anti-angiogenic drugs: from bench to clinical trials

Anti-Angiogenic Drugs:from Bench to ClinicalTrials

Ana R. Quesada,1 Ramon Munoz-Chapuli,2 Miguel A. Medina1

1Department of Molecular Biology and Biochemistry, Faculty of Science, University of Malaga,

29071 Malaga, Spain2Department of Animal Biology, Faculty of Science, University of Malaga,

29071 Malaga, Spain

Published online 1 May 2006 in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/med.20059

!

Abstract: Angiogenesis, the generation of new capillaries through a process of pre-existing

microvessel sprouting, is under stringent control and normally occurs only during embryonic and

post-embryonic development, reproductive cycle, and wound repair. However, in many

pathological conditions (solid tumor progression, metastasis, diabetic retinopathy, hemangioma,

arthritis, psoriasis and atherosclerosis among others), the disease appears to be associated with

persistent upregulated angiogenesis. The development of specific anti-angiogenic agents arises as

an attractive therapeutic approach for the treatment of cancer and other angiogenesis-dependent

diseases. The formation of new blood vessels is a complex multi-step process. Endothelial cells

resting in the parent vessels are activated by an angiogenic signal and stimulated to synthesize

and release degradative enzymes allowing endothelial cells to migrate, proliferate and finally

differentiate to give rise to capillary tubules. Any of these steps may be a potential target for

pharmacological intervention. In spite of the disappointing results obtained initially in clinical

trials with anti-angiogenic drugs, recent reports with positive results in phases II and III trials

encourage expectations in their therapeutic potential. This review discusses the current approaches

for the discovery of new compounds that inhibit angiogenesis, with emphasis on the clinical

developmental status of anti-angiogenic drugs. � 2006 Wiley Periodicals, Inc. Med Res Rev, 26, No. 4,

483–530, 2006

Key words: angiogenesis; inhibitors; cancer; macular degeneration; clinical trials

1 . I N T R O D U C T I O N T O A N T I - A N G I O G E N I C T H E R A P Y

Angiogenesis, the formation of new blood vessels from the existing vascular bed, has been described

as one of the hallmarks of cancer, playing an essential role in tumor growth, invasion, andmetastasis.

Correspondence to: Dr. Ana R.Quesada, Department of Molecular Biology and Biochemistry, Faculty of Science, University of

Ma¤ laga,E-29071Ma¤ laga,Spain.E-mail: [email protected]

Medicinal Research Reviews, Vol. 26, No. 4, 483^530, 2006

� 2006 Wiley Periodicals, Inc.

Furthermore, many other diseases are also dependent on upregulated angiogenesis. When resting

endothelial cells are activated by an angiogenic signal, they are stimulated to release degrading

enzymes allowing endothelial cells to migrate, proliferate, and finally differentiate to form new

vessels. Figure 1 is a scheme of the angiogenic process.Anyof the steps involved in angiogenesismay

be a potential target for pharmacological intervention of angiogenesis-dependent diseases. This is the

main reasonwhy angiogenesis has attracted recent attention in the field of pharmacological research.

Anti-angiogenic therapy, directed to activated endothelial cells, presents a series of advantages

when compared to traditional therapies, mainly targeted to tumor cells. First, endothelial cells are

homogeneous, diploid, genetically stable targets. Spontaneous mutations occur very rarely in them.

This is in contrast to the high genetic instability, heterogeneity, and mutation rate of tumor cells,

which favors the selection of subpopulations that become resistant to the anti-tumoral drugs, resulting

in the appearance of chemotherapy resistance phenomena. Second, the turnover of endothelial cells

in tumoral tissues may be 50-fold higher than that of quiescent endothelia in normal tissues. The

activated endothelium overexpresses specific markers, such as integrin avb3, E-selectin, Tie, andvascular endothelial growth factor (VEGF) receptors that could be used for the development of

specific therapies directed to these cells. On the other hand, the contact between endothelial cells and

blood makes this target easily accessible to systemically administered drugs, therefore avoiding the

problem of low penetration of the anti-tumoral drugs into the solid tumors.1 Finally, due to the fact

that a singlevascular netmay support the growth of different populations of tumor cells, the inhibition

of the vascular growth may affect the survival of many tumor cells.2

There are different strategies for angiogenesis intervention, based on the modulation of any of

the different steps of the angiogenic process (Fig. 2). Therefore, some angiogenesis modulators may

Figure 1. Scheme of the steps of the angiogenic process. An activating, proangiogenic signal1elicits the switching on of the

angiogenic phenotype of resting endothelial cells, which become activated.2These activated cells acquire an invasive potential,

involving basal membrane degradation,3extracellular matrix remodeling, proliferation, and migration.

4Finally, morphogenesis

contributes to thealignmentofendothelial cells forminganewmicrovessel, and this is stabilizedbyeventual recruitmentofpericytes

andacquisitionofanewbasalmembrane.5

484 * QUESADA, MUN‹ OZ-CHA¤ PULI, AND MEDINA

act on the angiogenic signal, or on one or several specific functions of activated endothelial cells

(proliferation, adhesion to extracellular matrix, protease production, migration, invasion or

differentiation). In this article, the main angiogenesis inhibitors will be classified according to the

step of angiogenesis they affect.

Figure 2. Thereare different strategies to inhibitangiogenesis, basedoneffectiveandspecificblockageofanyof the different stepsof the angiogenic process. Accordingly, anti-angiogenic drugs can be classified in the several groups described in the present

work, includingdrugs inhibiting the angiogenic factor (AF) andone or several functions ofactivated endothelial cells (proliferation,

adhesion, proteaseproduction, migration, invasion, anddifferentiation).

ANTI-ANGIOGENIC DRUGS * 485

2 . A G E N T S T H A T I N T E R F E R E W I T H T H E A N G I O G E N I C S I G N A L

Among the growth factors that are involved in the angiogenesis process, VEGF and basic fibroblast

growth factor (bFGF) seem to play the most relevant roles. For this reason, a number of therapeutic

strategies are aimed at neutralizing the activation of endothelial cells by these factors. The following

is a brief summary of such studies.

A. Agents Acting on the Angiogenic Factor

The production of neutralizing antibodies anti-VEGF stands out. One specific and prominent

example is the humanized recombinant monoclonal antibody rhu Anti-VEGF Mab (Bevacizumab,

Avastin, developed by Genentech, South San Francisco, CA; www.genentech.com). This drug was

generated by engineering the VEGF-binding residues of a murine neutralizing antibody into the

framework of a normal human immunoglobulin G (IgG). The humanization provides a longer half-

life and less immunogenicity. Bevacizumab neutralizes the biologically active forms of VEGF that

interact with VEGF receptors 1 and 2 (VEGFR1 andVEGFR2), which results in the neutralization of

the biological properties of human VEGF, including endothelial cell mitogenic activity, vascular-

permeability-enhancing activity, and angiogenic properties.3 The anti-tumor activity of this antibody

has been extensively demonstrated in pre-clinical studies in several tumor models. These studies

found that single-agent therapy with bevacizumab or its murine equivalent (A4.6.1) resulted in tumor

growth inhibition of a number of human tumor cell lines xenografted in rodentmodels, irrespective of

the route of administration or tumor location. The assayed tumor types included rhabdomyosarcoma,

glioblastoma, leiomyosarcoma, colon adenocarcinoma, hepatoblastoma, neuroblastoma, Wilms’

tumor, and ovarian, prostate or breast carcinomas.4 Studies with in vitro cell lines did not reveal a

direct effect of the antibody on the growth of tumor cells, suggesting that tumor suppression is

mediated through inhibition of neovascularization.5 Several studies have shown that anti-VEGF

therapy also prevented the growth of tumor cells at metastatic sites.4

Clinical data obtained to date indicate that most patients tolerated bevacizumab well when

administered alone or in combination with the traditional chemotherapy, although serious adverse

events have been observed. Side effects can include high blood pressure, nosebleeds, fever, headache,

rash, and chills. Occasionally, bevacizumab treatment could be associated with blood clots and

bleeding,which could be life threatening. From a safety point of view, themechanismof bleeding and

thrombosis associated with bevacizumab treatment remains to be clarified.6 Bevacizumab is being

evaluated in combination with standard therapies in phases II and III clinical trials of colorectal

cancer (CRC), renal cell carcinoma (RCC), breast cancer, Kaposi’s sarcoma, pancreatic cancer,

ovarian cancer, lymphoma, myeloma, melanoma, head and neck cancer, leukemia, liver cancer,

sarcoma, mesothelioma, and lung cancer. The most encouraging results have been obtained in the

treatment of metastatic CRC, RCC, breast cancer, and non-small cell lung cancer (NSCLC) patients.

The possibility of using bevacizumab in combination with chemo or radiotherapy seems very

promising,7 as suggested by results obtained in the treatment of advanced colon cancer.8 In a

randomized, open-label phase II assay, carried out with 104 metastatic cancer patients, subjects with

previously untreated metastatic cancer, were treated with a combination of bevacizumab and the

traditional chemotherapy (5-FU/LV). The results indicate that this combination therapy was clearly

superior to those obtainedwith chemotherapy alone. The first study showing phase III data validating

an anti-angiogenesis strategy for treating human cancers was presented in a communication that

received a lot of attention in the 2003American Society of Clinical Oncology (ASCO)Meeting.9 The

authors found that the bevacizumab/IFL (irinotecan/5FU/LV) combination led to significantly

prolonged survival. It also had a better ability to shrink tumors than standard IFL alone, without

statistically significant increases in adverse events in patients with metastatic colorectal cancer. The

results were based on 412 patients in the IFL/placebo arm and 403 patients in the IFL/bevacizumab


arm. The presence of bevacizumab in the treatment produced remarkable and statistically very

significant increases in all the four survival and response parameters determined: median survival,

progression-free survival, objective response, and duration of response.10 Based on these data,

bevacizumab received the US Food and Drug Administration (FDA) approval in 2004 for use in

combination with fluorouracil-based chemotherapy as first-line treatment for metastatic cancer of

the colon or rectum. Bevacizumab has also received a positive recommendation from the European

Committee for Medicinal Products for Human Use in 2004. In early 2005, preliminary results

from a large, randomized clinical trial for patients with advanced colorectal cancer who had

previously received treatment were made public. Those results indicate that bevacizumab

administered in combination with oxaliplatin-based chemotherapy (FOLFOX4) is well tolerated

and increases survival in advanced CRC patients previously treated with irinotecan and a

fluoropyrimidine.11,12

Bevacizumab has also been recently tested in metastatic renal cancer, a tumor with high

expression of VEGF resulting from inactivation of the von Hippel-Lindau (VHL) tumor suppressor

gene.13 In a randomized, double-blind, phase II trial therewas a significant prolongation of the time to

progression of disease in the high-dose-antibody group as compared with the placebo group

(4.8 months vs. 2.5 months).14 Recent studies in combination with the standard treatment, interferon

(IFN), or with erlotinib, a low molecular weight inhibitor of the endothelial growth factor receptor

(EGFR) tyrosin kinase are generating promising data in RCC treatment. In a phase II trial including

58 patients with recurrent RCC that underwent nephrectomy, 87% of patients treated with erlotinib/

bevacizumab achieved a clinical benefit, with 21% of patients achieving at least a 50% reduction in

their cancer size and 66% showing either a disease stabilization of aminor anti-cancer response. Only

13% of patients had a progression of their cancer while on treatment. One year later, 81% of patients

were still alive. These rates are some of the highest rates ever achieved in patients with this stage of

RCC. Side effects caused by treatment were generally mild, with the most common side effects

being high blood pressure, diarrhea, rash, nausea, and vomiting.15,16 An Intergroup phase III trial

investigating the addition of bevacizumab to initial systemic therapy in RCC is being evaluated to

see whether the addition of anti-VEGF therapy to IFN alpha would prolong survival in untreated

metastatic RCC patients.17

A randomized phase III trial comparing the efficacy and safety of capecitabine with or without

bevacizumab, in patients withmetastatic breast cancer previously treatedwith an anthracycline and a

taxane failed to show improvement in survival in bevacizumab treated patients.18 However, in April

2005 a press release from Genentech reported an interim analysis of a phase III study in first-line

metastatic breast cancer, showing bevacizumab plus paclitaxel improved progression-free survival

when compared with chemotherapy alone. This trial enrolled 722 women with previously untreated

metastatic breast cancer, who were randomized to receive paclitaxel with or without bevacizumab.

