Lymphedema People Logo



ANGIOGENESIS AND CANCER - Information Page Two

--------------------------------------------------

Angiogenesis (blood vessel growth), lymphangiogenesis (lymph system growth) are all intrinsically connected with lymphedema and share many of the same genes. We have several pages on both processes.

Pat O'Connor

May 23, 2008

--------------------------------------------------

Angiogenesis and Cancer Control: From Concept to Therapeutic Trial

Steven Brem, MD


Current research into angiosuppressive agents is providing cautious optimism
in the field of clinical cancer research.

Background:  There is extraordinary interest in developing angiosuppressive agents for cancer treatment. Several new agents appear promising for the treatment of a variety of human cancers. Current concepts and new agents in clinical trials are the focus of this article. In particular, the introduction of a new treatment for human brain tumors is presented in detail, using an antiangiogenic agent, penicillamine, and depletion of an obligatory cofactor of angiogenesis, copper.
Methods:  The explosive increase in literature on antiangiogenesis is reviewed using computerized search, findings presented at the recent national cancer and angiogenesis meetings. A specific protocol, NABTT 97-04, "Penicillamine and Copper Reduction for Newly Diagnosed Glioblastoma," is presented as an example of angiotherapeutic drug discovery.
Results: A number of promising molecular approaches are being introduced to suppress tumor angiogenesis. Major categories of angiogenesis antagonists include protease inhibitors, direct inhibitors of endothelial cell proliferation and migration, suppression of angiogenic growth factors, inhibition of endothelial-specific integrin/ survival signaling, chelators of copper, and inhibitors with specific other mechanisms. The preliminary results of early trials offer a glimpse into how antiangiogenesis therapy will be integrated into future care of the patient with cancer.
Conclusions:  Thirty-five antiangiogenesis therapies are currently being evaluated in clinical trials. As we learn more about the fundamental mechanisms of angiogenesis, eg, the role of copper in growth factor activation, effective methods of cancer control will be implemented.

Introduction

One of the great scientific questions of this century remains: What controls the growth of blood vessels? The answer to the biological control of angiogenesis is critical to the clinical control of cancer and other angiogenesis-dependent diseases.

Judah Folkman introduced a sweeping hypothesis in 1971.1 A quarter century later, the paradigm has evolved (Table 1) to accommodate the stream of discoveries coming from not only the Harvard laboratory, but also the National Cancer Institute (NCI) and research centers worldwide. In recent years, with the pace of discovery increasing, the search for an effective, nontoxic, antiangiogenesis therapy appears to be within the grasp of oncology. Antiangiogenic therapy is a high priority for the NCI, especially in the wake of the description of angiostatin and endostatin2-5

Research in antiangiogenesis therapy currently is robust with numerous promising avenues. More time, however, is required to be able to identify the appropriate inhibitor, to meld angiosuppression with the current therapeutic modalities, and to design optimal formulations, routes of administration, and dosing schedules. The results of the current clinical trials6 will shape Food and Drug Administration approval of antiangiogenesis therapy in the near future. Certain drugs, eg, thalidomide, are being used clinically on a limited, "off-label" basis.

Evolution of the Paradigm of Tumor Angiogenesis

The major changes in the paradigm of tumor angiogenesis are summarized in Table 1.

Table 1. — Evolution of the Paradigm of Tumor Angiogenesis
 
Original (1971)
Current (1999)
Angiogenesis Dependency
Tumor growth is angiogenesis-dependent; each increment of tumor growth requires an increment of capillary growth.

Concept validated in hundreds of experiments, using genetic, pharmacological, and physiological control mechanisms.

Concept extended to other "angiogenesis diseases," eg, diabetic retinopathy, atherosclerosis, rheumatoid arthritis, and psoriasis.

Cell-Cell Signaling
Tumor cell stimulates endothelial
cell Proliferation

Tumor cell stimulates endothelial cell proliferation. Also, there is paracrine, reciprocal, bi-directional cellular activation.

Endothelial cell stimulates tumor cell growth.

Molecular Mediator ("Factor") of Angiogenesis
"Tumor angiogenesis factor" produced by tumor cell.

"Dormancy therapy" validated in numerous animal models.

Regression inhibitors observed when combination of angiogenesis inhibitors is used or when antiangiogenesis is used in combination with classic cytotoxic/radiation therapy.

Switch to Malignant Phenotype
Switch to malignancy is characterized by onset of angiogenesis.

Switch to malignant phenotype is linked to angiogenesis.

Numerous signaling systems are involved in oncogenesis, and many of these involve angiogenesis stimulators.

Control of Angiogenesis
Predominantly under the control of an angiogenesis factor produced
by tumor.
Tightly coordinated "balancing act." Numerous oncogenes/suppressor genes, growth factors, and endogenous inhibitors, proteases and protease inhibitors, and trace elements that can switch angiogenesis "on" or "off."
Control of Solid Tumor Growth
Solid tumors larger than 2 mm require blood supply for further growth.
Antiangiogenesis therapy limits solid tumor growth but also may be valuable for control of leukemia and myeloma.
Role of Apoptosis
Angiogenic inhibitors block endothelial cell proliferation.
Angiogenic inhibitors can inhibit endothelial cell proliferation, but also can induce apoptosis in the endothelial cell population and/or increase apoptosis in the neighboring tumor cell population, even if microvascular density is unchanged.
Angiogenesis and Invasion
Invasion and metastatic spread are angiogenesis-dependent.

Angiogenesis and tumor cell invasion/metastatic spread are closely linked. Both require proteolytic degradation of extracellular matrix.

Angiogenesis antagonists suppress invasiveness and metastatic spread.

 

Angiogenesis Dependency of Tumor Growth

The central concept that tumor growth is "angiogenesis dependent"7 is well accepted today, with more than 2,500 scientific reports showing angiogenesis linked to tumor growth.8 Stated concisely, "every increment of tumor growth requires an increment of vascular growth.9

Tumor Angiogenesis Factors

Before the term "cell-cell signaling" became fashionable, Folkman postulated the existence of a specific protein that tumor cells secrete to stimulate capillary endothelial cell proliferation. Such a molecule, if identified, could be a target for therapy. Numerous angiogenic genes and gene products, from both neoplastic and normal tissues, have been isolated, purified, cloned, and produced using recombinant DNA technology (Table 2).

Table 2. — Endogenous Stimulators and Inhibitors of Angiogenesis
   
Stimulators
Inhibitors
Growth Factors Angiogenin
Angiotropin
Epidermal growth factor
Fibroblast growth factor (acidic and basic) Granulocyte colony-stimulating factor
Hepatocyte growth factor/scatter factor
Platelet-derived growth factor-BB
Tumor necrosis factor-alpha
Vascular endothelial growth factor
 
 
Proteases and
Protease Inhibitors
  Cathepsin
Gelatinase A, B
Stromelysin
Urokinase-type plasminogen activator
(uPA)
 

Tissue inhibitor of metalloprotease
(TIMP-1, TIMP-2)

Plasminogen activator-inhibitor-1
(PAI-1)

 
Trace Elements   Copper   Zinc
 
Oncogenes   c-myc
ras
c-src
v-raf
c-jun
  p53 Rb
 
Signal Transduction Enzymes   Thymidine phosphorylase
Farnesyl transferase
Geranylgeranyl transferase
   
 
Cytokines  

Interleukin-1
Interleukin-6
Interleukin-8

  Interleukin-10 Interleukin-12
 
Endogenous Modulators   Alpha v Beta 3 integrin
Angiopoietin-1
Angiostatin II (AT1 receptor)
Endothelin (ETB receptor)
Erythropoietin
Hypoxia
Nitric oxide synthase
Platelet-activating factor
Prostaglandin E
Thrombopoietin
  Angiopoietin-2
Angiotensin
Angiotensin II (AT2 receptor)
Caveolin-1, caveolin-2
Endostatin
Interferon-alpha
Isoflavones
Platelet factor-4
Prolactin (16 Kd fragment)
Thrombospondin
Troponin-1
 

It is now recognized that the endothelial cell, by paracrine mechanisms, produces growth factors that stimulate the proliferation of the tumor cell population. Thus, there is a bi-directional reciprocal signaling of endothelial and tumor cell growth. The major targets of pharmacologic therapies are vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Overall, angiogenesis can be viewed as the result of a complex "yin-and-yang" balance of tightly regulated oncogenes and suppressor genes, stimulatory and inhibitory peptides, proteases and endogenous inhibitors, and microenvironmental factors such as the level of oxygen or copper ion.

