosine kinasesVEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4). Proc NatlAcad Sci USA 1998; 95: 548-53.

[20] Partanen TA, Arola J, Saaristo A, et al . VEGF-C and VEGF-Dexpression in neuroendocrine cells and their receptor, VEGFR-3,in fenestrated blood vessels in human tissues. FASEB J 2000; 14:2087-96.

[21] Achen MG, Mann GB, Stacker SA. Targeting lymphangio-genesis to prevent tumour metastasis. Br J Cancer 2006; 94:1355-60.

[22] Folkman J. Tumor angiogenesis: therapeutic implications. NEngl J Med 1971; 285: 1182-86.

[23] Li J, Huang S, Armstrong EA, et al. Angiogenesis and radiationresponse modulation after vascular endothelial growth factorreceptor-2 (VEGFR2) blockade. Int J Radiat Oncol Biol Phys2005; 62: 1477-85.

[24] Brown LF, Detmar M, Claffey K, et al. Vascular permeabilityfactor/vascular endothelial growth factor: a multifunctionalangiogenic cytokine. Exs 1997; 79: 233-69.

[25] Dvorak HF, Detmar M, Claffey KP, et al. Vascular permeabilityfactor/vascular endothelial growth factor: an important mediatorof angiogenesis in malignancy and inflammation. Int ArchAllergy Immunol 1995; 107: 233-35.

[26] Folkman J. Role of angiogenesis in tumor growth and metastasis.Semin Oncol 2002; 29: 15-18.

[27] Hicklin DJ, Ellis LM. Role of the vascular endothelial growthfactor pathway in tumor growth and angiogenesis. J Clin Oncol2005; 23: 1011-27.

[28] Dvorak HF. Vascular permeability factor/vascular endothelialgrowth factor: a critical cytokine in tumor angiogenesis and apotential target for diagnosis and therapy. J Clin Oncol 2002; 20:4368-80.

[29] Griffioen AW, Molema G. Angiogenesis: potentials for pharma-cologic intervention in the treatment of cancer, cardiovasculardiseases, and chronic inflammation. Pharmacol Rev 2000; 52:237-68.

[30] Hanahan D. Signaling vascular morphogenesis and maintenance.Science 1997; 277: 48-50.

[31] Folkman J. Incipient angiogenesis. J Natl Cancer Inst 2000; 92:94-95.

[32] Korpelainen EI, Alitalo K. Signaling angiogenesis andlymphangiogenesis. Curr Opin Cell Biol 1998; 10: 159-64.

[33] Tortora G, Bianco R, Daniele G. Strategies for multiple signalinginhibition. J Chemother 2004; 16: 41-43.

[34] Rosen LS. VEGF-targeted therapy: therapeutic potential andrecent advances. Oncologist 2005; 10: 382-91.

[35] Millauer B, Shawver LK, Plate KH, et al . Glioblastoma growth

inhibited in vivo by a dominant-negative Flk-1 mutant. Nature1994; 367: 576-79.

[36] Drevs J, Hofmann I, Hugenschmidt H, et al. Effects of PTK787/ZK 222584, a specific inhibitor of vascular endothelialgrowth factor receptor tyrosine kinases, on primary tumor,metastasis, vessel density, and blood flow in a murine renal cellcarcinoma model. Cancer Res 2000; 60: 4819-24.

[37] Strawn LM, McMahon G, App H, et al. Flk-1 as a target fortumor growth inhibition. Cancer Res 1996; 56: 3540-45.

[38] Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK 222584, anovel and potent inhibitor of vascular endothelial growth factorreceptor tyrosine kinases, impairs vascular endothelial growthfactor-induced responses and tumor growth after oraladministration. Cancer Res 2000; 60: 2178-89.

[39] Wedge SR, Ogilvie DJ, Dukes M, et al. ZD4190: an orally activeinhibitor of vascular endothelial growth factor signaling withbroad-spectrum antitumor efficacy. Cancer Res 2000; 60: 970-75.

[40] Saleh M, Stacker SA, Wilks AF. Inhibition of growth of C6glioma cells in vivo by expression of antisense vascularendothelial growth factor sequence. Cancer Res 1996; 56: 393-401.