An additional ‘late-breaking’ session was created in the 41st Annual Meeting of the American

Society of Clinical Oncology (Orlando, FL, May 2005) to accommodate the presentation of these

results. Data presented at ASCO 2005, based on 355 events observed, showed that progression-free

survival (primary endpoint) was 6.11 months for paclitaxel and 10.97 months for the combination

arm, for a hazard ratio of 0.498 (95% CI, 0.401–0.618), and a P-value of <0.001. A trend for

improvement in overall survival was also noted, with a hazard ratio of 0.674 (95%CI, 0.495–0.917),

P ¼ 0.01. In terms of toxicity, both regimens were well tolerated. This is the first time an anti-

angiogenic agent has been found to be successful in metastatic breast cancer.19

In the same way, promising results have been recently reported for the treatment of lung cancer

patients with bevacizumab in combination with standard chemotherapy or erlotinib.20,21 A large,

randomized phase II/III clinical trial sponsored by the USNational Cancer Institute (NCI) focuses in

patients with previously untreated advanced non-squamous, NSCLC. Preliminary results from this

trial show that adding bevacizumab to paclitaxel and carbiplatin compared with paclitaxel/

carboplatin by itself improved survival 2 months.22


The use of soluble receptors is a different strategy to sequester the angiogenic factor. The

production of a soluble form of the receptor VEGFR1 by human endothelial and tumor cells has been

described, as well as its presence in plasma and serum from healthy donors.23 These observations

suggest that these soluble receptors may play a physiological role in the regulation of the biological

activity of VEGF. As an application of this principle to the pharmacological modulation of

angiogenesis, it has been reported that the use of extracellular domains of VEGFR1, VEGFR2, and

Tie-2 potently inhibited corneal neovascularization induced by tumor cell conditioned media, and

reduced mammary tumor growth and vascularization when they were administered into a rat

cutaneous tumor window chamber.24,25 Based on the results obtained with cancer cells infected with

adenovirus vectors encoding soluble VEGFR1, it has been suggested that anti-angiogenic gene

therapy using soluble VEGFR1 might be an effective approach for pancreatic or lung cancer

treatment. The tumor growth of the immuno-depressedmice thatwere inoculatedwith the genetically

modified cancer cells was significantly suppressed. The microvessel density in this group was also

significantly lower.26,27 Moreover, when adenovirus expressing the entire extracellular domain of

VEGFR1 was injected into the mice skeletal muscle and uninfected lung cancer cells were injected

s.c., the soluble receptor was detectable in the circulating blood. This was accompanied by the

cessation of tumor growth, and later a decrease of tumor size. Histological examination of the animal

models revealed that intratumor angiogenesis was markedly suppressed, and apoptosis was

enhanced. These results suggest that adenovirus-mediated overexpression of a soluble VEGF

receptor in a remote organ may have the potential to be an effective strategy for cancer treatment.27

The success of this experimental strategy can be helped by the possible synergy between VEGF and

bFGF soluble receptors, as well as their possible production in remote organs, therefore maintaining

their anti-angiogenic activity.27,28 Soluble forms of VEGFR1 suffer from poor pharmacokinetic

properties, which seem to correlate with their non-specific interactions with extracellular matrix.

By determining the requirements to maintain high affinity while extending in vivo half-life, a

very strong high-affinity VEGF blocker that has markedly enhanced pharmacokinetic properties

has been engineered. The VEGF-Trap (Aventis and Regeneron Pharmaceuticals, Tarrytown, NY

www.regeneron.com) is a derivative of VEGFR1, comprising portions of the extracellular domains

of both VEGFR1 and VEGFR2 fused to an Fc segment of IgG and therefore minimizing the

possibility of being immunogenic in humans. The combination of high affinity and improved

pharmacokinetics apparently contributes toward making VEGF-Trap one of the most potent and

efficacious VEGF-blockers available, showing picomolar affinity for both mouse and human

VEGF.29 Systemic administration of VEGF-Trap dramatically inhibited the growth and

vascularization of a variety of tumor cell lines, including mouse melanoma, human rhabdomyo-

sarcoma, and rat glioma.29 In a xenografted model of neuroblastoma, treatment with high-dose

VEGF-Trap resulted in stunted tumors, which were virtually avascular and in which only rare co-

opted glomeruli were present, suggesting that the co-opted vasculature had regressed.30 Remarkably,

VEGF blockade resulted in dramatic remodeling of the blood vessels in disseminated ovarian

carcinoma, prevented ascites accumulation, and inhibited the growth of disseminated cancer.31 Some

data suggest that blockade of VEGF may not only impede cancer progression, presumably by

preventing neoangiogenesis, but could also produce tumor regression. It has been reported that

high-affinity blockade of VEGF, using the VEGF-Trap, abolishes mature, pre-existing vasculature

in established xenografts. Eradication of vasculature is followed by marked tumor regression,

including that of lung micrometastases. These results support the importance of VEGF as a target in

cancer therapy and suggest that anti-VEGF strategies may not only halt tumor growth but also

produce actual regression. Such results also suggest that potent blockade ofVEGFmay be effective in

the treatment of patients with metastatic, bulky cancers, as well as those with minimal residual

disease.32 On the other hand, VEGF-Trapmay also provide a new agent for treatment of patients with

choroidal neovascularization and diabetic macular edema, since VEGF plays a central role in the

development of both diseases.33 VEGF-Trap is being evaluated in phase I in patients with solid


tumors and non-Hodgkin’s lymphomas, and for the treatment of age-related macular degeneration

(AMD).34

A different approach for therapeutic inhibition of angiogenesis employs drugs that inhibit the

angiogenic factor production. Taking this into account, it has been reported that interferon alpha

(IFN-a) (Schering Corp., Kenilworth, NJ, www.schering.com) decreases the expression of bFGF,

very widespread in hemangiomas,35 and inhibits VEGF transcription through an Sp1-transcription

and/or Sp3-dependent inhibition of VEGF promoter activity.36 IFN-a has been generally well

tolerated by children suffering from severe hemangiomas, and has given positive results in the

treatment of hemangiomas and other childhood tumors, as well as in metastatic melanoma

patients.37–48 The combination of temozolomide/IFN proved quite tolerable in a phase II trial

evaluating their efficacy in patients with advanced renal cell carcinoma. This regime appeared

inactive in terms of response in this population with poor prognosis. It was more promising in those

patients with diseases remaining stable for more than 6 months.49 The use of aggressive supportive

measures and appropriate dose-reduction strategies may lessen the toxicity of high doses of IFN.50

IFN-a is being evaluated in phase III for renal cell carcinoma, squamous cell carcinoma of the head

and neck, and in phase II for unresectable carcinoid tumors, multiple myeloma, metastatic

melanoma, and high grade gliomas. There are more than 60 clinical studies with IFN-a under course.Since bFGF biological activity depends on extracellular heparin concentration, bFGF activity

can be modulated in vivo by the use of heparin analogs. Among others, suramin, a polysulphonated

naphtylurea first synthesized as an agent against trypanosomiasis and onchocerciasis, should be

mentioned. Suramin was found to inhibit the binding of a number of peptide growth factors such as

PDGF and bFGF to their receptors by binding to the growth factor and by inducing a conformational

change and therefore inhibiting their biological signal.51–53 Suramin interacts with VEGFR2 and is a

potent VEGF antagonist.54 In vitro, suramin inhibited multiple control points of angiogenesis,

including endothelial cell migration, proliferation, and proteolytic activities.55 In vivo, suramin

inhibited angiogenesis in a number of experimental models, including the chick chorioallantoic

membrane (CAM) assay, the rat corneal and rat aortic ring assays, and gelatin sponges loaded with

bFGF and implanted subcutaneously in mice.55–57 Suramin reduced tumor growth and tumor

induced angiogenesis in the murine M5076 reticulosarcoma model, a tumor producing significant

levels of bFGF.57 Suramin has been extensively evaluated over the last 15 years as an anti-cancer

agent, with the most interest being the treatment of prostate cancer. Early clinical results were

promising with response rates of up to 70% being reported. However, a recent double-blind study

showed only modest palliative effect in patients with androgen-independent prostate cancer. In

retrospect, it appears that those initial reports failed to control for confounding variables such as anti-

androgen withdrawal and hydrocortisone.58 The clinical use of suramin is being complicated by a

broad spectrum of toxic effects, neurotoxicity being the most significant complication and appearing

to be related to the intensity of the dosage.59–61 Although suramin clinical use is limited by the

appearance of the mentioned toxicities, an improvement in the treatment regime has produced

enhanced response with decreased toxicity in a phase II trial in 58 patients with hormone refractory

prostate cancer.62 Suramin is currently being evaluated in phase I in patients with NSCLC or bladder

cancer and in phase II in patients with NSCLC or breast cancer.

Suramin toxicity could be overcome by the synthesis of less toxic compounds that maintain

suramin anti-angiogenic properties.63–65 Suradista NSC 651016 is a distamicine sulfonated

derivative of suramin that is also effective at complexing and inactivating growth factors and

cytokines while remaining relatively non-toxic. NSC 651016 is more effective than suramin in

inhibiting mitogenesis in normal or transformed cells, as well as the growth of xenografts grown in

athymic nude mice.66 NSC 651016 acts as an inhibitor of stromal cell-derived factor 1 alpha

(CXCL12)-mediated angiogenesis in a variety of in vitro and in vivo angiogenesis assays, including

endothelial cell migration, capillary-like tube formation, aortic ring, and matrigel plug assay.67 NSC

651016 has been evaluated in phase I for solid tumors and in phase II for recurrent primary brain


tumors. The promising anti-angiogenic properties of NSC 651016 evaluated with serum bFGF as the

surrogate end point have not been achievable with i.v. administration in patients with solid tumors.68

