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

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.

Full text

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 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,*
1Department 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 deve-
lopment, angiogenesis, vascular permeability, tumor progres-
sion 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 perme-
ability 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, VEGF-
E, 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, VEGF145, VEGF165, VEGF183, VEGF189, VEGF206)
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
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

60 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 Chen et al.
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 in-
utero [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 ligand-
dependent tyrosine phosphorylation. The major phosphory-
lation 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 disulfide-
linked 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 tumor’s 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 up-
regulated on vascular and lymphatic endothelial cells during
tumor angiogenesis in particular [29-32]. Thus anti-angio-
genic 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 Köhler 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

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), 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, pharmaco-
kinetic, 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 VEGF-
induced 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 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 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 naïve 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 VEGF-
Trap 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 Wilm’s 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 (RTK’s) have typically
been classified into families based on structural features of
their respective extracellular domains. While the extracel-
lular portion of each receptor expresses unique ligand
binding, the intracellular portion of RTK’s are generally
architecturally very similar. Furthermore, favorable pharma-
cokinetic 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 (IC50’s 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

62 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 Chen et al.
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)-6-
methoxy-7-[(1-methylpiperidin-4-yl)methoxy]quinazolin-4-
amine), was formed by incorporating a basic nitrogen in the
C7 side chain (2) [75]. Hennequin et al. showed that anilino-
quinazolines 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 charac-
teristics 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]-6-
methoxy-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 H-
bond 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-methy-
lation 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-(aryl-
amino) 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, Phar-
macia announced that they would end clinical development
of 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 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-β 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 = 2.1µM) 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-diethylamino-
ethylamido 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-β phosphorylation with IC50 = 10 nM for both
RTKs. It was also shown that SU11248 inhibited VEGF-
induced 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]-phthala-
zine 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 autophosphory-
lation assay (IC50 = 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

VEGF and VEGFR Inhibitors for Cancer Treatment Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 63
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 trans-
form 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 αC helix, and the backbone of the aspartate of the Asp-
Phe-Gly (DFG) motif [104]. AAL993 has been shown to
inhibit VEGF-induced proliferation of HUVECs (IC50 = 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 [116-
118], 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 AG’s patents included pyridylethyl analogues,
pyridylethylene and pyridylethyne derivatives with aryl or
hetercyclic amide substitutions. Schering’s discovery that
cyclic 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
N
NNN
N
HN
FMeO
O
Cl
N
N
N
O
N
FMeO
ON
H
N
H
N
CO2H
O
H
N
H
N
O
H
N
H
N
HN
O
F
O
NEt2
1
ZD4190 2
ZD6474
3
AZD2171
4
SU5416
5
SU6668 6
SU11248

64 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 Chen et al.
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 six-
membered 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 inhibi-
tion. 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 phos-
phorylation resulting in decreased vascular density and
tumor growth [130]. Phase I clinical studies revealed effica-
cious 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-
Fig. (2). Phthalazines & Anthranilamides.
N
N
HN
N
Cl
O
O
HO
OH
NH
HN
N
CF3
ONH
HN
N
N
O
NH
HN
N
H
O
O
CF3
NH
HN
N
N
H
O
O CF3
O
O-
NH
HN
N
N
O
N
O
O
H
ONH
HN
N
N
H
7
PTK787
8
AAL993
9
10
ABP309
12
11
13

VEGF and VEGFR Inhibitors for Cancer Treatment Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 65
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]. Indeno-
pyrrolocarbazole 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
Cephalon’s 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). Pharma-
cokinetic 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 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). 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 µM
[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 pyrazolo-
pyrimidines 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 anti-
angiogenic 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 nonspe-
cific 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 signifi-
cant 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 modifi-
cation, delivery, and production [177-181]. Their signature
anti-angiogenic product is an anti-Flt-1 ribozyme known as
Angiozyme [182]. Preclinical testing revealed strong

66 Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 Chen et al.
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 conti-
nued 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), 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 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 anti-
angiogenic 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
Fig. (3). Isothiazoles, Pyrroloindolocarbazoles & 2-Amino-(thiazol-2-yl) pyridines.
F
FBr
NS
H2N
NH
NN
O
O
H
N
H
N
OH
O
O
N
H
N
O
O
O
N
O
N
H
N
HO
N
N
NO
S
N
HO2C
CO2HHO
OH
N
N
HN S
N
R1
N
N
N
R2R3
HN S
N
R3
N
R2N
N
R4
N
R4
NO
HN
N
H
R5
H
NNH
O
N
N
S
O O
14
CP-547632 15
CEP-5214
16
CEP-7055
17
18
:
R1 = alkyl, O-alkyl, halogen, OH
R4 = SO2-alkyl, CONH-alkyl
R5 = NHCONH-alkyl
19
20

VEGF and VEGFR Inhibitors for Cancer Treatment Recent Patents on Anti-Cancer Drug Discovery, 2007, Vol. 2, No. 1 67
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 combina-
torial 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 anti-
angiogenic 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 angio-
genesis, 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, integrin-
αvβ3, has been shown to be up-regulated on cytokine-
activated vascular endothelial cells, smooth muscle cells, and
blood vessels in tumor, wound, and granulation tissue [199-
203]. MEDI-522, a mAb against human αvβ3 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 αvβ3 called EMD-121974 or Cilengitide. Phase II clinical
trials have revealed positive results for suppressing tumor
vascularization and growth [204,205]. Several other candi-
dates are also in various phases of development; these
include 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 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 multiface-
ted approach, targeting different aspects of tumor develop-
ment. 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.
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