[41] Oku T, Tjuvajev JG, Miyagawa T, et al. Tumor growthmodulation by sense and antisense vascular endothelial growthfactor gene expression: effects on angiogenesis, vascularpermeability, blood volume, blood flow, fluorodeoxyglucoseuptake, and proliferation of human melanoma intracerebralxenografts. Cancer Res 1998; 58: 4185-92.

[42] Prewett M, Huber J, Li Y, et al. Antivascular endothelial growthfactor receptor (fetal liver kinase 1) monoclonal antibody inhibitstumor angiogenesis and growth of several mouse and humantumors. Cancer Res 1999; 59: 5209-18.

[43] Kong HL, Hecht D, Song W, et al. Regional suppression of tumor growth by in vivo transfer of a cDNA encoding a secretedform of the extracellular domain of the Flt-1 vascular endothelial

growth factor receptor. Hum Gene Ther 1998; 9: 823-33.[44] Goldman CK, Kendall RL, Cabrera G, et al. Paracrine expression

of a native soluble vascular endothelial growth factor receptorinhibits tumor growth, metastasis, and mortality rate. Proc NatlAcad Sci USA 1998; 95: 8795-800.

[45] Kuo CJ, Farnebo F, Yu EY, et al. Comparative evaluation of theantitumor activity of antiangiogenic proteins delivered by genetransfer. Proc Natl Acad Sci USA 2001; 98: 4605-10.

[46] Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGFblocker with potent antitumor effects. Proc Natl Acad Sci USA2002; 99: 11393-98.

[47] Willett CG, Boucher Y, di Tomaso E, et al. Direct evidence thatthe VEGF-specific antibody bevacizumab has antivasculareffects in human rectal cancer. Nat Med 2004; 10: 145-47.

[48] Inai T, Mancuso M, Hashizume H, et al . Inhibition of vascularendothelial growth factor (VEGF) signaling in cancer causes lossof endothelial fenestrations, regression of tumor vessels, andappearance of basement membrane ghosts. Am J Pathol 2004;

165: 35-52.[49] Kohler G, Milstein C. Continuous cultures of fused cells

secreting antibody of predefined specificity. Nature 1975; 256:495-97.

*[50] Carter, P., Presta, L.: US20006054297 (2000).[51] Presta LG, Chen H, O'Connor SJ, et al. Humanization of an anti-

vascular endothelial growth factor monoclonal antibody for thetherapy of solid tumors and other disorders. Cancer Res 1997;57: 4593-99.

[52] Margolin K, Gordon MS, Holmgren E, et al. Phase Ib trial of intravenous recombinant humanized monoclonal antibody tovascular endothelial growth factor in combination with




chemotherapy in patients with advanced cancer: pharmacologicand long-term safety data. J Clin Oncol 2001; 19: 851-56.

[53] Gordon MS, Margolin K, Talpaz M, et al. Phase I safety andpharmacokinetic study of recombinant human anti-vascularendothelial growth factor in patients with advanced cancer. J ClinOncol 2001; 19: 843-50.

[54] Hurwitz H, Fehrenbacher L, Novotny W, et al., Bevacizumabplus irinotecan, fluorouracil, and leucovorin for metastaticcolorectal cancer. N Engl J Med 2004; 350: 2335-42.

[55] Asano M, Yukita A, Suzuki H. Wide spectrum of antitumoractivity of a neutralizing monoclonal antibody to human vascular

endothelial growth factor. Jpn J Cancer Res 1999; 90: 93-100.[56] Kaisheva, E., Gupta, S., Flores-Nate, A.: WO04039337(2004).[57] Jayson GC, Mulatero C, Ranson M, et al . Phase I investigation

of recombinant anti-human vascular endothelial growth factorantibody in patients with advanced cancer. Eur J Cancer 2005;41: 555-63.

[58] Jayson GC, Zweit J, Jackson A, et al. Molecular imaging andbiological evaluation of HuMV833 anti-VEGF antibody:implications for trial design of antiangiogenic antibodies. J NatlCancer Inst 2002; 94: 1484-93.

[59] Zhu Z, Witte L. Inhibition of tumor growth and metastasis bytargeting tumor-associated angiogenesis with antagonists to thereceptors of vascular endothelial growth factor. Invest NewDrugs 1999; 17: 195-212.

[60] Zhu Z, Rockwell P, Lu D, et al. Inhibition of vascularendothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodiesfrom a phage display library. Cancer Res 1998; 58: 3209-14.