Pentosan polysulfate (PPS) is a semi-synthetic pentasaccharide heparinoid derived from beech

wood shavings that has been used as anti-coagulant for more than 30 years. PPS is an effective

inhibitor of heparin-binding growth factors in vitro and can effectively inhibit the establishment and

growth of tumors in nude mice. Pentosan inhibited the growth of rat prostate adenocarcinoma when

treatment was started when the tumor was not palpable, but had little effect against established

tumors. PPS inhibited endothelial cell motility and tubule formation in vitro as well as capillary

formation in the CAM assay.69 PPS has been evaluated in phase I/II in advanced cancer and Kaposi’s

sarcoma, but its clinical application has been limited by the high anti-coagulant activity at doses

needed to inhibit the angiogenesis.70,71 A mild response has been reported in a phase II clinical trial

with patients with AIDS-related Kaposi’s sarcoma treated with PPS.72 New interest may arise from

its use in the treatment of inflammatory diseases and osteoarthritis.73,74

Finally, some attempts are being devoted to the development of chimerical molecules where

toxins are bound to VEGF or their receptors. Targeted toxins, developed by recombinant methods by

fusing VEGF165 or VEGF121 to the diphtheria toxin were found to be highly toxic to proliferating

endothelial cells but not to vascular smooth muscle cells. These fusion proteins completely inhibited

the bFGF-induced growth of new blood vessels in the CAM assay. Furthermore, the fusion toxin

substantially retarded the growth of Kaposi’s sarcoma tumors in mice.75 Dividing endothelial cells

overexpressing VEGFR2 have been reported to be approximately 60-fold more sensitive to a fusion

protein containing VEGF121 linked to the toxin gelonin. Endothelial cells overexpressing VEGFR1

were not sensitive to this fusion protein. Treatment of humanmelanomaor human prostate xenografts

with the fusion construct demonstrated a reduction in tumor volume and caused thrombotic damage

to tumor vessels with extravasation of red blood cells in the tumor bed.76 Whereas these preliminary

studies show a potential use of fusion protein in a variety of disorders in which angiogenesis plays a

role, certain limitations are inherent to the use of bacterial proteins due to the generation of antibodies

and the appearance of non-targeted toxicities. The development of less immunogenic immunotoxins

could permit repetitive dosing, improving their therapeutic performance.77

B. Agents Acting on the Receptor of the Angiogenic Factor

Some authors think that inhibiting VEGF receptors may be more effective than blocking their

ligands, sincemany cellular types can express VEGF, and this factor can be stored in the extracellular

matrix and in the tissular interstitial spaces. VEGF levels may therefore be very variable in space and

time, and this makes its neutralization difficult. On the contrary, the number of VEGF receptors in the

activated endothelial cells is relatively low, and, at least in theory, more easily saturable, since less

antagonist molecules would be needed to block all the receptors.78

Away to inhibit the expression of a receptor is by means of a specific ribozyme. A ribozyme is a

catalytic RNA able to hydrolyze a given RNA. Its specificity is derived from the matching between

the ribozyme binding arms, and the sequences flanking the cleavage site in the target RNA.

Ribozymes for clinical use are designed to resist in tissues and biological fluids that are rich in

nucleases. They have an adequate pharmacokinetic profile and have demonstrated their effectiveness

in in vitro and in vivo pre-clinical studies.79 Significant inhibition of angiogenesis using ribozymes

against both VEGFR1 and VEGFR2 has been reported. In animal tumor models, anti-tumor effects

were most pronounced with the anti-VEGFR1 ribozyme known as Angiozyme (Ribozyme

Pharmaceuticals, Boulder, CO, www.rpi.com). Angiozyme has demonstrated anti-angiogenic

effects in a rat cornea model of VEGF-induced angiogenesis and anti-tumor and anti-metastatic

activity in several murine tumor models.80–82 Extensive pre-clinical studies in mice and monkeys

have demonstrated no significant toxicities of angiozyme.83,84 Angiozyme has beenwell tolerated by

humans in phase I clinical studies, and has been evaluated in phase II trials in advanced and/or


metastatic cancer patients alone or in combination with traditional chemotherapy.85–87 However,

regardless of the phase II trial results, the company has announced that it does not intend to develop

angiozyme independently. This compound might be licensed to a third party partner, if found.

In a less advanced stage of development, we could mention the possible use of peptides to

interfere the binding site of VEGF and its receptor. These studies will throw light on the interactions

of growth factor and their receptor.88,89

Another experimental strategy to pharmacologic intervention of angiogenesis consists of

receptor blockade by antibodies generated against the receptor extracellular domain. The use of

monoclonal antibodies generated against the different VEGF receptors inhibits tumor angiogenesis

and human tumor growth in immunodepressed mice.3,90–92 The humanized anti-VEGFR2 IMC-

1C11 antibodies (Imclone, New York, NY, www.imclone.com) bind specifically to the endothelial

cell surface extracellular domain of VEGFR2, blocks its interaction with VEGF, and prevents VEGF

activation of the intracellular tyrosine kinase pathway.93 IMC-1C11 inhibits VEGF-stimulated

proliferation of human endothelial cells and migration of VEGFR2þ leukemia cells in vitro. When

administered in vivo, these antibodies significantly prolonged survival of NOD-SCID mice

inoculated with VEGFR2þ human leukemia cells. IMC-1C11 has been well tolerated in phase I in

patients suffering from colorectal carcinomawith livermetastasis, but no objective tumor regressions

were observed.92,94 IMC-1121B is a fully human monoclonal antibody that targets VEGFR2,

produced as a second-generation agent, which is expected to be non-immunogenic for chronic

administration as a single agent and in combination with chemotherapy or radiation. IMC-1121B

binds to VEGFR2 with high affinity and efficiently blocks its interaction with VEGF. IMC-1121B

also inhibits VEGF-stimulated VEGFR2 activation and endothelial cell migration and prolifera-

tion.95,96 In January 2005, Im Clone Systems has initiated a phase I clinical trial of IMC-1121B in

patients with solid tumors.

The angiogenesis inhibitory activities of antibodies anti-VEGFR1 and anti-VEGFR3 are

currently being studied, but they have not reached the clinical phases of development yet.97 A

bispecific antibody, a diabody, using the variable domain genes of IMC-1121B (specific to VEGFR2)

and hF4-3C5 (specific to VEGFR3) as the ‘‘building blocks,’’ has recently been constructed. The

diabody binds to both VEGFR2 and VEGFR3 in a dose-dependent manner and blocks interaction

between these receptors and VEGF. In cell-based assays, the diabody neutralized both VEGF-

stimulated endothelial cell migration and activation of the receptors and p42/p44 mitogen-activated

protein kinases (MAPK) in endothelial cells. These results suggest that a simultaneous dual blockade

of both VEGFR2 and VEGFR3 may represent a more potent approach to effective cancer therapy.98

C. Agents Interfering the Signaling Pathway of the Angiogenic Factor

As mentioned above, there is no doubt about the main role played by VEGF and its receptors in the

angiogenic process. For this reason, a number of efforts have been devoted to the search of drugs that

are able to inhibit the VEGF receptors signaling pathways, and in particular their autophos-

phorylation.

Among the compounds that inhibit VEGFR2we can remark SU5416 and SU6668 (Fig. 3), which

have received a high clinical interest. Both compounds compete with ATP for the receptor tyrosine

kinase domain.99 In addition, SU5416 (Semaxanib, SUGEN/Pharmacia, South San Francisco, CA,

www.sugen.com) inhibits tyrosine kinase activity of PDGF receptors, VEGFR1, VEGFR3, and c-

kit.78 The first results obtainedwith this compound in vitro and in vivowere impressive, showing anti-

tumoral activity even when the drug was administered once the tumor had reached a size that was

detectable in the animal. SU5416 has been shown to inhibit VEGF-dependent mitogenesis of human

endothelial cellswithout inhibiting the growth of a variety of tumor cells invitro. In contrast, systemic

administration of SU5416 at non-toxic doses in mice resulted in inhibition of subcutaneous tumor

growth of cells derived from various tissue origins.99 SU5416 inhibited metastases, microvessel


formation, and increased tumor and endothelial cell apoptosis in mice injected with CT-26 colon

cancer cells to generate metastases.100 Reduction in tumor growth in a human neurogenic sarcoma

explant xenograft model was due to decreased tumor angiogenesis, leading to reduction of tumor cell

proliferation and increased apoptosis.101 SU5416, when combined with irradiation, has an additive

effect over treatment with irradiation alone in a human glioblastoma xenograft. Permanent vascular

changes were seen after combined treatment resulting in complete remission.102 Its long lasting

effects in vivo suggested the possibility of a weekly administration in the clinical assays.103

Unfortunately, the results arising from the clinical studies have not confirmed these expectations. The

use of SU5416 in phases I and II clinical studies for the treatment of sarcoma,melanoma, von-Hippel

Lindau disease, and head and neck, breast, ovary, prostate andmetastatic renal cancers has given only

partially positive results in some cases.104–112 Phase III assays of co-administration of SU5416,

5-FU, and LV tometastatic colon cancer patients were a failure, casting doubts on the design of these

clinical trials. Among other undesired effects, risk of thromboembolisms after the SU5416

administration should be mentioned.113 All of these problems caused the total interruption of the

mentioned clinical trial in 2002. Lately, a phase II study conducted to investigate the safety and

efficacy of SU5416 for patients with advanced soft tissue sarcomas, has shown that this agent was

relativelywell tolerated, but did not demonstrate significant anti-tumor activity.114 Phase II studies of

single agent SU5416 showed a modest clinical activity in patients with refractory acute myeloid

leukemia (AML) or myelodysplastic syndromes, and in patients with refractory multiple

myeloma.115,116 Although prolonged remission was observed in a few patients with c-kit-positive

AML that was resistant to standard chemotherapy, the overall observed response rate was low and

responses consisted mainly of partial remissions of short duration.117 No clinical benefit was shown

in phase II studies in patients with RCC, advanced soft tissue sarcoma, advanced melanoma or

hormone refractory prostate cancer.118–121

Figure 3. Inhibitors of theactivationofangiogenic factor receptors.


SU6668 (SUGEN, Inc., co-developed with Taiho Pharmaceutical Co., Tokyo, Japan) is another

VEGFR2 inhibitor, which also inhibits bFGF and PDGF receptors.122 SU6668 has competitive

inhibitory properties with respect to ATP, inhibits phosphotyrosine levels in VEGFR2 and platelet-

derived growth factor receptor (PDGFR) b in vivo, and rapidly induces tumormicro vessel apoptosis,

followed by apoptosis of tumor cells.123 In cellular systems, it inhibits receptor tyrosine

phosphorylation and mitogenesis in a dose-dependent manner after stimulation of cells by

appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant

growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon,

ovarian, and epidermoid origin. Furthermore, intravital multi-fluorescence video microscopy of C6

glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed

tumor angiogenesis. SU6668 treatment induced striking regression of large established human tumor

xenografts.124 SU6668 increased median survival by 58% (P < 0.001). It also led to a progressive

increase in tumor cell and endothelial cell apoptosis that increased over time in BALB/c mice that

underwent splenic injectionwith CT-26 colon cancer cells to generate livermetastases.125 SU6668 in

combination with paclitaxel inhibited ovarian carcinoma progression in the peritoneal cavity, by

blocking ascites formation and tumor spread, in ovarian carcinoma xenograft models transplanted in

the peritoneal cavity of nude mice.126 The simultaneous inhibition of several receptors that are

involved in the angiogenic response of endothelial cells might increase the possibilities for the

pharmacological development of this compound. SU6668 induced a tumoral regression in lung and

colon experimental tumors, even when the treatment was initiated when the tumor mass was

considerably high.122,123 SU6668 has been assayed in phase I/II clinical studies with advanced

tumors and multiple myeloma patients, but no objective responses have been observed.126–128 A

recent study about the effects of SU5416 and SU6668 on receptor phosphorylation in tumor

xenografts and in paired tumor biopsies obtained in three clinical trials in patients with advanced

solid malignancies showed that both compounds displayed biological activity in xenografts.

However, neither drug produced marked biological activity in patients with primary tumors.129

Outstanding interest has been reached by the development of the new compound SU11248

(Fig. 3) (Sutent, sunitinib malate, Pfizer, Inc., NewYork, NY, www.pfizer.com), that is able to inhibit

the tyrosine kinase activity of a number of receptors including PDGFR, VEGFR2, Flt-3, and c-Kit.