Dormancy Therapy

Experiments where tumors were transplanted into an avascular environment proved that angiogenesis was a control point in tumor growth. Tumors deprived of angiogenesis remained dormant indefinitely; rapid logarithmic growth followed the acquisition of a blood supply.10 "Dormancy therapy" remains a powerful concept4,11-13

Solid Tumors and Hematopoietic Tumors

Recent data suggest that antiangiogenesis not only will be useful in the control of solid tumors,14 but also may be valuable as therapy for patients with leukemia and myeloma.15-17

Switch to the Malignant Phenotype

In numerous models, the original concept that the switch from premalignant to malignant lesions is linked to angiogenesis18,19 is validated with elegant models demonstrating a cohort of specific angiogenic genes, growth factors, peptides, and proteases activated during malignant transformation.20-22

Role of Apoptosis

Angiogenic inhibitors suppress endothelial cell proliferation,23,24 but another important control mechanism is the induction of endothelial cell apoptosis. Furthermore, even without changes in microvascular density, apoptosis of tumor cells occurs in proximity to endothelium following treatment with angiogenesis inhibitors.22

Angiogenesis and Invasiveness

Initially, invasion and metastatic spread were viewed as angiogenesis-dependent events.1,14 Reinforcing this concept is the increasing attention placed on the role of matrix proteases that control endothelial cell migration and tubule formation as well as tumor cell migration and spread. From a practical perspective of drug discovery and therapy, most angiogenesis inhibitors also act as anti-invasive or antimetastatic compounds.25,26

Deciphering the Angiogenesis Code

The list of known angiogenic stimulators and inhibitors grows yearly.27 Listed in Table 2 are 35 activators and 18 angiosuppressants. The activators can be divided into seven categories: (1) growth factors, (2) proteases, (3) trace elements, (4) oncogenes, (5) cytokines, (6) signal transduction molecules, and (7) endogenous inducers.

The principal growth factors driving angiogenesis are VEGF, bFGF, and hepatocyte growth factor/scatter factor.28-30 Other positive regulators are angiopoietin-1, angiotropin, angiogenin, epidermal growth factor, granulocyte colony-stimulating factor, interleukin-1 (IL-1), IL-6, IL-8, platelet-derived growth factor (PDGF), and tumor necrosis factor-alfa (TNF-alpha).27,31 Matrix proteins such as collagen and the integrins are critical to angiogenesis. Several proteolytic enzymes critical to angiogenesis and tumor spread include cathepsin, urokinase-type plasminogen activator, gelatinases A/B, and stromelysin.26,32

Angiogenesis is physiologically suppressed by one or more of the known endogenous inhibitors, including angiopoietin-2, angiostatin, endostatin, interferon-alfa, kringle-5, platelet factor-4, prolactin (16kD fragment), thrombospondin, tissue inhibitors of metalloproteinase (TIMP-1, TIMP-2 and TIMP-3), and troponin I.27,33,34

TNF-alpha, transforming growth factor-beta (TGF-beta), or IL-4 are bifunctional modulators. These molecules are either stimulators or inhibitors depending on the amount, the site, the microenvironment, the presence of other cytokines, etc.

Recent attention has been focused on the role of the p53 tumor-suppressor gene in angiogenesis. The p53 gene is inactivated in over 50% of all human cancers. Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and malignant progression.35 Exogenous expression of wt-p53 inhibits angiogenesis in vivo resulting in the formation of dormant tumors.13 By inhibiting angiogenesis, p53 indirectly induces apoptosis in vivo but not in vitro and can revert tumors to a dormant phenotype.13

One of the earliest genetic alterations, for example, in human astrocytoma progression is mutation of the p53 tumor suppressor gene; transfection of wild-type p53 into glioblastoma cells leads to angiosuppression. The transfected cells secrete a factor that neutralizes bFGF and the angiogenicity of the parent glioblastoma cells.36 As nonmalignant fibroblasts progress to tumorigenicity, cells become fully angiogenic in two steps. First, there is loss of both alleles of wild-type p53, which causes a 20-fold drop in secreted thrombospondin and a fourfold increase in secreted VEGF.20 Second, angiogenic activity increases again on transformation by activated ras due to a further twofold increase in secreted protein levels an in overall angiogenic activity. Thus, there is a step-wise change in the angiogenic phenotype in response to oncogene activation and tumor suppressor gene loss involving a decrease in the secretion of inhibitors and the sequential up-regulation of inducers of angiogenesis.20

Other recently discovered examples of endogenous inhibitors include caveolin-1 and -2. These molecules are highly expressed in endothelial cells. Angiogenic growth factors (bFGF, HGF/SF, VEGF) each down-regulate the expression of caveolin. Angiogenesis inhibitors (including angiostatin, fumagillin, and thalidomide) block VEGF-induced caveolin expression.37

Angiogenesis Drug Discovery

The basic algorithm we use for translating a lead compound from the laboratory to the clinic requires five steps (Fig 1). (1) First, there is a defined molecular target, eg, a growth factor receptor, or interference with signal transduction, or inhibition of a critical enzyme. The putative inhibitor would be tested for functional activity, eg, receptor binding, or inactivation of mRNA, or neutralization of enzymatic activity. Such assays could also be used for rapid throughput for screening of potential compounds. For example, Gross et al38 hypothesized that an angiogenic inhibitor, to be effective, would likely inhibit the plasminogen-plasmin system, particularly urokinase (uPA) activity. This strategy of using uPA as a pharmacological target has been used successfully in our laboratory (Fig 2) to identify several inhibitors (eg, penicillamine, suramin, diaminoanthraquinone [DAAQ])39-42 of clinical relevance because of the overexpression of uPA in human brain tumors.43 (2) The next step is to determine in vitro if endothelial cell migration (Fig 3) and proliferation are inhibited. (3) Further studies are done with chick chorioallantoic membrane (Fig 4) that has proved to be a useful, inexpensive screening test. The "gold standard" for an angiogenic activator or inhibitor is the corneal assay. The normally avascular cornea enables quantitation of the number of vessels and the rate of vascular ingrowth. A slow-release polymer containing the inhibitor can be placed in a cornea micropocket, and it can be titrated against a known agonist such as bFGF.40 (5) Finally, a rigorous test of an angiogenesis inhibitor is to evaluate it in a vascularized organ such as the brain (Fig 5).

Fig 1. — Flow diagram for evaluation of a potential pharmacological inhibitor and translation to clinical trial, used in the authorŐs laboratory for drug discovery of angiogenesis antagonists.
Fig 2. — Suramin inhibits protease (urokinase-type plasminogen activator) activity produced by bFGF. Protease activity can be used as a molecular screen to evaluate lead compounds. From Takano S, Gately S, Neville ME, et al. Suramin, an anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation, and induction of urokinase-type plasminogen activator. Cancer Res. 1994;54:2654-2660. Reprinted with permission.
Fig 3. — Endothelial cell migration assay. In response to an angiogenesis antagonist, eg, suramin, endothelial cells fail to migrate. Left, control, without suramin. Right, shows the effect of adding the inhibitor, suramin, 500 µg/mL. The bar equals 100 µm. From Takano S, Gately S, Neville ME, et al. Suramin, an anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation, and induction of urokinase-type plasminogen activator. Cancer Res. 1994;54:2654-2660. Reprinted with permission.
Fig 4. — Chick chorioallantoic membrane assay. Note the central avascular zone where an invisible methylcellulose disc contains an angiogenesis inhibitor (suramin) causing the vessels to regress and grow away from the disc. The assay is an inexpensive in vivo screening test for an angiogenesis inhibitor.

Fig 5. — Effect of copper depletion and penicillamine on angiogenesis in a rabbit brain tumor model. Note the control (left) shows numerous coiled vessels growing into the tumor, resembling the hypervascularity of a human malignant brain tumor. By contrast (right), the treated rabbit has a normal appearing vasculature on the cerebral cortex with a pattern of delicate, orderly blood vessels. From Brem S, Zagzag D, Tsanaclis AM, et al. Inhibition of angiogenesis and tumor growth in the brain. Suppression of endothelial cell turnover by penicillamine and the depletion of copper, an angiogenic cofactor. Am J Pathol. 1990;137:1121-1142. Reprinted with permission.

Strategies for Therapeutic Angiosuppression

Strategies for therapeutic angiosuppression generally involve either interference with the activators of angiogenesis or amplification of the endogenous suppressors. The five classes of angiogenesis antagonists in current clinical trials include inhibitors of proteases (Table 3), endothelial cell migration and proliferation (Table 4), angiogenic growth factors (Table 5), matrix proteins on the endothelial cell surface, such as integrins (Table 6), copper (Table 7) and inhibitors with unique mechanisms (Table 8).