*[61] Lemischka, I.: US5747651 (1998).[62] Zhu Z, Lu D, Kotanides H, et al. Inhibition of vascular

endothelial growth factor induced mitogenesis of humanendothelial cells by a chimeric anti-kinase insert domain-containing receptor antibody. Cancer Lett 1999; 136: 203-13.

[63] Posey JA, Ng TC, Yang B, et al. A phase I study of anti-kinaseinsert domain-containing receptor antibody, IMC-1C11, inpatients with liver metastases from colorectal carcinoma. ClinCancer Res 2003; 9: 1323-32.

[64] Rockwell, P., Goldstein, N.: US20046811779 (2004).[65] Miao HQ, Hu K, Jimenez X, et al. Potent neutralization of

VEGF biological activities with a fully human antibody Fabfragment directed against VEGF receptor 2. Biochem BiophysRes Commun 2006; 345: 438-45.

[66] ImClone S, ImClone Systems Commences Patient Treatment in

U.S. Phase I Clinical Trial Of IMC-1121B. 2005.[67] Konner J, Dupont J. Use of soluble recombinant decoy receptor

vascular endothelial growth factor trap (VEGF Trap) to inhibitvascular endothelial growth factor activity. Clin ColorectalCancer 2004; 4: S81-85.

*[68] Cedarbaum, J.: WO05123104 (2005).[69] Frischer JS, Huang J, Serur A, et al., Effects of potent VEGF

blockade on experimental Wilms tumor and its persistingvasculature. Int J Oncol 2004; 25: 549-53.

[70] Byrne AT, Ross L, Holash J, et al. Vascular endothelial growthfactor-trap decreases tumor burden, inhibits ascites, and causesdramatic vascular remodeling in an ovarian cancer model. ClinCancer Res 2003; 9: 5721-28.

[71] Fukasawa M, Korc M. Vascular endothelial growth factor-trapsuppresses tumorigenicity of multiple pancreatic cancer celllines. Clin Cancer Res 2004; 10: 3327-32.

[72] Kim ES, Serur A, Huang J, et al. Potent VEGF blockade causesregression of coopted vessels in a model of neuroblastoma. ProcNatl Acad Sci USA 2002; 99: 11399-404.

[73] Boyle, F.: US20026346633 (2002).

[74] Hennequin LF, Thomas AP, Johnstone C, et al. Design andstructure-activity relationship of a new class of potent VEGFreceptor tyrosine kinase inhibitors. J Med Chem 1999; 42: 5369-89.

[75] Underiner TL, Ruggeri B, Gingrich DE. Development of vascular endothelial growth factor receptor (VEGFR) kinaseinhibitors as anti-angiogenic agents in cancer therapy. Curr MedChem 2004; 11: 731-45.

[76] Hennequin LF, Stokes ES, Thomas AP, et al. Novel 4-anilinoquinazolines with C-7 basic side chains: design andstructure activity relationship of a series of potent, orally active,

VEGF receptor tyrosine kinase inhibitors. J Med Chem 2002; 45:1300-12.

[77] Morelli MP, Cascone T, Troiani T, et al . Anti-tumor activity of the combination of cetuximab, an anti-EGFR blocking mono-clonal antibody and ZD6474, an inhibitor of VEGFR and EGFRtyrosine kinases. J Cell Physiol 2006; 208: 344-53.

[78] Wedge, S., Ryan, A.: WO04071397 (2004).[79] Drevs J, Konerding MA, Wolloscheck T, et al. The VEGF

receptor tyrosine kinase inhibitor, ZD6474, inhibits angiogenesisand affects microvascular architecture within an orthotopicallyimplanted renal cell carcinoma. Angiogenesis 2004; 7: 347-54.

[80] Miller KD, Trigo JM, Wheeler C, et al . A multicenter phase IItrial of ZD6474, a vascular endothelial growth factor receptor-2and epidermal growth factor receptor tyrosine kinase inhibitor, inpatients with previously treated metastatic breast cancer. ClinCancer Res 2005; 11: 3369-76.

[81] ZD6474 headed for phase III trials in the fall. Oncology(Williston Park) 2005; 19(9): 1140-42.

[82] Wedge SR, Kendrew J, Hennequin LF, et al. AZD2171: a highlypotent, orally bioavailable, vascular endothelial growth factorreceptor-2 tyrosine kinase inhibitor for the treatment of cancer.Cancer Res 2005; 65: 4389-400.