SU11248 can be administered orally and exhibits an interesting anti-tumoral activity, as shown in

pre-clinical studies. SU11248 exhibited broad and potent anti-tumor activity causing regression,

growth arrest, or substantially reduced growth of various established mouse xenografts derived

from human or rat tumor cell lines.130 SU11248 dramatically regressed FLT3-ITD tumors in

the subcutaneous tumor xenograft model and prolonged survival in the bone marrow engraft-

ment model.131 SU11248 treatment potently regressed growth of mammary cancers in mouse

mammary tumor virus-v-Ha-ras transgenic mice and 7,12-dimethylbenz(a)anthracene-induced

mammary tumors in rats. This agent also inhibited tumor growth in human breast cancer xenografts of

s.c. MX-1 tumors. SU11248 treatment in combination with docetaxel effectively prolonged survival

of mice.132 It has been suggested that SU11248 might be an effective and tolerated therapy to inhibit

growth of breast cancer bone metastases, with the additional advantage of inhibiting tumor-

associated osteolysis.133 SU11248 enhanced radiation-induced endothelial cytotoxicity, resulting in

tumor vascular destruction and tumor control when combined with fractionated radiotherapy in

murine tumor models. A synergistic interaction of SU11248 and the traditional anti-leukemic agents

cytarabine or daunorubicin in leukemia cells has been reported.134,135 Recent results from a phase I

clinical study show that SU11248 monotherapy induced partial remissions of short duration in AML

patients.136

In a phase II clinical trial involving 63 renal cancer patients who had already tried IL-2 or IFN

without success, as a result a 40% had a partial response. Tumors did not progress for more than 3

months in an additional 28% of patients, indicating that 68% of patients benefited from SU11248

treatment. In addition, the average time to tumor progression for patients in this studywas 8.7months,


and the median overall survival was 16.4 months. A second phase II study of 106 patients

demonstrated an objective response rate of 39% in patients treatedwith SU11248. In addition, 23%of

patients experienced tumor stabilization. Taken together, a total of 62% of patients benefited from

treatment with SU11248.137–139 Results from these two studies suggest that SU11248 has substantial

anti-tumor activity inmetastatic renal cell carcinoma as second-line therapy and form the basis for the

launch of a large-scale phase III program to determine the potential benefits of SU11248 in earlier

stage disease.

A phase I study in Gastrointestinal Stromal Tumor (GIST), which had stopped responding to

other treatment gave encouraging results. In a phase I/II study, SU11248 therapy induced objective

responses in patients with several mutational variants of GIST displaying different genomic

mechanisms of resistance to imatinib mesylate (IM). Long-term follow-up data from this study have

demonstrated that SU11248 extended overall survival to nearly 20 months in patients whose cancer

had progressed despite treatment with other standard therapies. In addition, the median time to tumor

progression in this study was 7.8 months for all patients, with some specific subtypes of patients

benefiting even more dramatically than would be expected with IM.140,141 These data were the basis

for a phase III trial to further define the activity of SU11248 in patients with IM-resistant GIST. In

early 2005, Pfizer reported that this phase III randomized study has successfully met its efficacy

endpoint. Results from this double-blind phase III study of more than 300 patients showed SU11248

significantly prolonged the time to tumor progression (6.3 months on SU11248 vs. 1.5 months for

controls) and reduced the risk of death by approximately 50% compared to placebo.142These results

substantiate the concept that multi-targeted molecular therapy can overcome resistance to other

targeted drugs in cancer. SU11248 is currently being tested in phase II clinical trials in patients with

previously treated metastatic breast cancer and in patients with unresectable neuroendocrine

tumors.143,144

PTK787/ZK222584 (Fig. 3) (vatalanib, Novartis Pharma AG, Basel, Switzerland, www.

novartis.com, co-developed with Schering AG, Berlin, www.schering.de) is an orally available

aminophthalazine derivative that strongly inhibits all known VEGFR tyrosine kinases expressed on

endothelial cells. It also inhibits other class III kinases, such as the PDGFR beta tyrosine kinase, c-

Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor

families, such as EGFR, FGFR1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and

protein kinase C-a. The compound inhibits the endothelial cell proliferation, migration, and survival

in the nanomolar range in cell-based assays. After oral administration, it induced a dose-dependent

inhibition of VEGF and PDGF-induced angiogenesis in a growth factor implant model, and it

inhibited the growth of several human carcinomas, grown s.c. in nude mice, as well as a murine renal

carcinoma and its metastases in a syngeneic, orthotopic model. It did not affect wound healing.145

Oral administration of PTK787 alone or in combination with gemcitabine decreased the growth and

metastasis of human pancreatic carcinoma growing orthotopically in nude mice.146,147 Although the

clinical development of this drug was initially interrupted because of the appearance of some

undesired toxicities, it was reinitiated after checking that those toxicities were reversible and of

benign nature. In various phase I studies, daily oral administration was well tolerated.148,149 A phase

I/II study has shown that PTK787 can be safely combined with FOLFOX4 (oxaliplatin, fluorouracil,

folinic acid) in patients with metastatic CRC. At the higher doses of PTK787, dizziness and

neurologic toxicities were seen.150 Ongoing randomized phase III trials (CONFIRM-1 and

CONFIRM-2) are evaluating the efficacy and safety of PTK787 in combination with FOLFOX4 in

patients with newly diagnosed or relapsed advanced stage CRC.151 In March 2005, the company

announced that the analysis of CONFIRM-1 results indicated that pre-planned analysis of

progression-free survival achieved statistical significance. At the same time, analysis of primary

endpoint of progression-free survival assessed by central review did not achieve statistical

significance. Independent data monitoring board has recommended CONFIRM-1 trial to continue to

allow analysis of overall survival (Second Primary Endpoint). Novartis and Schering have now


anticipated filing for approval with the US FDA and the European Medicines Agency (EMEA) in

early 2007. Moreover, encouraging anti-tumor activity was seen with PTK787 monotherapy as well

as in combination with temozolomide or lomustine in a phase I/II study in patients with recurrent

glioblastoma multiforme.152 PTK787 is also currently being evaluated in Phase II studies for the

treatment of myelodysplastic syndromes, malignant mesothelioma, and von Hippel-Lindau disease.

Von Hippel-Lindau disease (VHL) is a familiar hereditary cancer caused by mutations in the VHL

suppressor gene germ line. It is characterized by the appearance of very vascularized tumors,

overexpressing VEGF, in the retina and in the central nervous system. For these reasons, they are

good candidates for the anti-angiogenic therapies directed to block VEGF action.

The drug ZD4190 (Astra Zeneca Pharmaceuticals LP, Macclesfield, Cheshire, UK, www.

astrazeneca.com) is a 4-anilinoquinazoline (Fig. 3) that inhibits the tyrosine kinase activities of

VEGFR2 andVEGFR1, as well as theVEGF stimulated human endothelial cell proliferation in vitro.

It shows anti-tumoral activity in breast, lung, prostate, and ovarian human tumor xenografts; upon

withdrawal of therapy, tumor growth resumed after a short delay.153 ZD6474 (Fig. 3) is a soluble

anilinoquinazoline that selectively inhibits the VEGFR2 tyrosine kinase and inhibits, to a lesser

extent, the activity of other tyrosine kinases such as those of the EGFR and the PDGFR. ZD6474

blocks VEGF-stimulated endothelial cell migration and proliferation. Chronic, daily oral

administration of ZD6474 produced significant broad-spectrum anti-tumor activity on human tumor

xenografts implanted in nude mice.154 It has also been reported that ZD6474 has the potential to

enhance the effect of radiotherapy and potentiates the anti-tumoral activity of taxanes in vitro and

in vivo.155,156 Recent studies have demonstrated that the inhibition of EGFR phosphorylation by

ZD6474 can contribute significantly to the anti-tumor efficacy of this agent.157 Phase I clinical

evaluation has shown that ZD6474 is generally well tolerated, and tumor responses in NSCLC have

been documented.158,159 Phase II clinical studies of ZD6474 oral administration to patients of lung

cancer or multiple myeloma are in progress.160,161

Sorafenib (BAY 43-9006, Bayer Pharmaceuticals Corporation, Leverkusen, Germany, www.

pharma.bayer.com, andOnyxPharmaceuticals, Inc., Emerville, CA,www.onyx-pharm.com) is a novel

bi-aryl urea that inhibits tumor cell proliferation and tumor growth in several human tumor xenograft

models by targeting the RAF/MEK/EKR signaling pathway at the level of RAF kinase.162,163 In

addition, BAY 43-9006 demonstrated significant activity against several receptor tyrosine kinases

involved in neovascularization and tumor progression, including VEGFR2, VEGFR3, PDGFR beta,

Flt-3, and c-KIT. Once daily oral dosing of sorafenib demonstrated broad-spectrum anti-tumor

activity in colon, breast, and NSCLC xenograft models. Analyses of microvessel density and

microvessel area in the same tumor sections using anti-murine CD31 antibodies demonstrated

significant inhibition of neovascularization in all three of the xenograft models.164

Sorafenib has beenwell tolerated and showed anti-tumoral activity in phase I studies in advanced

refractory solid tumors.165–168 A phase II randomized discontinuation study showed a significantly

better progression-free survival rate with sorafenib in renal cancer.169–171 In May 2005, Bayer and

Onyx announced that an ongoing multi-national, phase III study in patients with RCC or kidney

cancer had met its surrogate endpoint, resulting in statistically significant longer progression-free

survival in those patients administered with sorafenib. More than 900 patients, who had previously

failed one prior systemic therapy, were randomized one-to-one to receive either 400 mg sorafenib or

placebo twice a day. Results from the study—the largest randomized controlled trial ever conducted

in advanced renal cell cancer—were presented in an oral session during the ASCO 2005 meeting in

Orlando. Disease progression was significantly delayed in those patients who received sorafenib. As

assessed by independent radiologic review, progression-free survival was doubled to a median value

of 24 weeks in patients receiving sorafenib as compared to 12 weeks for patients receiving placebo

(P-value < 0.000001).172 In July 2005, the companies completed the submission of a new drug

application (NDA) with the FDA for sorafenib for patients with RCC, or kidney cancer. Discussions

are also underway with regulatory agencies about registration in other territories. A phase III study


has been recently initiated in patients with advanced liver cancer, and an additional phase III study

evaluating sorafenib in combination with carboplatin and paclitaxel to treat patients with advanced

metastatic melanoma will begin soon. In 2005, a phase II clinical trial sponsored by the NCI has

begun to evaluate how well sorafenib works alone and paired with gemcitabine and anti-metabolite,

currently the standard chemotherapy for pancreatic cancer.