Table 3. — Angiogenesis Inhibitors in Current Clinical Trials: Protease Inhibitors
Drug
Mechanism
Sponsor
Trial
Marimastat Synthetic matrix metalloprotease
inhibitor (MMPI)
British Biotech
Phase III for cancer of breast, lung (non-small cell), pancreas, malignant glioma
 
Bay 12-9566   Synthetic MMPI and inhibitor
tumor growth
 
Bayer
  Phase III for carcinoma of lung, ovary, and pancreas
 
AG3340   Synthetic MMPI  
Agouron/Warner-Lambert
  Phase III for lung (NSCLC);
phase III for prostate cancer
 
CGS 27023A   Synthetic MMPI  
Novartis
  Phase I/II
 
CGS 27023A   Synthetic MMPI  
Novartis
  Phase I/II
 
COL-3   Synthetic MMPI; Tetracycline derivative  
Collagenex; NCI
  Phase I
 
Ć-941 (Neovastat)   Naturally occurring MMPI  
Ćterna
  Phase III for colon and NSCLC
(to start in 1999)
 
BMS-275291   Synthetic MMPI  
Bristol-Myers Squibb
  Phase I
 
Penicillamine   Urokinase inhibitor  
NCI-NABTT; commercially available
  Phase II for glioblastoma
 
Table 4. — Angiogenesis Inhibitors in Current Clinical Trials:
Direct Inhibitors of Endothelial Cell Proliferation/Migration
Drug
Mechanism
Sponsor
Trial
TNP-470 (fumagillin derivative) Inhibits endothelial
cell growth
TAP Pharmaceuticals TAP Pharmaceuticalslymphomas, and acute leukemias; phase II for advanced, adult, solid tumors
 
Squalamine   Inhibits sodium-hydrogen exchanger, NIHE3   Magainin   Phase III for lung (NSCLC); phase III for
prostate cancer
 
Combretastatin   Induction of apoptosis in proliferating endothelial cells   Oxigene   Phase I; phase II to start late 1999
 
Endostatin   Inhibition of endothelial cells   EntreMed   Phase I solid tumor to start late 1999
 
Penicillamine   Blocks endothelial cell migration and proliferation   NCI – NABTT; commercially available   Phase II for glioblastoma
 
Farnesyl Transferase Inhibitor (FTI)   Blocks endothelial cell migration and proliferation   NCI – NABTT   Phase I for solid tumors and glioblastoma
to start 1999 – 2000
-L-778,123   Merck  
-SCH66336   Schering-Plough  
-R115777   Janssen  
 
Table 5. — Angiogenesis Inhibitors in Current Clinical Trials:
Antagonists of Angiogenic Growth Factors
Drug
Mechanism
Sponsor
Trial
Anti-VEGF Antibody Monoclonal antibody that inactivates VEGF Genentech Phase II/III for cancers of the lung, breast, prostate, colorectal, and renal
 
Thalidomide   Blocks activity of angiogenic growth factors (bFGF, VEGF, TNF-alpha)   Celgene   Phase II for Kaposi's sarcoma, glioblastoma, cancer of the prostate, lung, and breast
 
SU5416   Blocks VEGF receptor (Flk-1/KDR) signaling (tyrosine kinase)   Sugen-NCI   Phase I/II for Kaposi's sarcoma; phase I/II for metastatic colorectal cancer; phase I/II for advanced malignancies
 
Ribozyme (Angiozyme)   Attenuates mRNA of VEGF receptors   Ribozyme Pharmaceuticals, Inc   Phase Ia studies completed to establish pharmacokinetics
 
SU6668   Blocks VEGF, bFGF, and PDGF receptor signaling   Sugen   Phase I for advanced cancer
 
PTK787/ZK22584   Blocks VEGF receptor signaling   Novartis   Phase I for advanced cancers (Germany and UK); phase I for glioblastoma and Kaposi's sarcoma; phase I/II for von Hippel-Lindau disease
 
Interferon-alpha   Inhibition of bFGF and VEGF production   Commercially available   Phase II/III
 
Interferon-alpha   Inhibition of bFGF and VEGF production   Commercially available   Phase II/III
 
Suramin   Blocks binding of growth factor to its receptor   NCI – NABTT   Phase II for glioblastoma
 
Table 6. — Angiogenesis Inhibitors in Current Clinical Trials:
Drugs That Inhibit Endothelial-Specific Integrin/Survival Signaling
Drug
Mechanism
Sponsor
Trial
Vitaxin Antibody to alpha-v-beta3 integrin present on endothelial cell surface Ixsys Phase II for leiomyosarcoma
 
EMD121974   Small molecule blocker of integrin present
on endothelial cell surface
  Merck KGaA   Phase I/II for Kaposi's sarcoma, brain tumors, and solid tumors (to begin in 1999)
 
Table 7. — Angiogenesis Inhibitors in Current Clinical Trials: Chelators of Copper
Drug
Mechanism
Sponsor
Trial
Penicillamine
Sulfhydryl group binds copper; clears copper through urinary excretion
NCI-NABTT Brain Tumor Consortium Phase II for glioblastoma
 
Tetrathiomolybdate   Thiol groups tightly bind copper, inactivate copper available to tumor   University of Michigan Cancer Center   Phase I/II trial for advanced metastatic cancer, multiple tumor types
 
Captopril   Chelates copper and zinc; also, inhibitor of MMP and angiotensin converting enzyme   Northwestern University   Phase I/II clinical trial
 
Table 8. — Angiogenesis Inhibitors in Current Clinical Trials:
Angiogenesis Antagonists with Distinct Mechanisms
Drug
Mechanism
Sponsor
Trial
CAI Inhibitor of calcium influx
NCI
Phase II/III for ovarian, non-small cell lung, and renal cell cancers
 
ABT-627   Endothelin receptor antagonist  
Abbott/NCI
  Phase I, refractory prostate and other malignancies; phase II, glioblastoma and prostate
 
CM101/ZDO101   Group B Strep toxin that selectively disrupts proliferating endothelium by interaction with the (CM201) receptor  
CarboMed/Zeneca
  Phase I trials completed; phase II trials
to start
 
Interleukin-12   Induction of interferon-gamma  
M.D. Anderson Cancer Center/Temple University
  Phase I trials for ovarian, renal cell, melanoma, and gastrointestinal cancers; phase I/II for Kaposi's sarcoma, and solid tumors
  Down-regulation of IL-10
Temple University
 
  Inhibits production of matrix metalloproteases
Genetics Institute
 
  Induction of IP-10
Hoffman LaRoche
 
 
IM862   Blocks production of VEGF and bFGF; increases production of the inhibitor
IL-12
 
Cytran
  Phase III for AIDS-related Kaposi's sarcoma
 
PNU-145156E   Blocks angiogenesis induced by
Tat protein
 
Pharmacia and Upjohn
  Phase I trial for solid tumors; phase II planned
 

Angiogenic Inhibitors in Current Clinical Trials

Protease Inhibitors

Marimastat (BB-2516) is the first matrix metalloproteinase (MMP) inhibitor to be tested in humans.44,45 Given orally, marimastat has excellent bioavailability. Phase I studies involved healthy volunteers who received short courses of marimastat; these were well tolerated. Symptoms experienced by many patients with various malignancies included severe joint and muscle pain that were debilitating in more than 60% of patients at doses greater than 50 mg bid. These symptoms were reversible when the drug was stopped. The incidence of fibromyalgia has been decreased by using 10 mg bid of marimastat, the dose used in current studies. Phase II studies involved the use of serum tumor markers as surrogate indicators of antitumor activity. Six studies in patients with colorectal, ovarian, and prostate cancer have been completed. Pooled analysis demonstrates a dose-dependent biological effect; 58% of patients respond at doses greater than 50 mg bid. Alterations of tumor markers correlated with increased survival. Small phase II studies suggest potential activity in pancreatic and gastric cancer. These studies also demonstrate the safety of combining cytotoxic chemotherapeutic agents with marimastat. Ongoing phase III studies are investigating the effects of marimastat in addition to chemotherapy in the treatment of small cell lung cancer, gastric, and pancreatic carcinoma.