[83] Buzzetti F, Brasca MG, Crugnola A, et al. Cinnamamide analogsas inhibitors of protein tyrosine kinases. Farmaco 1993; 48: 615-36.

[84] Sun L, Tran N, Tang F, et al. Synthesis and biologicalevaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity towardparticular receptor tyrosine kinases. J Med Chem 1998; 41: 2588-603.

[85] Andrews, S., Wurster, J., Wang, E., Malone, T.: US20060063940

(2006).[86] App, H., McMahon, G., Tang, P., Gazit, A., Levitzki, A.:

US19985712395 (1998).[87] Tang, P., Sun, L., McMahon, G.: US5792783(1998).[88] Lockhart AC, Cropp GF, Berlin JD, et al. Phase I/pilot study of

SU5416 (semaxinib) in combination with irinotecan/bolus 5-FU/LV (IFL) in patients with metastatic colorectal cancer. Am JClin Oncol 2006; 29: 109-15.

[89] Fong TA, Shawver LK, Sun L, et al. SU5416 is a potent andselective inhibitor of the vascular endothelial growth factorreceptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis,tumor vascularization, and growth of multiple tumor types.Cancer Res 1999; 59: 99-106.

[90] Mendel DB, Laird AD, Smolich BD, et al. Development of SU5416, a selective small molecule inhibitor of VEGF receptor

tyrosine kinase activity, as an anti-angiogenesis agent.Anticancer Drug Des 2000; 15: 29-41.

[91] Ning S, Laird D, Cherrington JM, Knox SJ. The antiangiogenicagents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation. Radiat Res 2002; 157: 45-51.

[92] Sepp-Lorenzino L, Thomas KA. Antiangiogenic agents targetingvascular endothelial growth factor and its receptors in clinicaldevelopment. Expert Opin Investig Drugs 2002; 11: 1447-65.

[93] Corporation, P., Pharmacia Announces Closing of SU5416(semaxanib) Clinical Trials, in PRNewswire-FirstCall. 2002:PEAPACK, N.J.

*[94] Hirth, K., Schwartz, D., Mann, E., Shawver, L., Keri, G.,Szekely, I., Bajor, T., Haimichael, J., Orfi, L., Levitzki, A.,Gazit, A., Ullrich, A., Lammers, R., Kabbinavar, F., Slamon, D.,Tang, P.: US5990141 (1999).

[95] Sun L, Tran N, Liang C, et al. Design, synthesis, and evaluationsof substituted 3-[(3- or 4-carboxyethylpyrrol-2-yl)methyli-denyl]indolin-2-ones as inhibitors of VEGF, FGF, and PDGF

receptor tyrosine kinases. J Med Chem 1999; 42: 5120-30.[96] Sukbuntherng J, Shawver L, Wagner W, Antonian L. Proc Am

Assoc Cancer Res 2000; 41(abstract 815).[97] Laird AD, Christensen JG, Li G, et al. SU6668 inhibits Flk-

1/KDR and PDGFRbeta in vivo, resulting in rapid apoptosis of tumor vasculature and tumor regression in mice. FASEB J 2002;16: 681-90.

[98] Kuenen BC, Levi M, Meijers JC, et al . Analysis of coagulationcascade and endothelial cell activation during inhibition of vascular endothelial growth factor/vascular endothelial growthfactor receptor pathway in cancer patients. Arterioscler ThrombVasc Biol 2002; 22: 1500-05.




[99] Mendel DB, Laird AD, Xin X, et al. In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascularendothelial growth factor and platelet-derived growth factorreceptors: determination of a pharmacokinetic/pharmacodynamicrelationship. Clin Cancer Res 2003; 9: 327-37.

*[100] Tang, P., Sun, L., McMahon, G., Hirth, K., Shawver, L.:US20006147106 (2000).

[101] Foran J, Paquette R, Cooper M, et al. Proc. Am. Soc. Hemat.2002; 44 (abstract 2195).

[102] Raymond E, Faivre S, Vera K, et al. Proc Am Soc Clin Onol2003; 22 (abstract 769).

[103] *Bold, G., Frei, J., Traxler, P., Altmann, K., Mett, H., Strover,D., Wood, J.: WO9835958 (1998).