Some efforts are being devoted to the development of inhibitors of bFGF receptors, although

they are still in preliminary stages of development. Among others, we could point out the drugs

SU4984, SU5402, and PD166866, that inhibit the FGFR kinase, and PD173074, which inhibits

FGFR and VEGFR2.89

The overexpression of EGF or its receptor plays a main role in essential processes for tumor

progression, such as cell proliferation, apoptosis metastasis, and resistance to anti-tumoral drugs. In

addition, some experimental evidence supports the role for EGF in angiogenesis, probably by

modulating VEGF production.173 For these reasons, there has been a considerable interest in the

search and development of drugs that are selective inhibitors of EGFR, which could be good

candidates for anti-tumoral drugs. The advanced stage of development of the humanized monoclonal

antibodies cetuximab (IMC-225, erbitux, ImClone Systems Incorporated, New York, NY,

www.imclone.com) should be emphasized. It binds competitively and with high affinity to EGFR,

competing for natural ligand-binding sites and causing receptor internalization and down-

regulation.174 Pre-clinical studies using human cancer xenografts growing either s.c. or

orthotopically in nude mice have demonstrated that cetuximab inhibits tumor growth, increasing

the survival of mice, and inhibiting tumor-induced angiogenesis.173 Clinical efficacy of IMC-C225

appears to involve multiple mechanisms, including inhibition of cell cycle progression, induction of

apoptosis, inhibition of angiogenesis, inhibition of metastasis, and enhancement of the response to

chemotherapy and radiotherapy.173 Very recently, it has been reported that cetuximab inhibits VEGF

secretion in cell culture and in animal models, and that this inhibition occurs at the level of

transcription in response to a reduced level of hypoxia inducing factor (HIF)-1a. These results justifyfurther testing of therapeutic strategies that combine cetuximab with approaches inhibiting the

function of VEGF or the VEGF receptor.175

Phase I studies of cetuximab both as a single agent or in combination with conventional

chemotherapy or radiotherapy confirmed a lack of immunogenicity of this compound, that is

generally well tolerated, with skin rashes and allergic reactions being the most clinically important

adverse events reported.173 Cetuximab has demonstrated in phase II/III studies that it can make the

cancer cells more sensitive to chemotherapy and radiation. Results of a large phase II study have

shown response when used in combination with irinotecan in 22.9% of patients with EGFR-

expressing, irinotecan-refractory CRC.176 Cetuximab was approved in February 2004 for this

indication by the US FDA, and for use in Switzerland, Iceland, Norway and the member states of the

European Union. Very recently, in a phase II study cetuximab in combination with carboplatin has

demonstrated clinical activity and an acceptable safety profile in heavily pre-treated patients with

recurrent or metastatic nasopharyngeal carcinomawho had previously experienced treatment failure

with platinum-based therapy.177 Other phase II and III studies showed significant response to

treatment invariable proportions of patients with RCC, carcinoma of the head and neck, NSCLC, and

pancreatic cancer, when cetuximab is used first or second line in combination with chemother-

apy.178–183 Results of an international, randomized phase III clinical trial of 424 patients who

received radiation � cetuximab for advanced head and neck cancer demonstrated a near doubling of

median survival for patients treated with radiation plus cetuximab, 54 months versus 28 months for

patients treatedwith radiation alone.184 Thus, cetuximab is emerging as avery promising new therapy

to be used in combination with existing therapies for the treatment of a spectrum of solid tumors.

Cetuximab is being currently evaluated in phase III in combination with standard chemotherapy in

CRC, and pancreatic cancer, in phase II/III in patients who have unresectable liver metastases from

colorectal cancer, and in phase II in NSCLC, CRC, ovarian and metastatic pancreatic cancer.185–189


The encouraging data obtainedwith the EGFR inhibitor iressa (gefitinib,ZD1839, Astra Zeneca,

London, UK, www.astrazeneca.com) in phase II trials in advanced CRC, NSCLC, breast cancer,

glioblastoma, and head and neck cancer gave an impulse to the clinical development of this

compound, with more than 35 clinical studies under course in mid-2004.190–200 Iressa is being

evaluated in phase III inNSCLCand in squamous cell carcinoma of the head and neck, and in phase II

in ovarian, breast or skin cancer, metastatic bladder cancer, CRC, glioblastoma, progressive

metastatic neuroendocrine tumors, metastatic synovial sarcoma, recurrent or progressive CNS

tumors, and childhood brain stem glioma. Iressa has been used in over 200,000 patients since its first

launch in Japan in 2002 and is licensed in more than 30 other countries. In May 2003, it received the

US FDA accelerated approval for the third-line treatment of NSCLC. However, in December 2004,

AstraZeneca notified that a large clinical trial of nearly 1,700 patients comparing iressa with placebo

in patients with NSCLC who had failed other therapies failed to show a survival benefit

(www.fda.gov). The trial was looking for increased survival in patients, but despite statistically

significant improvement in tumor shrinkage, this did not translate into a significant survival benefit.

Recent data suggests that only a small subgroup of non-smoker patients with bronchioloalveolar

pathologic subtype showed a better response to iressa.201 The response rates significantly increased

among oriental patients, probably due to the existence of somatic mutations in the EGFR kinase

domain in those patients who responded well to iressa.202–204 Thus it is likely that ultimate approval

of iressa in lung cancer will only be for a small identifiable subset of patients with NSCLC.

The EGFR inhibitor erlotinib (OSI-774, tarceva, OSI Pharmaceuticals, Inc., Melville, NY,

www.osip.com), was Iressa’s closest competitor and is now in a very dominant market position.

Erlotinib received fast track status from the FDA in May 2002 for treatment of chemotherapy-naıve

advanced-stage NSCLC, which was later extended to include second- or third-line monotherapy in

patientswith incurableNSCLC failing standard chemotherapy. InNovember 2004, erlotinib received

US FDA approval for the treatment of NSCLC and gained the distinction of being the first EGFR

inhibitor to show a survival benefit in lung cancer patients. The FDA based its approval decision for

erlotinib on results from a randomized, double-blind, placebo-controlled pivotal phase III trial of

patients with second- and third-line advanced NSCLC. The trial included 731 patients with advanced

NSCLC for whom one or more chemotherapy regimens had failed. Patients receiving erlotinib

showed an increase in median survival by 42% compared to those in the placebo arm (6.7 months vs.

4.7 months), an improvement of 2 months.205 There was also a significant increase in both the length

of time before patients disease symptoms deteriorated and the time when patients were stable and

therewas no progression of their cancer. A 45% improvement in survival at 1 year was observedwith

erlotinib, with benefits being shown in a broad spectrum of patients. Similarly to the significant

survival benefit in NSCLC, erlotinib has also shown survival benefit in a phase III study in locally

advanced ormetastatic pancreatic cancer patients. In early 2005OSI Pharmaceuticals announced that

a multi-center, randomized, double-blind, placebo-controlled phase III trial evaluating erlotinib plus

gemcitabine chemotherapy in 569 patients with locally advanced ormetastatic pancreatic cancer met

its primary endpoint by demonstrating a statistically significant 23.5% improvement in overall

survival when compared to patients receiving gemcitabine plus placebo.206,207 Erlotinib is the first

drug shown in a phase III trial to prolong survival when added to the standard of care (gemcitabine) in

the treatment of patients with previously untreated advanced pancreatic cancer. Other indications in

which erlotinib has produced objective evidence of anti-tumor activity in patients failing standard

chemotherapy include ovarian cancer as well as cancers of the head and neck. Erlotinib is currently

being evaluated in an extensive clinical development program by a global alliance among OSI

Pharmaceuticals, Genentech, and Roche, and is being currently evaluated in more than 35 clinical

studies, including phase II in patients with NSCLC, glioblastoma multiforme, recurrent breast, head

and neck, ovarian, renal or colon cancer, glioblastomamultiforme, liver cancer, advanced esophageal

cancer or stomach cancer,metastaticmalignant peripheral nerve sheath tumor, and recurrent cervical

cancer.190,208–218


A final commentary to the existence of drugs acting on sites of the signaling pathways that are

located downstream the angiogenic factor receptors, and that also exhibit anti-angiogenic properties.

Among others, we could mention LY317615 (Eli Lilly, Indianapolis, IN, www.lilly.com), which

inhibits the protein kinase C b, an enzyme that is involved in the signaling pathway of VEGFR2, and

that exhibits anti-angiogenic activities in the rat cornea assay and anti-tumoral activities in vivo in

glioblastoma and gastric, renal, colorectal, breast, ovary, and lung tumors.219–223 Phase I studies in

patients with solid tumors have been completed, and phase II studies in patients with glioma or

lymphoma are in progress.224

3 . A G E N T S T H A T I N H I B I T E N D O T H E L I A L C E L L P R O L I F E R A T I O N

The first experimental strategy for the screening of new anti-angiogenic compoundswas the search of

endothelial cell growth inhibitors. One of the firstly described inhibitors of angiogenesis, fumagillin

(Fig. 4) was isolated from an endothelial cell culture that had been contaminated by the fungus

Aspergillus fumigatus fresenius. Fumagillin inhibits endothelial cells proliferation in vitro, and

angiogenesis and tumor growth in experimental models. A prolonged administration of fumagillin is

limited by a severe weight loss. This fact aimed at the synthesis of new derivatives with improved

anti-angiogenic activities and lower undesired effects.225 The fumagillin analog AGM-1479/TNP-

470 (Fig. 4) (TAP Pharmaceuticals, Inc., Deerfield, IL) is more selective to endothelial cells than

fumagillin.226 AGM1470 mechanism of action seems to be related to the inhibition of type 2

methionine amino peptidase, and cyclin-dependent kinases.227,228 This drug is active in several

animal tumor models, and in a tumorogenesis model in transgenic mice, exhibiting therapeutic

Figure 4. Inhibitors ofendothelial cell proliferation.


synergies with ionizing radiation in human glioblastoma multiforme xenographs.229,230 AGM1470

anti-tumor activity has brought it to phase II/III clinical studies with a series of solid tumors, and

phase I studies in acute leukemia, lymphoma, and Kaposi’s sarcoma. The appearance of secondary

toxicities (encephalopathy and ataxia) seems to be reversible after treatment interruption, and new

approaches to develop polymer conjugates may diminish drug-related toxicities.231 Partial responses

have been described in patients of renal carcinoma, cervix cancer, and Kaposi sarcoma.232–235 Some

partial responses have also been obtained after co-administration of AGM1470 and paclitaxel in the

treatment of lung cancer patients.236

Squalamine (MSI-1256F, Magainin Pharmaceuticals, Detroit, MI, www.magainin.com,

Genaera Corporation, PlymouthMeeting, PA, www.genaera.com) is an aminosterol (Fig. 4) isolated

from the liver of the shark Squalus acanthias. Squalamine inhibits angiogenesis and tumor growth in

multiple animal models. This effect is mediated, at least in part, by blocking mitogen-induced

proliferation and migration of endothelial cells, thus preventing neovascularization of the tumor.

Squalamine has no observable effect on unstimulated endothelial cells, is not directly cytotoxic to

tumor cells, does not alter mitogen production by tumor cells, and has no obvious effects on the

growth of newborn vertebrates. Squalamine was also found to have remarkable effects on the

primitive vascular bed of the CAM.237 Squalamine is an inhibitor of Naþ/Hþ interchanger protein.238

Evenwhen no limiting toxicities have been found in phase I studies, no objective responses have been

reported for squalamine as a single agent. Some positive results have been obtained in phase II studies

in lung cancer in combination with standard chemotherapy. Strong clinical results, including

improved vision for the phase I/II trial have been reported for the treatment of aged-related macular

degeneration. Squalamine is currently being evaluated in phase II studies in solid tumors (NSCLC

and prostate) co-administered with chemotherapy in ovarian cancer patients. Its big potential as a

non-invasive anti-angiogenic therapy for blinding eye disease, is being evaluated in aged-related

macular degeneration, being granted in October 2004with the Fast Track designation by FDA for the

treatment of this disease.239,240

Rapamycin (sirolimus, Fig. 4), a well-known immunosuppressor, inhibits tumor growth and

angiogenesis in in vivo models. This anti-angiogenic activity has been related to the inhibition of

endothelial cell proliferation after VEGF stimulation, and with a decrease in the production of this

angiogenic factor.241 Rapamycin is an inhibitor or the mammalian target of rapamycin (mTOR), a

downstream effector of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signaling

pathway,whichmediates cell survival and proliferation.As a result of its positionwithin this pathway

and its central role in controlling cellular growth, mTOR is viewed as an important target for anti-

cancer therapeutics development.242 Everolimus (RAD-001, SDZ RAD, Certican; Fig. 4) (Novartis

Pharma, Basel, Switzerland, www.novartis.com), is an analog of rapamycin that can be administered

orally, and exhibits more favorable pharmacokinetic properties.243 This compound is very selective

to endothelial cells, with a significantly lower activity on non-endothelial cell lines.244 Everolimus

presents a limited toxicity in patients. It is being developed as an anti-proliferative agent and has been

approved in Europe as an immunosuppressive agent in the solid organ transplantation setting.242–246

In the last few years, several tumor-produced endogenous molecules with angiostatic activity

have been described. Their discoverywas based on the observation that some primary tumors seemed

to inhibit their own metastases; the primary tumor removal provoked the growth of the metastases.