Because marimastat inhibits the growth and spread of pancreatic cancer in animal models, Rosemurgy et al46 prospectively studied 64 patients with advanced carcinoma of the pancreas in whom standard treatments failed. Marimastat was well tolerated. Musculoskeletal pain, stiffness, and tenderness emerged as dose-limiting toxicities. The overall median survival was 160 days, with a 1-year survival of 21%. Marimastat was associated with an acceptable toxicity profile, and the preliminary data suggest that long-term oral administration is feasible and safe. Doses of 5, 10, and 25 mg twice daily were identified as the optimal doses to be tested in larger randomized studies.46

BAY 12-9566 is a selective, nonpeptidic oral inhibitor of MMP-2 and MMP-9 angiosuppressive compound. Expression of these two MMPs correlates with a poor prognosis for cancers of the ovary, lung, breast, and colon.47 Clinical pharmacology in phase I studies48 shows that a dose level of 800 mg po bid produced steady-state plasma levels (>100 mg/L) sufficient for preclinical activity, and it shows evidence of antitumor activity in humans, without musculoskeletal toxicities. In 18 of 29 patients treated for an average of 2.5 months, the disease was stabilized.48 The main toxicities were transient dose-related thrombocytopenia and transaminase elevations.

A phase I study determined that BAY 12-9566 could be given safely with standard doses of paclitaxel and the combination of paclitaxel and carboplatin.47 Currently, BAY 12-9566 is in phase III clinical trials for small cell carcinoma of the lung, non-small cell carcinoma of the lung, metastatic pancreatic carcinoma, and recurrent ovarian carcinoma.48

AG 3340 is a novel, synthetic, rationally designed, oral, selective MMP inhibitor that is potent with Ki values against specific gelatinases in the low picomolar range. Because it is not a broad-spectrum MMP inhibitor, it is believed that its clinical toxicity will be limited. It is effective to inhibit tumor growth and angiogenesis in a variety of preclinical models including cancers of the colon, lung (NSCLC), and breast49 as well as human U87 gliomas.50 The combination of AG3340 and several chemotherapeutic agents have been shown to have synergistic effects. Phase I studies, in combination with paclitaxel and carboplatin, showed the combination to be safe and well tolerated.51 The drug is currently in phase III clinical trials for patients with non-small cell lung cancer in combination with paclitaxel and carboplatin, as well as in advanced hormone refractory prostate cancer in combination with mitoxantrone.52,53 The most common side effects are in the musculoskeletal system (occasional joint stiffness, swelling, and limitation of mobility) and fatigue. These side effects cease after the treatment is stopped. Because a reduced rate of growth associated with MMP inhibition may extend beyond the time of disease progression, patients enrolled in these studies may continue therapy with placebo tablets in a blinded manner during second- and third-line therapies. 53

Neovastat (Ć-941) is a naturally occurring (shark cartilage extract), oral agent that shows angiosuppressive and anti-MMP activities in vitro and in the chorioallantoic membrane (CAM) assay.54 It is currently in phase I/II clinical trials for refractory lung, breast, and prostate cancers. In clinical studies with more than 540 patients, there were no serious clinical toxicity or laboratory abnormalities. The main side effects are gastrointestinal (nausea, vomiting), but overall, Neovastat demonstrates a favorable toxicity profile in a wide several clinical studies.54,55 It will be evaluated by the NCI for pivotal phase III trials for treatment of colon and lung carcinomas.

Inhibitors of Endothelial Cell Migration and Proliferation

TNP-470 (fumagillin analog) is a derivative of fumagillin, a naturally occurring inhibitor of angiogenesis and tumor growth.56-58 TNP-470 is not cytotoxic in vitro, but it inhibits bFGF- and PDGF-stimulated endothelial proliferation. In vivo, TNP-470 inhibits angiogenesis in the chick CAM assay, the corneal assay, and the surgically implanted Matrigel assay. In mice, TNP-470 reduces the concentration of bFGF in surgically induced wounds; it does not alter the repair process if given before or 5 days after wounding.57

To date, TNP-470 has been used in more than 188 patients in phase 1 trials and in 127 patients in phase II studies.6 At doses of 70 mg/m2, dose-related, reversible CNS toxicities (encephalopathy, dizziness, ataxia, and imbalance) occasionally occur. Fatigue, a nonspecific symptom, is not dose-related. Current phase II trials are ongoing for patients with glioblastoma (50 mg/m2), stage II/III pancreatic cancer (60 mg/m2), cervical cancer (60 mg/m2), and renal cell carcinoma (60 mg/m2). TNP-470 shows evidence of efficacy in the treatment of cervical cancer59 and Kaposi’s sarcoma.60

Squalamine (MSI 1256 F), originally derived from the liver of a dogfish shark, is a novel noncytotoxic aminosterol with potent antiangiogenic properties in vivo and in vitro.61 Squalamine prevents the neovascularization of tumors by suppression of endothelial cell migration and proliferation. A phase I study was performed to evaluate the toxicity at doses ranging from 6 to 255 mg/m2 per day. Steady-state concentrations in patients treated with 24 to 66 mg/m2 per day resulted in plasma concentrations of 0.37 to 0.51 mg/mL, approximating those required for antiangiogenic effects in vitro. There were no serious (NCI grade 3 or 4) toxicities in a study of 12 patients.62

A second phase I study determined the maximum tolerated dose-limiting toxicity and pharmacokinetics when given as a 120-hour continuous intravenous infusion to patients with advanced cancer.63 Dose-limiting toxicities were grade 3 elevations of transaminase (2 of 16 patients) at a dosage of 538 mg/m2. Other toxicities noted were a grade 2-3 (fatigue), and a grade 1-2 (nausea, anorexia, or myalgia with numbness). Steady-state concentrations (150 to 300 ng/mL) were reached within 24 hours at doses of 6 to 12 mg/m2 per day. Fourteen of 16 patients experienced disease progression after two to nine courses of treatment. Two patients continue to receive treatment. There was no objective tumor response in patients with advanced cancer of the lung (3), ovary (3), melanoma (2), breast (2), pancreas (1), colorectal (1), sarcoma (1), and cholangiocarcinoma (1). Patient accrual is ongoing to define the optimal dose and duration of treatment for phase I/II trials of squalamine in combination with cytotoxic chemotherapy.

Combretastatins are small organic molecules found in the bark of the African bush willow, the Combretum Caffrum. Combretastatins not only suppress proliferating endothelium, but also specifically target tumor endothelium. The combretastatin A-4 prodrug is a derivative of combretastatin, which is activated by a phosphatase selectively amplified in proliferating endothelial cells. Combretastatin A-4 induces apoptosis in human endothelial cells.64 In tumor-bearing mice, combretastatin A-4 significantly enhanced the antitumor effects of radiation therapy.65

Endostatin is a 20kDa C-terminal fragment of collagen XVIII discovered by O’Reilly et al.5 Zinc-binding of endostatin is essential for its antiangiogenic activity.66 Endostatin specifically inhibits endothelial proliferation and potently inhibits angiogenesis and tumor growth. Primary tumors regress to dormant microscopic lesions. There is no known toxicity in animal models. Furthermore, the concept of dormancy therapy4 is being extended to endostatin using cycles of therapy. Endostatin was given to mice bearing Lewis lung carcinoma, T241 fibrosarcoma, or B16F10 melanoma; treatment was stopped when tumors regressed. Tumors were then allowed to regrow. Following resumption of endostatin, after multiple treatment cycles, the tumors did not reappear after discontinuation of therapy. Remarkably, no drug resistance occurs with endostatin or angiostatin.67,68 When angiostatin and endostatin are combined, the tumors do not recur once treatment is suspended. Thus, antiangiogenesis therapy could represent first-line treatment.

There has been great anticipation among oncologists and their patients since the reports of angiostatin and endostatin appeared in the media.3 The NCI staff acknowledge that they had difficulty confirming the results in their own laboratories, but when they visited the laboratory at Harvard and repeated the experiments, they found striking inhibition of Lewis lung carcinoma in mice. The NCI has concluded that there are significant differences in storage, handling, and purification techniques that can alter the activity of endostatin. Preliminary data of the NCI confirm that endostatin is safe and well tolerated in animals. Full-scale toxicology studies of endostatin are underway.

As an outgrowth of the laboratory studies, the NCI plans to initiate phase I trials in patients with solid tumors, including cancers of the lung, breast, colon, prostate, as well as lymphoma. The first phase I trials, with enrollments of 15 to 25 patients, are planned to start in 1999 at the M.D. Anderson Cancer Center and at the University of Wisconsin.