[104] Dumas J, Dixon J. VEGF receptor kinase inhibitors:phthalazines, anthranilamides, and related structures. ExpertOpin Ther Patents 2005; 647 - 658.

[105] Morgan B, Thomas AL, Drevs J, et al. Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for thepharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases,in patients with advanced colorectal cancer and liver metastases:results from two phase I studies. J Clin Oncol 2003; 21: 3955-64.

[106] Siemeister, G., Haberey, M., Thierauch, K.: WO0197850(2001).[107] Wood, J., Brandt, R., Bold, G., Traxler, P.: WO0241882 (2002).[108] Tadja, P., Remiszewski, S., Trogani, N.: WO103358(2004).[109] Brazzell, R., Wood, J., Campochiaro, P., Kane, F.: WO009098

(2000).[110] Giles, F.: WO033042 (2004).[111] Hohneker, J.: WO043459 (2004).[112] Furet P, Bold G, Hofmann F, et al. Identification of a new

chemical class of potent angiogenesis inhibitors based onconformational considerations and database searching. BioorgMed Chem Lett 2003; 13: 2967-71.

[113] Altmann, K.H., Bold, G., Furet, P., Manley, P., Wood, J., Ferrari,S., Hofmann, F., Mestan, J., Huth, A., Krueger, M., Seidelmann,D., Menrad, A., Haberey, M., Theirauch, K.: WO0027820(2000).

[114] Huth, A., Seidelmann, D., Thierauch, K., Bold, G., Manley, P.,Furet, P., Wood, J., Mestan, J., Brueggen, J., Ferrari, S., Krueger,M., Ottow, E., Menrad, A., Schirner, M.: WO0027819 (2000).

[115] Seidelmann, D., Krueger, M., Ottow, E., Huth, A., Theirauch, K.,Menrad, A., Haberey, M.: WO0185691 (2001).

[116] Manley, P., Bold, G.: WO0155114 (2001).[117] Seidelmann, D., Krueger, M., Petrov, O., Huth, A., Thierauch,

K., Menrad, A., Haberey, M.: WO0185715 (2001).[118] Baenteli, R., Zenke, G., Cook, N., Duthaler, R., Thoma, G., Von

Matt, A., Honda, T., Matsurra, N., Nonomura, K., Ohmori, O.,Umemura, I., Hinterding, K., Papageorgiou, C.: US20060100227(2006).

[119] Bold, G., Manley, P.: HK1052500 (2006).[120] Altmann, K., Bold, G., Furet, P., Manley, P., Wood, J., Ferrari,

S., Hofmann, F., Mestan, J., Huth, A., Kruger, M., Seidelmann,D., Menrad, A., Haberey, M., Thierauch, K.: US20060074112(2006).

[121] Huth, A., Seidelmann, D., Thierauch, K.: WO0181311 (2001).[122] Krueger, M., Huth, A., Petrov, O., Seidelmann, D., Thierauch,

K., Haberey, M., Menrad, A., Ernst, A.: WO0185671 (2001).[123] Ernst, A., Huth, A., Krueger, M., Thierauch, K., Menrad, A.,

Haberey, M.: WO02090349 (2002).[124] Huth, A., Krueger, M., Zorn, L., Ince, S., Theirauch, K., Menrad,

A., haberey, M., Hess-Stumpp, H.: DE10328036 (2005).[125] Huth, A., Krueger, M., Zorn, L., Ince, S., HBohlmann, R.,

Theirauch, K., Menrad, A., Haberey, M., Hess-Stumpp, H.:WO111005 (2004).

[126] Amgen Inc.: WO02066470 (2002).[127] Xi, N.: MXPA05000584. (2005).[128] Elbaum, D., Potashman, M., Van der Plas, S., Askew, B.,

Germain, J., Habgood, G., Kim, J., Harmange, J., Patel, V., DiPietro, L.: CA2518909 (2004).

[129] Bohlmann, R., Haberey, M., Huth, A., Ince, S., Krueger, M.,Thierauch, K., Hess-Stumpp, H., Reichel, A.: US20060116380(2006).

[130] Beebe JS, Jani JP, Knauth E, et al. Pharmacologicalcharacterization of CP-547,632, a novel vascular endothelialgrowth factor receptor-2 tyrosine kinase inhibitor for cancertherapy. Cancer Res 2003; 63: 7301-09.