Recent evidence suggests that physiological levels of endogenous inhibitors of angiogenesis can

serve as endothelium-specific tumor suppressors.247 The search of circulating factors produced by

tumors brought to the isolation of angiostatin and endostatin (EntreMed, Rockville, MD,

www.entremed.com).248,249 Angiostatin was isolated from the urine of lung cancer bearing mice.

Those mice only suffered from metastasis when the primary tumor was removed. Angiostatin has

been suggested to be produced by partial hydrolysis of plasminogen, probably by means of a

macrophage metalloelastase or other proteases produced by tumor cells.250,251 Endostatin was

initially found in the medium conditioned by hemangioendothelioma cells, and it can be originated


by proteolysis of collagen XVIII, frequently present in the basal membranes. A variety of murine

tumors as well as human breast, prostate, and colon tumors in human xenograft models regressed

when treatedwith angiostatin or endostatin. Regression of tumors upon systemic treatmentwith these

proteins was in part due to increased tumor cell apoptosis. Repeated cycles of endostatin therapy led

to prolonged tumor dormancy without further treatment and were not associated with any apparent

toxicity or acquired drug resistance.252 Endostatin-mediated apoptosis is associated with several

apoptotic signaling pathways including overloading of intracellular magnesium and calcium, as well

as regulation of p53 and Bcl-2 expression.253 Endostatin was the first angiogenesis inhibitor in

reaching the clinical studies. Phase I clinical studies indicated that endostatin is well tolerated by

patients, but the results were not as satisfactory as expected, since evenwhen some evidence ofminor

anti-tumor activity was observed, no objective response was obtained in those studies.254–257 New

hopes are arising fromvery recently reported phase III results showing that the addition of endostar (a

new recombinant human endostatin developed by Medgenn Co., Yantal, Shandong, China,

www.medgenn.com) to vinorelbine and cisplatin (NP) regimen resulted in significant and clinically

meaningful improvement in response rate, median TTP, and clinical benefit rate compared with NP

alone in advanced NSCLC patients.258 Angiostatin has been well tolerated in phase I clinical studies,

and is being evaluated in phase II for the treatment of lung cancer patients in combination with

paclitaxel and carboplatin.259,260 A gene-based angiostatin treatment for vision loss is also being

explored.261 Efforts are being devoted to the development of alternative ways of administration to

increase angiostatin or endostatin efficacy. They include the use of osmotic pumps for the continuous

administration of endostatin, and intravenous administration of angiostatin and endostatin genes

complexed to cationic liposomes.262–264

4 . A G E N T S T H A T I N H I B I T E N D O T H E L I A L C E L L I N V A S I O NA N D M O T I L I T Y : P R O T E A S E I N H I B I T O R S

Drugs that inhibit matrix metalloproteinases (MMPs) have been recently compared to a mirage, a

tantalizing possibility that keeps always out of reach. In theory,MMPs are one of the most promising

targets to fight cancer and other diseases, but the clinical failures of their inhibitors do not support this

idea.265,266 The discovery of the relation between MMPs overexpression and tumor invasion awoke

the interest in the search of inhibitors ofMMPs that, in theory, could act on at least two steps of tumor

progression: angiogenesis and metastasis. But time has demonstrated that things are not so simple:

MMPs are not only involved in the extracellular matrix, basement membrane, and connective tissue

remodeling, but they play a relevant role in the control of other molecules that regulate cell

proliferation, migration and differentiation, angiogenesis and apoptosis. This may make the

prediction of the consequences derived from the inhibition of a given protease difficult, since normal

cell processes can be affected.267

This complex panorama was not known when the first MMPs inhibitors were described. They

were designed to contain a hydroxamate group that would complex the metalloprotease Zn2þ,

essential for its activity. Batimastat (Fig. 5) (BB94, British Biotech, Oxford, UK, www.britishbio-

tech.com), awide spectrum, competitive and reversible inhibitor ofMMPs, was the first compound of

this family to reach clinical studies.268 Its development was interrupted in 1996 to favor that of

marimastat (Fig. 5) (BB2626,BritishBiotech, licensed to Schering-PloughCorporation,Kenilworth,

NJ, www.schering-plough.com, and Tanabe Seiyaku Co, Osaka, Japan, www.tanabe.co.jp), a

compound that maintained the MMP inhibitory properties of batimastat, but with an increased

solubility that could allow an oral administration. After promising results in pre-clinical and early

clinical studies, undesired effects including fatigue and cumulative poliarthritis forced to reduce the

doses in subsequent studies. Unfortunately, the newdoseswere not high enough to reach a therapeutic

efficacy, and results obtained in phase III clinical studies were very disappointing (marimastat failed


to show superior efficacy over either standard chemotherapy or placebo in pivotal phase III trials in

glioblastoma, breast, ovarian, and small and non-small cell lung cancer).269,270 In two recent phase III

studies with SCLC and metastatic breast cancer patients, marimastat treatment did not only fail in

improving survival, but had a negative impact on quality of life.271,272

A similar hapless fate was that of the drug prinomastat (AG3340, Agouron Pharmaceuticals,

Inc., San Diego, CA, www.agouron.com), another synthetic derivative of hydroxamic acid (Fig. 5)

that exhibits a more selective MMPs inhibition profile. Pre-clinical studies showed that prinomastat

produced growth delays in a variety of tumors including neuroblastoma, as well as in cancers of the

lung, breast, colon, brain, and prostate. Prinomastat inhibited tumor angiogenesis and cell

proliferation in neuroblastoma and enhanced the efficacy of carboplatin and taxol in NSCLC.273,274

In spite of the fact that the compound seemed to be well tolerated and synergize with the traditional

chemotherapy in early clinical trials, the most frequent side effects being musculoskeletal pain and

stiffness, prinomastat development was almost interrupted in August 2000, after discouraging results

in phase III clinical studies for the treatment of prostate and lung cancer patients.275–280 Data from

over 1,000 patients with stage IIIB, IV, or recurrent NSCLC receiving gemcitabine/cisplatin or

paclitaxel/carboplatin showed that prinomastat doubled the risk of venous thromboembolisms in

patients with advanced NSCLC receiving chemotherapy.281 New hopes for the clinical usefulness of

this compound may derive from its use in combination with photodynamic therapy or in

ophthalmology and dermatology.282–286

The drug BAY12-9566 (Fig. 5) (Tanomastat, Bayer, Inc., Leverkusen, Germany, www.bayer.-

com) is structurally different to the above-mentionedMMP inhibitors. It is a butanoic acid derivative

that selectively inhibits MMP2, MMP3, and MMP9, which could explain its lower muscular

toxicity.287–289 Promising pre-clinical data included inhibition of tumor growth and metastasis in

murine melanoma and human colon tumor xenografts, and phase I clinical studies indicated that the

compound was relatively well tolerated.290 Even when the compound reached phase III clinical

Figure 5. Inhibitors ofmatrixmetalloproteinases.


studies for the treatment of lung, pancreas and ovary cancers, a decrease in the survival of the patients

treated with this compound provoked an immediate interruption of its clinical development.291

Nowadays the pharmaceutical companies do not slack in their effort for finding a new inhibitor of

MMPs with a wide clinical application. British Biotech is developing new inhibitors, such as

solimastat (BB-3644, Fig. 5), a wide spectrum inhibitor, orally administrable and with a series of

advantages when compared tomarimastat, that is, lower effects on skeletal muscle, and an additional

inhibition of the production of TNFa. BB-3644 has been evaluated phase I clinical studies for the

treatment of solid tumors that are refractory to chemotherapy. As dose-limiting musculoskeletal

toxicity was encountered at doses of BB-3644 unlikely to provide an advantage over currently

available MMPIs, further evaluation is not recommended.292,293

BMS-275291 (Fig. 5) (Bristol Myers Squibb, New York, NY, www.bristolmyers.com) is a

peptidomimetic soluble MMPI that contains a chemically novel mercaptoacyl zinc-binding group

and that was rationally designed to inhibitMMPs activities, but not those of other proteases that could

be involved in the release ofmolecules including TNFa, L-selectine, and the receptors of TNFa or IL-6. In vitro, BMS-275291 is a strong inhibitor ofMMP1,MMP2,MMP7,MMP9, andMMP14. BMS-

275291 inhibits tumor growth and metastasis in murine models, and inhibits angiogenesis in the

Matrigel plug angiogenesis model.294 BMS-275291 does not inhibit the sheddases, which are related

metalloproteinases that regulate pro-inflammatory cytokine and cytokine receptor shedding from the

cell surface.295 Shedding of tumor necrosis factor and its receptor (TNF-RII) were hypothesized to

play a role in MMPI-associated musculoskeletal toxicity. In early clinical trials, BMS-275291

therapywas not associatedwith arthralgia ormyalgia.296–297Recent results from a randomized phase

II trial in 80 hormone refractory prostate cancer patients with bone metastasis indicated that BMS-

275291 was well tolerated and had no dose-limiting arthritis.298 Phase II/III studies with BMS-

275291 in combination with paclitaxel and carboplatin for the treatment of advanced NSCLC

patients and in phase II in combination with zoledronate in patients with hormone refractory prostate

cancer are under curse. BMS-275291 plus paclitaxel/carboplatin was well tolerated and active in

advanced non-small cell lung cancer. Treatment with BMS-275291 was not limited by drug-related

arthrotoxicity, and tumor response was as expected. Patient accrual was continued to further

investigate the effect of BMS-275291 on overall and progression-free survival in a phase III

setting.299,300 Very recently reported results from this randomized phase III study of BMS-275291 in

combination with paclitaxel and carboplatin in 774 patients of advanced NSCLC, indicated that

BMS-275291 added to chemotherapy increased toxicity and did not improve survival in those

patients.301 A randomized phase II feasibility trial was performed to evaluate the safety, phar-

macokinetics and feasibility of incorporating BMS-275291 into adjuvant breast cancer therapy. The

pattern of arthralgia in BMS-275291-treated patients was consistent with matrix metalloproteinase

inhibitor toxicity, showing that an adjuvant trial in this patient population is not feasible. Although the

rate of arthralgia ormyalgiawas sufficient to require early termination of the trial, it was nevertheless

lower than reported for other MMPIs. Therefore, although the rational design of this MMPI to avoid

sheddase release did not eliminate arthralgia, it may have widened the therapeutic window. The

critical question, whether it is possible to completely separate inhibition of MMPs that are important

in cancer progression from those whose inhibition produces joint toxicity, still remains.302

MMI270 (Fig. 5) (CGS27023A, Novartis Pharma AG, Basel, Switzerland) is a potent, broad

spectrum, orally active MMPI that inhibits the gelatinases MMP2 and MMP9 and the stromelysin

MMP3 in low nanomolar concentrations. In rat tumor models of breast and endometrial cancer,

MMI270 as a single agent or in combination with megestrol acetate significantly reduced tumor

burden and number of lymph node and lung metastases.303 MMI270 inhibited the migration and

invasion of endothelial cells, in addition to tube formation in vitro.304 A single-agent phase I study of

MMI270 performed in patients with advanced solid cancer indicated that it was rapidly absorbed

following oral administration, reaching in plasma of patients those concentrations required for

optimum MMPI activity. The pattern of toxicity (arthralgia and/or myalgia) was similar to that


described with other broad-spectrumMMPIs.305 Considering that althoughMMPIs may be active as

single agents, it is likely that they will bemore effective in combination with chemotherapy. In fact, a

dose-finding study of MMI270 combined with 5-FU and folinic acid has already been conducted.