Angiostatin is a 38 kDa circulating, endogenous, antiangiogenic and antimetastatic protein.4,69,70 Angiostatin binds ATP synthase on the surface of human endothelial cells,71 induces apoptosis in endothelial cells72,73 and tumor cells,74 and inhibits endothelial cell migration and tubule formation, but it does not affect growth-factor-induced signal transduction.73 Furthermore, it activates the focal adhesion kinase, suggesting that it subverts the formation of endothelial cells adhesion plaques.73 Expression analysis in angiostatin-treated tumors indicates a decrease in mRNA expression for VEGF and bFGF.74 Angiostatin inhibits matrix-enhanced plasminogen activation to account, in part, for its angiosuppressive and anti-invasive properties.75 Of great interest, angiostatin is generated by free sulfhydryl donors (eg, D-penicillamine and captopril) that may explain, in part, their angiosuppressive properties.70

As a paradigm for potential clinical use, angiostatin is of interest for several reasons. First, it is highly synergistic with endostatin, suggesting that combination antiangiogenesis strategies may be more effective than monotherapy with a single agent. Second, like endostatin, it appears to be effective at specific stages of carcinogenesis.22 Third, it is synergistic with ionizing radiation in multiple tumor models, supporting the concept that angiosuppression will potentiate radiation therapy.76-78 Fourth, angiostatin may be of value in treating vascular malignant gliomas, either by systemic administration74 or by using retroviral or adenoviral vectors for gene-based therapies.79

Angiostatin is not available for human studies at this time. EntreMed Corp (Rockville, Md) is working with the NCI to bring angiostatin to the clinic for initial testing. Bristol-Myers Squibb Co (New York, NY) announced early in 1999 plans to discontinue its direct involvement to bring angiostatin to the clinic, largely due to practical difficulties of manufacturing, stability of the molecule, homogeneity, and purity of batches.

Penicillamine is a low-molecular-weight, antiangiogenetic, copper-chelating molecule discovered to be an effective inhibitor of intracerebral glioma growth, invasiveness, and angiogenesis.24,25,39,80 Penicillamine in vitro is a direct inhibitor of endothelial cell proliferation and migration in clinically relevant concentrations.23,81 Penicillamine has multiple functions in addition to copper chelation that could account for its antiangiogenic activity (Table 9). For example, Gross et al39 discovered that penicillamine is a dose-dependent inhibitor of urokinase-type plasminogen activator. Penicillamine by itself was found to inhibit brain tumor growth, but the inhibition was markedly enhanced when combined with copper depletion24 — another example of synergistic combination antiangiogenesis therapy

Table 9. — Angiosuppressive Mechanisms of Penicillamine and Copper Reduction
Steps in the Angiogenesis
Pathway
Copper Reduction
Endothelial cell (EC) migration Blocks EC migration Blocks copper-stimulation of EC migration
 
Endothelial cell proliferation   Blocks EC proliferation   Blocks copper-stimulation of EC proliferation
 
Collagen synthesis and cross-linking   Inhibits (separate from copper defect)   Inhibits (separate from penicillamine)
 
Growth factor binding factors (eg, bFGF, VEGF)     Copper is a cofactor of heparin and copper-binding growth
 
Growth factor function     Reversibly activates or suppresses growth factor-induced angiogenesis dependent on level of copper
 
Protease activity   Dose-dependent inhibitor of uPA, tPA, gelatinase B activity; stabilizes TIMP  
 
Angiostatin   Converts plasminogen (inert) to angiostatin (inhibitor) sulfhydryl donor  
 
Effect on tumor growth as monotherapy   Mild inhibition   Mild inhibition
 
Effect on tumor growth as combination (penicillamine + copper reduction)   Strong inhibition   Strong inhibition
 

 Farnesyl Transferase Inhibitors (FTIs): Activation of the ras oncogene is an important pathway to stimulate angiogenesis.20,21,82-84 Furthermore, the tumorigenicity of the ras oncogene may be VEGF dependent.85 At our institute, we tested the hypothesis that inhibitors of the enzymes farnesyl transferase and geranylgeranyl transferase would block angiogenesis in vitro.86 Not only did FTI-277 and GGTI-298 produce a dose-dependent inhibition of glioma cell growth, but also the inhibition of ras and related G-proteins directly inhibited endothelial cell proliferation. Thus, the FTI and GGTI compounds, in addition to blocking the proliferation of glioma cells directly, may also function as antiangiogenesis compounds.86

Other inhibitors of farnesyl transferase are being evaluated in clinical trials. These compounds could function in part as angiogenesis inhibitors. L-778,123 (Merck and Co, Inc, Whitehouse Station, NJ) was tested as a continuous 7-day infusion every 3 weeks in a phase I clinical trial.87 Sixteen patients received 35 courses at dose levels between 35 and 560 mg/m2 per day. There were no serious drug related toxicities; mild to moderate nausea and vomiting and fatigue were noted in a few patients. Pharmacokinetic studies showed steady-state concentrations were achieved in the plasma within 3 hours. At a dose of 280 mg/m2 per day, serum steady-state concentrations ranged from 3 to 5 mM, capable of significantly inhibiting the processing of Ras and the growth of tumors with ras mutations in preclinical models. Inhibition of farnesylation of the marker protein, hDJ2, in peripheral blood mononuclear cells paralleled plasma L-778,123 concentrations. The maximal inhibition was achieved on days 4 and 8 and returned to pretreatment levels by day 17. Taken together, the results indicate that L-778,123 is well tolerated at concentrations that produce relevant biological effects in preclinical studies and inhibit protein farnesylation in vivo.87

SCH66336 (Schering-Plough Corp, Madison, NJ) is an oral FTI that has broad antitumor activity in preclinical studies.88-90 H-, K-, and N-ras, most G-proteins, rho B, pre-laminin A, and many other proteins require the posttranslational addition of the isoprenoid farnesyl group (or the related geranylgeranyl group) for biological activity. Daily administration in 24 patients at levels of 25 to 300 mg twice daily (bid) showed a maximal tolerated dose of 200 mg bid.88,90 Reversible toxicities include fatigue, anorexia, nausea, vomiting, and diarrhea.88-90 Further clinical testing is underway.88

A phase I trial of an oral R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) in 12 patients with refractory solid tumors in doses of 60 to 420 mg/m2. Myelosuppression is the main dose-limiting toxicity using this schedule. The estimated maximal tolerated dose is 240 mg/m2 twice daily. Plasma levels of R115777 in the tolerable dose range are similar to effective in vitro concentrations.91

Phase II clinical trials of FTIs for glioblastoma and other malignancies, sponsored in part by the NCI, are in the planning stage.

Antagonists of Angiogenic Growth Factors

Anti-VEGF Antibody: VEGF is a critically important angiogenic growth factor. In animal models, an anti-VEGF monoclonal antibody inhibits tumor growth.92 A phase I trial93 of anti-VEGF antibody in 25 patients with metastatic cancer in doses ranging from 0.1 to 10.0 mg/kg intravenously over 90 minutes showed that the anti-VEGF antibody was well tolerated without grade 3 or 4 toxicities. There was a >=20% incidence of grade 1 or 2 adverse events including headache, asthenia, fever, nausea or vomiting, arthralgias, cough, dyspnea, and rash.93 Although there were no objective responses, a patient with renal cell carcinoma had a 39% reduction in tumor and 13 (52%) of 25 patients had stable disease after 10 weeks. The anti-VEGF dosing resulted in anticipated decreases in free VEGF concentrations. No patients developed an antibody to anti-VEGF antibody.93

A second phase I trial was performed to evaluate safety/toxicity using the antibody to VEGF in combination with cytotoxic chemotherapy; this trial was conducted due to antitumor synergisms in animal models when both were applied.94 The VEGF antibody, given once per week for 8 weeks, could be safely combined with chemotherapy (doxorubicin, carboplatin, or 5-fluorouracil with leucovorin). Neither the pharmacology of the antibody nor the clearance of the chemotherapeutic agent was affected. There were no identifiable late toxicities associated with long-term therapy. Three of 12 patients showed tumor regression, and two patients continue to receive chemotherapy plus the VEGF antibody 11 to 12 months after the start of the study. Randomized trials to determine efficacy are planned.94

Thalidomide: Thalidomide is a potent teratogen and sedative that inhibits angiogenesis induced by bFGF95 and VEGF.37 It is effective orally and has been found useful in the treatment of malignant recurrent gliomas, especially when given in combination with a cytotoxic chemotherapeutic drug such as carboplatin.96

In a phase I/II trial,96 71 patients with recurrent glioblastoma used a maximal tolerated dose of 300 mg/m2. At this dosage, 46 patients were evaluable for efficacy. Thirty-three patients had responses (5 partial, 28 stable disease), and 13 had progressive disease. The median survival was 40 weeks. The median response duration was 24 weeks. Toxicity attributed to thalidomide included drowsiness and constipation. The combination of carboplatin and thalidomide appears more effective than either agent alone for recurrent glioblastoma.96

In a phase II study of 28 patients with metastatic breast cancer, two dose levels (200 mg vs 800 mg per day) were compared for toxicity and efficacy.97 At the 200-mg level, two patients had stable disease and tolerable side effects after 8 weeks of treatment. At the 800-mg dose, side effects included somnolence and peripheral neuropathy. Less severe side effects that did not require dose reduction included fatigue, dry mouth, dizziness, nausea, anorexia, headaches, skin rash, and neutropenia. Overall, thalidomide as a single agent was not effective for this patient population.