[131] Gant, T., Williams, G.: US20046831091 (2004).[132] Larson, E., Noe, M., Gant, T.: US20016235764(2001).[133] Jani JB, Beebe JS, Emerson E, et al . Proc. Am. Assoc. Cancer

Res., 2002; (abstract 5354).[134] Jani JB, Beebe JS, Emerson E, et al . Proc Am Soc Clin Oncol

2002; (abstract 473).[135] Gingrich, D., Hudkins, R., WO020217914 (2002).[136] Gingrich DE, Reddy DR, Iqbal MA, et al. A new class of potent

vascular endothelial growth factor receptor tyrosine kinaseinhibitors: structure-activity relationships for a series of 9-alkoxymethyl-12-(3-hydroxypropyl)indeno[2,1-a]pyrrolo[3,4-

c]carbazole-5- ones and the identification of CEP-5214 and itsdimethylglycine ester prodrug clinical candidate CEP-7055. JMed Chem 2003; 46: 5375-88.

[137] Hudkins, R.: US20050107458 (2005).[138] Ruggeri B, Singh J, Gingrich D, et al. CEP-7055: a novel, orally

active pan inhibitor of vascular endothelial growth factorreceptor tyrosine kinases with potent antiangiogenic activity andantitumor efficacy in preclinical models. Cancer Res 2003; 63:5978-91.

[139] Gingrich, D., Yang, S., Albom, M., Angeles, T., Robinson, C.,Hunter, K., Chang, H., Ruggeri, B., Hudkins, R. MEDI-096 . in224th National Meeting of the American Chemical Society. 2002.Boston, MA.

[140] Hudkins, R., Reddy, D., Singh, J., Tripathy, R., Underiner, T.:HU0105363 (2002).

[141] Hudkins, R.: WO010114380 (2001).[142] Samizu, K., Hisamichi, H., Matsuhisa, A., Kinoyama, I., Hayak,

M., Taniguchi, N., Ideyama, Y., Kuromitsu, S., Yahiro, K.,Okada, M.: WO0202094809 (2002).

[143] Amino N, Ideyama Y, Yamano M, et al . YM-359445, an orallybioavailable vascular endothelial growth factor receptor-2tyrosine kinase inhibitor, has highly potent antitumor activityagainst established tumors. Clin Cancer Res 2006; 12: 1630-38.

[144] Bilodeau, M., Manley, P., Balitza, A., Rodman, L., Hartman, G.:WO0303011836 (2003).

[145] Fraley, M., Peckham, J., Arrington, K., Hoffman, W., Hartman,G.: WO030301183 (2003).

[146] Bilodeau, M., Hungate RW., Kendall, R., Rutledge, R., Thomas,K., Rubino, R., Fraley, M.: WO9854093 (1998).

[147] Bilodeau, M., Fraley, M., Hungate, R.: WO0053605(2000).[148] Bilodeau, M., Hungate, R., Cunningham, A., Koester, T.:

WO9916755 (1999).[149] Bilodeau, M., Hungate RW., Cunningham, A., Koester, T.:

WO00012089 (2000).[150] Arrington, K., Fraley, M., Hanney, B., Kim, Y., Spencer, K.:

WO03024969 (2003).[151] Fraley, M., Hambaugh, S., Hungate RW.: WO010128993(2001).[152] Fraley, M., Hartman, G., Hungate RW.: WO010162252 (2001).[153] Peckham, J., Hoffman, W., Arrington, K., Fraley, M., Hartman,

G., Kim, Y., Hanney, B., Spencer, K.: WO03020699 (2003).[154] Kim, Y., Hanney, B., SPencer, K., Hartman, G., Arrington, K.:

WO03020276 (2003).[155] Hartman, G., Tucker, T., Sisko, J., Smith, A., Lumma, W.,

WO03015778 (2003).[156] Hartman, G., Tucker, T., Sisko, J., Smith, A., Lumma, W.:

WO03015717 (2003).[157] Bilodeau, M., Hungate RW., Rodman, L., Hartman, G., Manley,

P.: WO0117995 (2001).[158] New 2-amino-(5-cyanothiazol-2-yl) pyridines: KDR inhibitors

with improved pharmacokinetic profile. Expert Opin TherPatents 2003; 1305-1312.