Results from this study indicate that MMI270 can be safely administered in conjunction with 5-FU

and folinic acid over a range of dose levels.306 However, no clear responses have been found in the

mentioned clinical trials.

The first positive results of clinical trials withMMPs inhibitors were reported in 2001. In a study

with 414 non-operable pancreas cancer patients, marimastat treatment resulted to be as effective as

the treatment with gemcitabine, the reference compound for this type of tumors.270 Nevertheless in

subsequent studies the therapeutic benefit of the use of marimastat in combination with gemcitabine

versus gemcitabine alone has not been established.307 The most hopeful results are derived from a

study with 367 patients of gastric cancer, where it could be demonstrated that the treatment with

marimastat increased patient survival. This therapeutic benefit was more pronounced in those

patients that had previously been treatedwith chemotherapy.308More recently, newhopes have arisen

after partial responseswere obtainedwith the combination ofmarimastat and standard chemotherapy

in the treatment of patients of glioblastoma or pancreas and gastric cancers.309,310

The drug COL-3 (Fig. 5) (Metastat, CollaGenex Pharmaceuticals, Inc., Newtown, PA, www.

collagenex.com), is a modified tetracyclin which is devoid of anti-microbial properties and is a

competitive and selective inhibitor ofMMP2 andMMP9 enzymes. COL-3 also inhibits the activity of

activated neutrophil gelatinase, and blocks the invasion of several types of tumors into Matrigel and

basementmembranematrices. In addition, impressivegrowth-inhibitory activity has been noted after

COL-3 treatment in a broad range of human tumor cell lines and xenografts, including human

melanoma and metastatic prostate cancer, probably due to the induction of apoptosis.311–315 The

main undesired effect of this compound is the photosensibilization induction, so that it has to be

administered in combination with the use of sun protection filters.316 Although no objective anti-

tumor responses were observed, apparent clinical benefit in several patients with soft tissue sarcoma

and with advanced solid malignancies that were unresponsive to standard therapy, as well as a 44%

overall response rate in patients with AIDS-related Kaposi’s sarcoma, suggest that COL-3 should

undergo additional evaluation for the treatment of various types of sarcoma and othermalignancies in

whichMMPs may play an important role in invasion, proliferation, and metastases.316–318 This drug

is being tested in phase I/II studies with astrocitoma/multiforme glioblastoma, and in phase II with

Kaposi sarcoma patients, in collaboration with the NCI (www.clinicaltrials.gov).

Neovastat (AE-941, Aeterna Laboratories Inc., Quebec, Canada, www.aeterna.com) is a shark

cartilage extract soluble in aqueous media, representing less than 5% crude cartilage. It is a multi-

functional anti-angiogenic product (containing several molecules with biological activity), which

affects multiple levels of the angiogenic cascade. Neovastat inhibits MMP2, MMP9, and MMP12,

interferes with VEGFR2 signaling pathway, stimulates tissue-type plasminogen activator enzymatic

activity, inhibits endothelial cell proliferation and tubulogenesis, and induces apoptosis in

endothelial cells.319,320 This pro-apoptotic activity is specific for endothelial cells and correlates

to an increase in caspases 3 and 8.321 Anti-angiogenic activity of the compound is also supported by

its inhibitory effect on neovascularization in the ex ovo CAM model as well as by its inhibition of

neovascularization induced in vivo by s.c. implants of Matrigel.322 Chronic administration of

Neovastat significantly inhibits tumor growth in vivo andmetastases invasion in an in vivoLewis lung

carcinoma model.314 Neovastat is active in the treatment of multiple myeloma, renal and lung

carcinoma, with an increase in the patients survival time, without the appearance of great

toxicities.323 A significant survival advantage has been observed in NSCLC patients receiving the

highest doses of neovastat in a phase I/II trial.324 The main activity has been on neovastat in clinical

trials for renal and NSCLC, both reaching phase III.325–327 Results from a phase III clinical trial

evaluating neovastat in 305 patients with advanced renal cell cancer refractory to immunotherapy did

not meet its primary endpoint of improving median survival time. Despite the drug’s failure,


significant survival advantage was observed in a subgroup of certain healthier patients. This

38-patient subgroup showed a median survival time of 26.3 months for those treated with Neovastat,

comparedwith 12.6months for patients receiving a placebo. The increase inmedian survival time for

the renal cell carcinoma subgroup, which had an earlier disease stage compared with the overall

group, seems to suggest that treatment with neovastat in earlier-stage cancer could be more

efficacious.328 Aeterna has decided to focus neovastat efforts in lung cancer only. A phase III trial,

sponsored by theNCI and scheduled to be finished in 2006, is evaluating neovastat inNSCLCpatients

in combination with other drugs and radiotherapy.329 Neovastat therapeutic potential is not restricted

to cancer, since good results have been obtained with this drug in phase II studies for the treatment of

psoriasis patients.330

Finally, a brief commentary about a new experimental approach to inhibit the proteolytic

activity, consisting of the use of drugs that may inhibit the expression, secretion or activation of

proteases, or increase those of their inhibitors. Among them, we could mention curcumin, a

polyphenol from the plant Curcuma longa of widespread use as spice or alimentary colorant,331,332

halofuginone, an alkaloid from the plant Dichroa febrifuga, that inhibits MMP2 expression by

interfering TGF-b signaling pathway,333 and aeroplysinin-1, isolated from the marine sponge

Aplysina aerophoba, that induces a shift in the proteolytic balance toward anti-proteolysis, and

therefore to less invasive phenotypes.334

5 . A G E N T S T H A T I N H I B I T E N D O T H E L I A L C E L L A D H E S I O N

Another strategy for the anti-angiogenic therapy consists of the inhibition of endothelial cell adhesive

interactions during their migration. Endothelial cell migration depends on adhesion to the

extracellularmatrix proteins bymeans of a number of cell adhesion receptors known as integrins. The

avb3 integrin, a receptor for extracellular matrix components that contains the peptidic motif

arginine-glycine-aspartic acid (RGD), is an attractive target for the anti-angiogenic therapy, since it is

only present in the cell surface of the activated endothelial cells, and not in quiescent endothelia and

other cell types. It was initially described that some peptides that are antagonist of RGD domain, and

monoclonal antibodies generated against the avb3 integrin (LM609) disrupted ongoing angiogenesis

on theCAM, leading to the rapid regression of histologically distinct human tumors transplanted onto

the CAM.335 Intravenous administration of LM609 in a SCID mouse/human chimerical model with

transplanted human skin containing avb3-negative human breast tumor cells, either prevented tumor

growth ormarkedly reduced tumor cell proliferationwithin themicroenvironment of the human skin.

These LM609-treated tumors not only contained significantly fewer human blood vessels but also

appeared considerably less invasive than tumors in control animals.336 The excellent results obtained

in animal models led to the evaluation of integrin antagonists in clinical trials. Those conducted with

vitaxin (MEDI-522, Scripps Research Institute, la Jolla, CA;MedImmune, Gaithersburg,MD; Ixsys,

La Jolla, CA) are remarkable. Vitaxin is a humanized form of the LM609 antibody that selectively

binds to sprouting blood vessels and suppresses tumor growth in several animal models.336,337 Based

on the selectivity of avb3 for the activated endothelial cells and on their anti-angiogenic activity in

vivo, some clinical assays have been carried out to establish vitaxin tolerability and pharmacokinetic.

Vitaxin antibodies have been well tolerated by cancer patients, giving positive results in some

cases.92,338Very recently, results from amulti-center, non-comparative phase II study have suggested

a potential clinical activity of vitaxin with or without dacarbazine in patients with metastatic

melanoma.339 Phase II studies for the treatment of leiomyosarcoma patients, and other clinical

studies for the treatment of inflammatory diseases and rheumatoid arthritis are in progress.340–342

A different way to block the avb3 receptor is by means of RGD-type peptides. Their use may

present some advantages when compared to that of antibodies, such as their easy synthesis, resistance

to proteolysis, and low immugenicity. Among others, we could mention the cyclic peptides


EMD12974 (cilengitide, RGDf-[NmeVal) and EMD270179 (RGDf-ACHA) (Merck KGaA,

Darmstadt, Germany, www.merck.de). EMD12974 has demonstrated efficacy in melanoma tumor

models as a single agent,343 and its combination with radio immunotherapy significantly increased

efficacy and tumor and endothelial cell apoptosis without apparent increases in toxicity.344

EMD12974 has reached phase II in NSCLC, pancreatic cancer, and glioblastoma.345–349 In January

2004, orphan designation was granted by the European Commission toMerckKGaA for EMD12974

for the treatment of glioma. This drug is currently being evaluated in phase I in patientswith advanced

tumors and lymphoma, and in children with refractory primary brain tumors and in phase II in

patients with metastatic prostate cancer and recurrent glioblastoma multiforme.

Some efforts are being invested in the search of non-peptidic, small size molecules that are

antagonist ofavb3. Severalof thesedrugshavedemonstrated agoodanti-angiogenic activity in invitro

and in vivo models, but they have not yet reached the clinical phases of development.350–354

Finally, we will make a reference to the recent controversy concerning the hypothesis that avb3and/or avb5 integrins are necessary for tumor angiogenesis, since their absence in knockout mice

causes increased angiogenesis and tumor growth. This apparent paradox suggests that integrins are

also negative regulators of angiogenesis and therefore, anti-integrin drugs may act mainly as agonist

of somenegative regulator functions. Thus, although integrinsmay still be considered as a valid target

for the pharmacological inhibition of angiogenesis, the design and development of the new anti-

integrin drugs will have to take into account all the molecular processes that could be affected by

them.355

6 . O T H E R A G E N T S T H A T I N H I B I T A N G I O G E N E S I S

A. Thalidomide

Thalidomide (a-(N-ftalimido)glutarimide, Fig. 6) (EntreMed, Ocknille, MD Celgene Corporation,

Warren, NJ, www.celgene.com) is an inhibitor of the bFGF- and VEGF-induced angiogenesis. It is a

chiral compound and the S(�) enantiomer presents strong teratogenic and anti-angiogenic

properties. It was initially used as sedative and anti-emetic in pregnant women, but it was given

up in 1961 because it provoked fetal malformations.356 However in 1965 its evaluation for the

treatment of inflammatory and autoimmune diseases was activated.357 The first reference of the anti-

angiogenic properties of thalidomide is due to D’Amato and colleagues, which found that this

compound inhibited the bFGF-induced angiogenesis in rabbit cornea.358 Even when thalidomide

mechanismof action is not clear, itmay be partially related to its capability to inhibit the expression of

some adhesion molecules, including VCAM-1 (CD106) and E-selectin, and mediated by ceramide

through depletion of VEGFRs, and antagonized by sphingosine-1-phosphate.359,360 Several clinical

studies have been conducted to evaluate the interest of thalidomide for the treatment of hematological

or solid tumors. Some positive results have been obtained with multiple myeloma patients, and

partially positive results have been obtained in the treatment of high grade glioma patients.361–364

Some hopeful results have been obtained in the treatment of Kaposi’s sarcoma, prostate cancer,

myelofibrosis withmyeloidmetaplasia, and neurofibromatosis.365–371 In a phase II study designed to

evaluate the anti-tumor activity and toxicity of thalidomide as a single agent in patients with

metastatic melanoma this compound showed poor activity, but acceptable toxicity.372 In spite of

some evidence pointing to the activity of thalidomide in renal carcinoma, the high toxicity of the

compound at doses needed for the anti-angiogenic activity makes its use unadvisable for the

treatment of this type of tumors.373–375 No positive results have been obtained in the treatment of

metastatic breast cancer patients.376 Recent results from a phase III study show that thalidomide does

not improve survival in patients receiving cranial irradiation (WBRT) for multiple brain

metastases.377 There are currently more than 50 clinical studies under curse with thalidomide. It is

being evaluated in combination with standard therapies in phase III in prostate, ovarian and renal


cancer, SCLC, and multiple myeloma, and in phase II in multiple myeloma, hematological cancer,

glioma, glioblastoma, liver cancer, myelodysplastic syndrome, uterine sarcoma, and SCLC.