Sugen 5416: SU 5416 is a novel synthetic compound, a specific VEGF receptor (Flk-1) antagonist that decreases VEGF-stimulated Flk-1 phosphorylation.98 Because it is a specific angiogenesis antagonist, SU5416 does not directly inhibit tumor cells in vitro. However, SU5416 shows broad antitumor efficacy in subcutaneously implanted tumor xenografts in athymic mice.98 In 63 patients with a variety of advanced cancers, their disease was generally stable after six months of treatment. The tumors included Kaposi’s sarcoma, non-small cell carcinoma of the lung, and colorectal, renal cell, adenoid cystic, and basal cell carcinomas.99 The pharmacokinetics indicated extensive tissue penetration and dose-independent clearance.99 The study is being expanded to treat at the recommended optimal dosage of 145 mg/m2.100

Antiangiogenic Ribozyme: The pharmacology and toxicology of an antiangiogenic ribozyme is being developed. The ribozyme Angiozyme (Ribozyme Pharmaceuticals, Inc, Boulder, Colo) inactivates mRNA for two VEGF receptors (Flt-1 and Flk-1 [KDR]), thereby disrupting the VEGF signaling pathway, inhibiting angiogenesis, and suppressing tumor growth in preclinical models (Lewis lung metastases and colon carcinoma). The ribozyme is well tolerated in animals. Phase Ia trials in humans are completed, showing good pharmacokinetics with a half-life of >2 hours when given subcutaneously.101

SU6668: SU6668 is a potent, broad-spectrum tyrosine kinase inhibitor that combines both antiangiogenic and antitumor properties.102 Given orally, there are measurable plasma levels for 24 hours to support once-daily oral schedule. Preliminary data show that analogs of SU6668 (and SU5416) inhibit glioma cell growth and the production of matrix metalloproteases associated with tumor growth and angiogenesis and that SU5402 (an analog of SU5416 and SU6668) inhibits the formation of matrix metalloproteases produced by cancer cells of the prostate.

Interferon Alpha: Interferon-alpha2a (IFN-alpha2a) was the first angiogenesis inhibitor to be used in clinical trials and was effective in children for the treatment of life-threatening hemangiomas.103-105 However, because of the occurrence of severe neurological toxicity (spastic diplegia), IFN-alpha must be used with caution for treatment of hemangiomas.105

Another clinical indication has been giant-cell tumor of the bone.106,107 Kaban et al106 reported the dramatic regression of a large, rapidly growing, recurrent giant-cell tumor of the mandible. The angiogenic protein bFGF was initially elevated in the urine, but after one year of treatment, the bone tumor regressed and disappeared, the urinary bFGF fell to normal levels, and the mandible regenerated. In a series of 10 patients with unresectable or metastatic giant-cell tumor treated with interferon-alpha2b,107 five patients had major responses including three of five patients with pulmonary metastases. Responses occurred slowly and continued after ceasing therapy. In one case, disease progressed throughout 6 months of therapy and only then started to regress. The median time to maximal response was 3.1 years. Four patients had rapidly progressive disease, and treatment was discontinued. One additional patient had stable disease for 7 months after discontinuation of therapy but then progressed.

These reports demonstrate some of the principles that are important in designing clinical trials of therapeutic angiosuppression for malignant disease. (1) Tumors that express bFGF (or other angiogenic factors such as VEGF) may successfully respond to IFN-alpha or other agents that suppress the expression of bFGF (or VEGF, etc), (2) angiosuppressive therapy, given uninterrupted for one year, can be safe and effective, (3) treatment can be continued for one or more years without the development of drug resistance, and (4) angiosuppressive therapy, in contrast to cytotoxic chemotherapy, requires prolonged treatment and follow-up. Responses can continue and may become noticeable after discontinuing treatment.

Suramin: Suramin is the prototype of a growth factor antagonist and discovered to inhibit the mitogenic action in vitro of numerous growth factors. Suramin inhibits multiple molecular control points of angiogenesis, including the production of urokinase-type plasminogen activator.40 Suramin is a polysulfonated molecule and interferes with the binding of the growth factor to its receptor.108 Suramin inhibits endothelial cell proliferation and migration (Fig 3) and is a dose-related inhibitor of angiogenesis in the chick CAM assay (Fig 4). It was also shown to be effective in the brains of animals where suramin inhibited the proliferation of both glioma and endothelial cells.40 In animals, it produced significant cerebral hemorrhages, possibly because of its heparin-like properties. Based on its angiosuppressive and antiglioma properties, suramin was evaluated by the NABTT Consortium. Using a dosing regimen similar to one found effective for prostate cancer, 12 patients with malignant gliomas were treated.109 Ten of the 12 patients were diagnosed with glioblastoma; 11 of 12 patients had received prior nitrosoureas. The actual drug-related toxicities were mild and reversible.109

The results underscore the challenges to evaluate clinical responses modified by cytostatic angiosuppressive molecules in contrast to classic cytostatic chemotherapeutic agents. One patient removed from the study due to "progression" at 10 weeks had a partial response 7 months later and remains free of dexamethasone with a Karnofsky Performance Status scale of 100%. This patient did not receive post-suramin antineoplastic therapy. Another patient showed disease stabilization and lived for 2.2 years without any other therapy. Pharmacokinetics were available for 11 patients. The target serum concentrations of 100 to 300 µg/mL were achieved, and 9 of the 11 patients were on p450-inducing anticonvulsants.109 In light of these findings, the NABTT CNS Consortium plans a further study of suramin for newly diagnosed malignant glioma patients in combination with radiation therapy. The angiogenesis inhibitor will be evaluated in patients expected to live sufficiently long to benefit from the cytostatic properties, and the primary end-point will be survival in contrast to tumor volumetric reduction.109

Inhibitors of Endothelial-Specific Integrin/Survival Signaling

Integrin Antagonists - Vitaxin: Cheresh110 discovered that the alpha-v-beta3 integrin is a critically important adhesion molecule in the regulation of angiogenesis110 and that it promotes endothelial and tumor cell survival.111 The vascular integrin has been used as a prognostic biomarker in breast cancer112 and can be used for tumor detection using magnetic resonance imaging (MRI).113 The LM609 antibody to the integrin (Vitaxin) has moved from phase I trials to phase II studies. Vitaxin has been proven to be safe and has led to the stabilization of disease in most of the patients treated thus far.114

Copper Antagonists/Chelators

If angiogenesis controls cancer growth, what controls angiogenesis? We are learning that the copper status is critical to the function of the angiogenic growth factors (Fig 6).

Fig 6. — Hierarchy of control mechanisms for tumor growth. The concept of tumor growth driven by angiogenesis is well accepted,7 but what drives angiogenesis? To translate from the molecular or ionic level to the clinical level involves "vertical reasoning."185 Based on several lines of evidence, it is reasonable to hypothesize that angiogenesis is dependent on the copper status.

 Recent work supports our hypothesis that copper mediates the "switch" (Fig 7) of the normally quiescent (G0) endothelium into a proliferative state by activation of the angiogenic growth factors.115 We observed that copper reduction, using a low-copper diet and a chelator of copper (penicillamine), inhibits the angiogenic activity of four structurally diverse angiogenic factors and cytokines (Fig 8).