[159] Bilodeau, M., Hartman, G.: WO03000687(2003).[160] Ki, S., Payack J., Treemaneekarn V., Wang, Y., Sato, Y.:

WO03088900 (2003).[161] Paruch, K., Guzi, T., Dwyer, M., Shipps, G.: US20060094706

(2006).[162] Read M, Stevenson M, Farrow P, et al . RNA-based therapeutic

strategies for cancer. Expert Opin Ther Patents 2003; 13(5): 627-38.

[163] Tafech A, Bassett T, Sparanese D, Lee CH. Destroying RNA as atherapeutic approach. Curr Med Chem 2006; 13: 863-81.

[164] Hannon GJ. RNA interference. Nature 2002; 418(6894): 244-51.[165] Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic

interference by double-stranded RNA in Caenorhabditis elegans.Nature 1998; 391: 806-11.




[166] Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in culturedmammalian cells. Nature 2001; 411: 494-8.

[167] Murata M, Takanami T, Shimizu S, et al. Inhibition of ocularangiogenesis by diced small interfering RNAs (siRNAs) specificto vascular endothelial growth factor (VEGF). Curr Eye Res2006; 31: 171-80.

[168] Reich SJ, Fosnot J, Kuroki A, et al. Small interfering RNA(siRNA) targeting VEGF effectively inhibits ocularneovascularization in a mouse model. Mol Vis 2003; 9: 210-16.

[169] Vargeese, C., Shaffer, C., Wang, W.: US20060036090 (2006).

[170] Pavco, P., Mcswiggen, J., Stinchcomb, D., Escobedo, J.:US20046818447 (2004).

[171] Jadhav V., Kossen, K., Zinnen, S., Vaish, N., McSwiggen, J.:US20050233998 (2005).

[172] McSwiggen, J., Beigelman, L., and Pavco, P.: US20050171039(2005).

[173] Shen J, Samul R, Silva RL, Akiyama H, et al. Suppression of ocular neovascularization with siRNA targeting VEGF receptor1. Gene Ther 2006; 13: 225-34.

[174] Mulkeen AL, Silva T, Yoo PS, et al. Short interfering RNA-mediated gene silencing of vascular endothelial growth factor:effects on cellular proliferation in colon cancer cells. Arch Surg2006; 141: 367-74.

[175] Yan RL, Qian XH, Xin XY, et al. Experimental study of anti-VEGF hairpin ribozyme gene inhibiting expression of VEGF andproliferation of ovarian cancer cells. Ai Zheng 2002; 21: 39-44.

[176] Pavco PA, Bouhana KS, Gallegos AM, et al. Antitumor andantimetastatic activity of ribozymes targeting the messengerRNA of vascular endothelial growth factor receptors. ClinCancer Res 2000; 6: 2094-103.

[177] Stinchcomb, D., Draper, K., McSwiggen, J.: US20030003469(2003).

[178] Thompson, J.: US20020192685 (2002).[179] Klimuk, S., Scherrer, P., Hope, K., Zhang, Y., Reynolds, M.,

Min, J., Semple, S.: WO9904819 (1999).[180] Beigelman, L., Matulic-Adamic, J., Karpeisky, A., Haeberli, P.,

Sweedler, D., Reynolds, M., Chaudhary, N., Min, J.:WO9905094 (1999).

[181] Thompson, J., Draper, K.: US20026492512 (2002).*[182] Pavco, P., McSwiggen, J., Stinchcomb, D., Escobedo, J.:

US20026346398 (2002).[183] Weng DE, Masci PA, Radka SF, et al. A phase I clinical trial of a

ribozyme-based angiogenesis inhibitor targeting vascularendothelial growth factor receptor-1 for patients with refractorysolid tumors. Mol Cancer Ther 2005; 4: 948-55.

[184] Kobayashi H, Eckhardt SG, Lockridge JA, et al. Safety andpharmacokinetic study of RPI.4610 (ANGIOZYME), an anti-VEGFR-1 ribozyme, in combination with carboplatin andpaclitaxel in patients with advanced solid tumors. CancerChemother Pharmacol 2005; 56: 329-36.

[185] de Castro Junior G, Puglisi F, de Azambuja E, et al.Angiogenesis and cancer: A cross-talk between basic science andclinical trials (the "do ut des" paradigm). Crit Rev OncolHematol 2006; 59: 40-50.