B. Carboxyamido-Triazole (CAI)

Carboxyamido-triazole (CAI) (Fig. 6) is a lowmolecular weight synthetic compound that inhibits the

cellular calcium input. It inhibits both endothelial cell proliferation and invasion, through inhibition

of endothelial adhesion and spreading, inhibition ofmigration and induction of apoptosis.378–381CAI

decreases the expression of MMP2 and VEGF in vitro, and that of VEGF and IL-8 in animal

models.382,383 CAI inhibits vascular tube formation in Matrigel and aortic ring assays and

angiogenesis in the chicken chorioallantoic membrane assay by blocking the calcium-dependent

nitric oxide synthase VEGF pathway.380–383 In clinical trials, CAI stabilizes and in some cases

reduces the tumoral mass and the metastatic spread at plasma concentrations that are in the range of

those that regulate the cell signaling in vitro.384,385 CAI anti-angiogenic and anti-metastatic

properties, together with its possible oral administration has led it to be tested in clinical trials, with

moderate positive results.385–388 CAI is currently being evaluated in combination with paclitaxel in

phase I in patients with advanced solid tumors or refractory lymphomas.

C. NM-3

NM-3 (Fig. 6) (ILEXOncology, Boston,MA,www.ilexonc.com) is an isocumarinic derivative orally

administrable that inhibits hypoxia-induced VEGF expression in human fibroblasts and a number of

tumor cell lines.389 NM-3 inhibits the proliferation of human umbilical vein endothelial cells in vitro,

Figure 6. Othermiscellaneous inhibitors ofangiogenesis.


at concentrations 10-fold less than those required to inhibit normal fibroblasts or tumor cells (HT29,

MKN28, andMCF-7), and this effect is mediated by the generation of reactive oxygen species.390,391

NM-3 inhibits endothelial sprouting and tube formation in vitro, and exhibits in vivo activity against

Lewis lung carcinoma and human tumor xenografts, potentiating the anti-tumor effects of

chemotherapy and radiotherapy without an increase in toxicity.391–393 NM-3 is currently being

evaluated in phase I in patients with advanced solid tumors.

D. Tecogalan

Tecogalan (DS-4152) (Daiichi Pharmaceutical Corp., Fort Lee, NJ) is a polysaccharid–

peptidoglycan complex, isolated from the culture medium of Arthrobacter bacteria. Tecogalan

inhibits the bFGF-stimulated proliferation, migration, and tube formation of bovine choroidal

endothelial cells in vitro.394 It inhibits angiogenesis in the CAM, the corneal neovascularization

induced by bFGF, and the murine tumor M5076 cell-induced s.c. angiogenesis.395 Tecogalan

significantly inhibited the growth of s.c.-implantedB16melanoma, and inhibited the growth of tumor

xenografts of human breast cancer cells.395,396 Tecogalan anti-angiogenic activity has been proposed

to be related to its ability to block the angiogenic factors that bind heparin (bFGF, among others) as

well as the inhibition of proteases (u-PA and MMP1).397 After being assayed in phase I in patients

bearing tumors that are refractory to chemotherapy, it has been discontinued.398

E. Aeroplysinin-1

Aeroplysinin-1 is a bromotyrosine that can be isolated from several marine sponges. It inhibits

angiogenesis in vitro and in vivo, mainly due to the inhibition of endothelial cell differentiation.

Aeroplysinin-1 induces apoptosis in proliferating endothelial cells, and modifies their proteolytic

balance toward less invasive phenotypes, which results in an inhibition of the endothelial cells

migration and invasion in vitro.334 Some studies devoted to the evaluation of the aeroplysinin anti-

tumoral and anti-angiogenic activities in vivo, as well as the determination of its possible mechanism

of action, are under course.

F. Use of ‘‘Traditional’’ Chemotherapeutic Agents as Inhibitors of Angiogenesis

The increasing interest in the search of new strategies for inhibiting angiogenesis has provoked the

evaluation for anti-angiogenesis of many cytotoxic compounds traditionally used in cancer

chemotherapy. In spite of many chemotherapeutic drugs that have been reported to exhibit anti-

angiogenic properties, the clinical relevance of these results is unclear.

Miller et al. have proposed some criteria to consider a given drug useful for anti-angiogenic

therapies: (i) the compound is toxic for angiogenic endothelial cells at doses lower than those

required for killing tumor cells, (ii) it interferes with any activated endothelial cell functions without

causing cell death, (iii) it interferes specifically with any of the angiogenesis steps, and (iv) it inhibits

angiogenesis in in vivo assays.399

Among the number of anti-tumoral compounds being evaluated for anti-angiogenic therapies,

the taxanes stand out. Two taxanes are currently being studied in the clinic.400–403 Paclitaxel (taxol)

and docetaxel (taxotere), Figure 6, have demonstrated anti-tumoral activity in several solid

tumors,403–405 acting as potent radiosensibilizators.406–409 It has been recently reported that the

endothelial cells are 10–100 times more sensitive than tumor cells to these compounds. In addition,

these compounds mainly inhibit the proliferation and differentiation of endothelial cells, inducing

apoptosis, with docetaxel being ten times more active than paclitaxel. All these data should be taken

into accountwhen designing new clinical trials with taxanes as angiogenesis inhibitors.410 Therefore,

while traditional chemotherapy is normally administrated at the maximum tolerated doses, followed

by long resting periods, the chemotherapy ‘‘anti-angiogenic administration’’ advises the use of

prolonged expositions to low concentrations of the drug.411


7 . C O N C L U D I N G R E M A R K S : T H E F A L L A N D R I S E O FA N T I - A N G I O G E N I C D R U G S

Angiogenesis inhibition has been proposed as a general strategy to fight cancer and other

angiogenesis-dependent diseases.412 However, in spite of the promising pre-clinical results, a first

generation of anti-angiogenic compounds gave poor results in clinical trials.413 Conceptual errors

and mistakes in the design of trials and in the definition of clinical endpoints could account for these

negative results.414 The discovery and pharmacological development of future generations of

angiogenesis inhibitors will benefit from further advances in the understanding of the mechanisms

involved in human angiogenesis. New styles of trials are necessary, to avoid missing potential

therapeutic effects. Different clinical endpoints, new surrogate biomarkers, and methods of imaging

will be helpful in this process.413–416 As shown in the case of the first relevant successful phase III

trial involving an anti-angiogenic compound,10 real efficacy in clinical trials may come with the

combined use of anti-angiogenic agents with conventional chemotherapy or radiotherapy, and

combinations of several anti-angiogenic compounds with different mechanisms of action.415

Several strategies to increase efficacy of anti-angiogenic therapy may be followed. They include

metronomic chemotherapy (continuous low-dose chemotherapy), and combination of anti-

angiogenic drugs and anti-HIF drugs, among others. Pre-clinical results indicating that anti-

angiogenic therapies are more effective in earlier than in later stages of the disease, could help to

understand the failure of so many clinical trials with patients in late-stage disease, and advised

new trials to be carried out with patients in earlier stages. However,more recently it has been reported

that potent VEGF blockade can cause regression of established tumors and metastases, indicating

that new generations of anti-VEGF drugs may be effective as single agents in the treatment of bulky

advanced-stage cancer.

On the other hand, one of the main concerns regarding the problems that could derive from an

anti-angiogenic therapy is now being discarded. By targeting endothelial cells, the anti-angiogenic

therapy could cause hypoxia therefore provoking an activation of cancer cells, and increasing their

capability to induce angiogenesis, to invade and metastasize. Recent results demonstrate that

although a shift from normoxia to hypoxia indeed activates cancer cells for aggressive behavior, most

cancer cells are constitutively activated by physiological hypoxia, and anti-angiogenic therapy may

not galvanize them further.417

As originally conceived, anti-angiogenic therapy would inhibit the formation of new blood

vessels without affecting the pre-existing ones. However, recent evidences indicate that some

angiogenesis inhibitors do not only block angiogenesis in tumors, but can also change or destroy

abnormal tumor vessels, causing vessel regression and normalization as well as stromal

maturation resulting in a reversion to a non-invasive tumor phenotype.418,419 The vascular

normalization process prunes immature vessels and improves the integrity and function of the

remaining vasculature by enhancing the perivascular cell and basement membrane coverage, and

induces a hydrostatic pressure gradient across the vascular wall, which leads to a deeper

penetration of molecules into tumor.420 In this way, certain anti-angiogenic agents could

transiently ‘‘normalize’’ the abnormal structure and function of tumor vasculature to make it more

efficient for oxygen and drug delivery.421 This mechanism might contribute to the frequently

observed potentiation of conventional therapies by anti-angiogenic agents. The normalization

process is mediated by angiopoietin-1 and matrix metalloproteinases and creates a window of

opportunity for improved sensitivity to ionizing radiation and the delivery of chemotherapeutic

drugs.422

Finally, the existing anti-angiogenic strategies should include other alternative and

complementary approaches, such as vascular targeting and angioprevention.423,424 The increasing

number of new anti-angiogenic drugs entering clinical trials clearly shows that the initial

disappointment has been overcome. Exciting new results are awaited in the near future.


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Ana R. Quesada is an Associate Professor of Biochemistry and Molecular Biology at University of Malaga,

Spain and Team Leader of a research group devoted to the search, screening, identification, and characterization

of new drugs with potential modulatory effects on angiogenesis.

RamonMunoz-Chapuli is a Full Professor of Animal Biology at University of Malaga, Spain and Team Leader

of a research group devoted to the study of the mechanisms involved in the control of cardiovascular

development, the embryonal origins of vascular and hematopoietic precursors, the epithelium-mesenchyme

transitions, and the evo–devo relationships of cardiovascular system.

Miguel Angel Medina is an Associate Professor of Biochemistry and Molecular Biology at University of

Malaga, Spain, and an active member of the research group leaded by ARQ. His research interests include

several aspects of basic oncology, including angiogenesis, but also amino acid and biogenic amine transport and

metabolism, as well as plasma membrane electron transport.


anti-angiogenic drugs: from bench to clinical trials

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