Fig 7. — Hypothetical scheme of a proposed "copper switch" that turns angiogenesis "on" (copper-sufficient) or "off" (copper deficient). Copper acts as an obligatory cofactor and is permissive to the angiogenic activator. Copper reduction blocks angiogenesis by "switching" endothelial cell into apoptosis pathway or quiescence (G0).
Fig 8. — Four structurally diverse angiogenesis cytokines/growth factors are inactivated by copper withdrawal. From Brem et al.115

The angiogenic activity of bFGF, vascular endothelial growth factor (VEGF), TNF-alpha, and IL-1 were found to be copper dependent.115 Furthermore, copper repletion switches angiogenesis back "on" when a copper-sufficient diet is restored, providing evidence for a novel, physiologic, and metabolic control pathway of angiogenesis. This mechanism could explain the inhibition of angiogenesis observed in the brains of animals by copper reduction.24,25 These observations led to the clinical trial of penicillamine and a low copper diet for patients with glioblastoma (Table 10).

Table 10. — Summary of NABTT Clinical Trial, 97-04: Phase II Clinical Trial of Penicillamine and Reduction of Copper for Angiosuppressive Therapy of Newly Diagnosed Glioblastoma
Sponsor
NCI (CTEP) and NABTT (New Approaches to Brain Tumor Therapy) Consortium
Rationale   Penicillamine
  (1) is a selective inhibitor of angiogenesis,
  (2) forms disulfide bonds that inactivates vascular growth factors,
  (3) contains a sulfhydryl group that converts plasminogen to angiostatin,
  (4) chelates and excretes copper,
  (5) inhibits collagen crosslinking,
  (6) is a protease inhibitor, and
  (7) has a low-molecular weight, active in CNS.
  Copper
  (1) is an obligatory cofactor of angiogenesis,
  (2) stimulates endothelial cell migration and proliferation,
  (3) activates vascular growth factors, and
  (4) is required for collagen synthesis.
Eligibility
At least 18 years old
No previous biological therapy, chemotherapy, or radiation therapy
Must be on stable corticosteroid regimen for at least 1 week
No allergy to penicillin
Histologically confirmed glioblastoma with or without measurable (macroscopic) tumor
Adequate baseline laboratory values
Treatment
Penicillamine (250 mg) is given orally over a 5-week escalation from 250 mg daily to 2 gm daily. Patients receive radiation therapy for 6 weeks beginning on the first day of penicillamine therapy. Patients also maintain a diet low in copper (0.5 mg/day) and receive vitamin B6 daily.
End-points
Primary end-point is time of survival. Secondary end-points are reduction of serum copper level, delay in time to progression, and reduction of tumor volume.
Principal Investigator
Steven Brem, MD, Moffitt Cancer Center, (813) 979-3063.

Copper and Angiogenesis: In an attempt to McAuslan isolate a peptide, endothelial stimulating growth factor, McAuslan and Reilly116 noted a high concentration of copper salts. They postulated that copper was the "active principal" in angiogenesis. Copper, but not other trace metals, stimulated the directional migration of endothelial cells. More recently, copper was found to stimulate directly the in vitro proliferation of endothelial cells.117 In a series of elegant experiments at the NCI, Gullino and co-workers118 discovered that the availability of copper in vivo is critical to the initiation and development of angiogenesis. Using a low copper diet and penicillamine therapy, prostaglandin E-stimulated angiogenesis was suppressed. Diverse angiogenic molecules show high affinity for copper.27 Copper-binding molecules (ceruloplasmin, heparin, and the tripeptide glycyl-histadyl-lysine) are non-angiogenic when free of copper, but they become angiogenic when bound to copper.119

Copper and Cancer: Copper metabolism is profoundly altered in neoplastic development in human cancer and in tumor-bearing animals.120,121 Ceruloplasmin, the principal copper-transporting protein, increases four- to eight-fold during malignant progression, often before tumors become palpable; tumor regression returns ceruloplasmin levels to normal.122,123 Serum copper levels correlate with tumor incidence, tumor burden, malignant progression, and recurrence in a variety of human cancers (Hodgkin’s lymphoma, sarcoma, leukemia, and cancer of the cervix, breast, liver, and lung)124-127 as well as brain tumors.128,129 In preclinical tests, Yoshida et al129 noted that penicillamine and copper reduction lowered the tissue levels of copper in the brain to nearly normal levels, reversing the copper toxicosis associated with a brain tumor.

Tetrathiomolybdate: Merajver et al130 recently reported that copper depletion prevents the development of mammary cancer in HER2/neu+ transgenic mice that were given oral tetrathiomolybdate, a potent and nontoxic chelator copper. When tetrathiomolybdate was given to animals with large tumors, the tumors shrank. Immunohistochemical measurements of factor VIII and apoptosis were used to establish intermediate end-points of antiangiogenesis.130 A phase I/II study of oral tetrathiomolybdate for patients with advanced metastatic cancers is encouraging. Preliminary results show that "in patients who survive long enough to become copper deficient, which takes about three to four weeks, their tumors have stopped growing."131

Captopril: Captopril is an orally administered drug already in widespread use to treat nonmalignant disease. Yet, it can antagonize several steps in the angiogenesis cascade, resulting in decreased tumor growth.132,133 Used commonly as an inhibitor of the angiotensin converting enzyme to treat hypertension, the sulfhydryl group enables captopril, like penicillamine, to convert plasminogen to angiostatin.70 Captopril is known to be a chelator of copper,134,135 or to function as a metalloprotease inhibitor.136 There is anecdotal evidence that captopril, combined with urokinase, causes regression of an advanced refractory malignancy.

Angiogenic Inhibitors With Distinct Mechanisms

Carboxyamido-Triazole (CAI) - An Inhibitor of Calcium Influx: CAI is an orally given, low-molecular-weight synthetic compound that inhibits calcium influx, endothelial cell proliferation, and neovascularization in physiologically attainable concentrations.6,137 The long-term half-life of CAI permits a once-daily schedule. The inhibitory effects of cell-cell signaling, proliferation, and invasion, using CAI, are reversible — similar to other cytostatic agents.32

Phase I clinical trials of CAI showed a safe therapeutic window.32 Disease stabilization was observed in patients heavily pretreated with advanced colorectal, pancreatic, renal cell, ovarian, and breast cancer.137,138 The primary toxicity observed included gastrointestinal intolerance. Serious side effects that were reversible with drug discontinuation included retinal hyperemia and a concentration-dependent cerebellar ataxia possibly associated with cognitive dysfunction.32 Phase II trials are planned for multiple primary disease sites, including patients with brain tumors.

A synergistic antiproliferative effect of CAI and paclitaxel (but not carboplatin) has been observed in vitro and is the basis for an ongoing clinical trial.6

ABT-627 - An Endothelin Receptor Antagonist: Endothelin-1 (ET-1) is a potent, vascular, smooth-muscle mitogen and vasoconstrictor; the expression of endothelin-1 correlates with tumor vascularity and malignancy of human astrocytomas.139 Experimental data support a role for endothelin-1 in the pathophysiology of adenocarcinoma of the prostate140 and the ovary.141 The level of endothelin-1 in plasma is significantly elevated in patients with colorectal cancer and nearly double in patients with colorectal metastases to the liver.142

ABT-627 is a selective receptor antagonist of the ETA receptor. In a dose-escalation study of 26 patients with hormone refractory prostate cancer,143 there were declines in the prostate-specific antigen and/or stabilization of the disease by computed tomography scan and bone scintigraphy in 19 patients (73%). There were no grade 3-4 toxicities. In 29 patients with refractory adenocarcinoma,144 given dosages ranging from 10 to 75 mg/day, the toxicity was minimal. The main side effects were transient grade 2 headache (35%), rhinitis (91%), mild anorexia (35%), and fatigue (35%). Encouraging findings were early tumor marker changes and improvement in pain, and there were no measurable responses in a brief phase I trial.144 Phase II trials are underway for treatment of prostate cancer. A phase II trial is planned for human glioblastoma using the NABTT Brain Tumor Consortium.

CM101 - A Bacterial Toxin That Selectively Attacks Proliferating Vessels: Hellerqvist and colleagues145 discovered that the group B streptococcus toxin (CM101) interacts with receptors selectively on proliferating blood vessels but not on nonproliferating vessels. CM101 induces a complement-activated, cytokine-driven inflammatory reaction targeting a specific receptor, CM201, found in proliferating endothelium. Phase I trials for patients with advanced cancer have been completed with encouraging results.145,146 The optimal dosage is 15 to 25 µg/kg every other week for 10 weeks. CM101 will be developed further by AstraZeneca, Inc, as ZDO101.