[186] Glade-Bender J, Kandel JJ, Yamashiro DJ. VEGF blockingtherapy in the treatment of cancer. Expert Opin Biol Ther 2003;3: 263-76.

[187] Ziche M, Donnini S, Morbidelli L. Development of new drugs inangiogenesis. Curr Drug Targets 2004; 5: 485-93.

[188] Miller KD, Chap LI, Holmes FA, et al. Randomized phase IIItrial of capecitabine compared with bevacizumab pluscapecitabine in patients with previously treated metastatic breastcancer. J Clin Oncol 2005; 23: 792-99.

[189] Chen X. Multimodality imaging of tumor integrin αvβ3

expression. Mini Rev Med Chem 2006; 6: 227-34.[190] Wu Y, Zhang X, Xiong Z, et al. microPET imaging of glioma

integrin αvβ3 expression using (64)Cu-labeled tetrameric RGDpeptide. J Nucl Med 2005; 46: 1707-18.

[191] Zhang X, Xiong Z, Wu Y, et al. Quantitative PET Imaging of Tumor Integrin αvβ3 Expression with 18F-FRGD2. J Nucl Med

2006; 47: 113-21.[192] Wu Y, Cai W, Chen X. Near-Infrared Fluorescence Imaging of

Tumor Integrin αvβ3 Expression with Cy7-Labeled RGDMultimers. Mol Imaging Biol 2006; 8: 226-36.

[193] Zhu AX, Blaszkowsky LS, Ryan DP, et al. Phase II study of gemcitabine and oxaliplatin in combination with bevacizumab inpatients with advanced hepatocellular carcinoma. J Clin Oncol2006; 24: 1898-903.

[194] Wainberg Z, Hecht JR. A phase III randomized, open-label,controlled trial of chemotherapy and bevacizumab with orwithout panitumumab in the first-line treatment of patients withmetastatic colorectal cancer. Clin Colorectal Cancer 2006; 5:363-67.

[195] Normanno N, Gullick WJ. Epidermal growth factor receptortyrosine kinase inhibitors and bone metastases: differentmechanisms of action for a novel therapeutic application? EndocrRelat Cancer 2006; 13: 3-6.

[196] Lee KS, Min KH, Kim SR, et al . Vascular Endothelial GrowthFactor Modulates Matrix Metalloproteinase-9 Expression inAsthma. Am J Respir Crit Care Med 2006; 174: 161-70.

[197] Kaplan RN, Riba RD, Zacharoulis S, et al. VEGFR1-positivehaematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 2005; 438: 820-27.

[198] Eriksson U, Alitalo K. VEGF receptor 1 stimulates stem-cellrecruitment and new hope for angiogenesis therapies. Nat Med2002; 8: 775-77.

[199] Brooks PC, Montgomery AM, Rosenfeld M, et al. Integrin αvβ3

antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels. Cell 1994; 79: 1157-64.

[200] Enenstein J, Kramer RH. Confocal microscopic analysis of integrin expression on the microvasculature and its sprouts in theneonatal foreskin. J Invest Dermatol 1994; 103: 381-86.

[201] Enenstein J, Waleh NS, Kramer RH. Basic FGF and TGF-betadifferentially modulate integrin expression of humanmicrovascular endothelial cells. Exp Cell Res 1992; 203: 499-

503.[202] Sepp NT, Li LJ, Lee KH, et al. Basic fibroblast growth factor

increases expression of the αvβ3 integrin complex on humanmicrovascular endothelial cells. J Invest Dermatol 1994; 103:295-99.

[203] Friedlander M, Brooks PC, Shaffer RW, et al. Definition of twoangiogenic pathways by distinct alpha v integrins. Science 1995;270: 1500-02.

[204] Burke PA, DeNardo SJ, Miers LA, et al. Cilengitide targeting of αvβ3 integrin receptor synergizes with radioimmuno-therapy toincrease efficacy and apoptosis in breast cancer xenografts.Cancer Res 2002; 62: 4263-72.

[205] Raguse JD, Gath HJ, Bier J, et al. Cilengitide (EMD 121974)arrests the growth of a heavily pretreated highly vascularisedhead and neck tumour. Oral Oncol 2004; 40: 228-30.

vegg as angiogenic recepts

Documents