Interleukin-12: IL-12 is a multifunctional cytokine discovered to be an antiangiogenic agent147-149 that enhances antitumor activity in preclinical models.150 The antiangiogenic activity of IL-12 is linked to the IFN-gamma-induced IP-10 molecule. Phase I trials using the subcutaneous and intravenous routes have shown responses in patients with renal cell cancer and melanoma. Dose-limiting toxicities encountered at doses of >=1,000 ng/kg, include leukopenia, stomatitis, and elevated transaminases. A phase I trial is underway to determine the maximum tolerated doses in patients with peritoneal carcinomatosis secondary to ovarian and gastrointestinal malignancies.151 Even at doses as low as 3 ng/kg, however, significant biological activity is observed, including modulation of angiogenesis-related molecules.151

IM862 - A Peptide That Blocks Production of VEGF and bFGF and Stimulates IL-12 Production: IM862 is a naturally occurring small peptide that inhibits angiogenesis in the CAM assay. The protein blocks production of VEGF and bFGF. It increases production of an immune-boosting cytokine and an inhibitor of angiogenesis, IL-12.152 When given as intranasal drops to AIDS patients with Kaposi’s sarcoma who were also taking protease inhibitors, there was a response rate of 37% (partial or complete remissions). Adverse effects to IM862 are mild and limited to transient headaches, fatigue, tingling, and nausea.152

PNU145156E - A Suramin Analog That Blocks Angiogenesis Induced by the Tat Protein: PNU145156E is a sulfonated distamycin A derivative and a suramin analog that demonstrates antiangiogenic and antitumor effects in preclinical models.153 It blocks the angiogenesis induced by the Tat protein.154 The recommended dose for phase II studies is 840 mg/m2. Like suramin, it is has a long half-life. In a phase I trial of 29 patients at dose levels of 100 to 1,050 mg/m2 given at 1-hour intravenous infusions every 6 weeks, there were no tumor responses observed nor any changes in serum levels of bFGF or VEGF.155

Emerging Concepts in Angiogenesis Research

Gene Therapies for Antiangiogenesis

Among the most promising of exciting new gene therapies are the regulators of angiogenesis.156 For example, it has been demonstrated that the transfection of antisense-VEGF-cDNA results in down-regulation of the endogenous VEGF and suppresses the ability of glioma cells to form tumors in mice.156 In addition, the transfer of antisense VEGF to U87 malignant glioma cells in vivo, by using an adenovirus (Ad5CMV-alpha), inhibits tumor growth.157 Use of the adenovirus-mediated wild-type p53 gene transfer to human colon carcinoma cells results in decreased levels of VEGF and decreased angiogenesis in vivo.158 Direct injection of the endostatin gene into mouse muscle is followed by local expression of endostatin and detectable levels of endostatin secreted into the bloodstream for up to 2 weeks following a single injection.159

Gene transfer of a cDNA coding for mouse angiostatin into murine fibrosarcoma cells suppresses primary and metastatic tumor growth in vivo. The transfected metastases are maintained in a prolonged dormancy state where high cell proliferation is balanced by apoptosis. The metastases remain avascular for several months.37

Cationic liposome-DNA complex-based intravenous gene delivery targets gene expression to vascular endothelial cells, macrophages, and tumor cells.37 Cationic liposome-DNA complex-based intravenous delivery of the p53 gene was as effective as the angiostatin gene in reducing tumor metastasis and angiogenesis37 and induced the expression of the thrombospondin gene. Combination delivery of multiple genes is as effective as a single gene, suggesting that the genes tested (p53, granulocyte-macrophage stimulating factor, and angiostatin) each inhibit a common angiogenesis pathway.

Recombinant adenovirus directing the secretion of an antagonist of cell-associated urokinase, blocking the attachment of uPA to its receptor (uPAR), was recently shown to control local malignant tumor growth and angiogenesis as well as distant metastases of the Lewis lung carcinoma or human colon carcinoma xenograft when given systemically or locally by injection into the tumor.160

A promising approach is the use of retroviruses encoded with a deficient VEGF receptor-2 (VEGFR-2). Survival time of rats with intracerebral tumors was significantly prolonged in a dose-dependent manner when the retroviruses carrying the VEGFR-2 were co-transplanted with tumor cells. Controls, cells without virus or the supernatant, failed to show an effect on survival. Tumors showed signs of impaired angiogenesis. Retrovirus-mediated gene transfer was found to be safe without signs of changes observed in other tissues.161 A similar approach is to transfect tumor cells with ribozymes that reduce mRNA of VEGF and cause a reduction of more than 70% in the VEGF expression level.162

Problems that bedevil gene transfer technology include insufficient distribution of vectors in human tumors and low transduction efficiency.163 The development of new strategies targeting tumor angiogenesis is one of the major steps to improve the efficacy of gene therapy, including the role of gene therapy as part of a combined treatment approach.163

End-points for Determination of Efficacy of Angiosuppressive Agents in Clinical Trials

The development of antiangiogenesis therapies would be accelerated by the availability of surrogate end-points to determine efficacy of treatment.164 Attempts to identify angiogenic proteins in the urine or serum,165 eg, bFGF or VEGF, are problematic and inconsistent. Angiogenesis is a tightly regulated local tissue phenomenon, and systemic metabolic changes such as changes in copper metabolism are not currently well understood. The use of immunohistochemical tissue markers (eg, thrombospondin, CD-31, factor VIII antigen, E-selectin, microvascular density, p53, uPA, uPA receptor) not only may correlate with survival, but also may predict responders and nonresponders to angiosuppressive therapy.166-173

No single, reproducible, specific, sensitive serum biomarker exists today, but a major effort remains to develop one. For example, a recent phase I study of SU5416 measured several putative angiogenesis biomarkers, TNF-alpha, tPA-Ag, IL-8, plasma tissue factor, VEGF, soluble E-selectin, and coagulation tests. There was no change in any of the biomarkers except for a transient decrease in VEGF within 8 hours following the dose.100

Noninvasive imaging techniques to monitor antiangiogenesis prospectively are being developed using MR spectroscopy, perfusion MR, vascular permeability changes, or other detectable changes in the microcirculation.174-176 These techniques may be sufficiently sensitive to detect a decrease in tumor metabolism and mitotic rate while undergoing angiosuppressive therapy.174 MRI contrast enhancement correlates with microvascular density,177 supporting our observation that angiogenesis to be a determinant of radiographic contrast enhancement.178 A standardized yet sensitive method of MRI to detect efficacy of antiangiogenesis therapy would represent a significant advance.

Comparison of Clinical Application of Standard Chemotherapy and Angiosuppressive Therapy

Notable differences between classic cytotoxic chemotherapy and cytostatic antiangiogenesis therapy are summarized in Table 11.

Table 11. — Comparison of Classic Cytotoxic Chemotherapy and
Cytostatic Angiosuppressive Therapy
   
Cytotoxic Chemotherapy
 
Cytostatic Antiangiogenesis
Onset of Effect
Rapid Slower
 
End-point for Tumor Response   Reduction in tumor volume   Time to progression/time of survival
 
Toxicity   Significant (anemia, leukopenia, gastrointestinal, immunological, alopecia)   Mild and well-tolerated; concerns of delayed wound-healing and infertility
 
Combination Therapy   Multiple agents used frequently. Utilized in conjunction with surgery and radiation therapy.   Synergistic with other antiangiogenic therapies, radiation therapy, cytotoxic chemotherapy, and as adjunct to cytoreductive surgery to prevent recurrence.
 
Molecular Target   Single or multiple   Single or multiple. Multiple targets (or "panstasis") may be desirable because of "biochemical redundancy" in the control of angiogenesis.
 
Staging   Selection of chemotherapy regimen determined by organ site and the
TNM classification
  Antiangiogenesis therapy is dependent on stage and size (ie, preventive for microscopic disease, intervention for small tumor, and adjuvant for large, end-stage tumor). Organ specificity is not established.
 

Less Toxicity: Because angiosuppressive molecules do not target proliferating epithelial cells, while the endothelium of skin and the gastrointestinal tract are quiescent, angiosuppressive drugs are unlikely to cause bone marrow suppression, gastrointestinal symptoms, or hair loss. However, as with standard chemotherapy or radiation therapy, wound healing could be delayed with antiangiogenesis therapy.57 Furthermore, angiosuppression could interfere with fertility because of the important role of angiogenesis in ovulation.179 Angiosuppressive molecules would also be potentially harmful to a fetus because of the role of neovascularization in embryogenesis.95

Tumor Regression vs Disease Stabilization: Because antiangiogenic drugs do not necessarily kill tumors or dissolve the already established microvasculature, the end-point of early clinical trials may be different than that of standard therapies. Rather than focusing on tumor volumetric response, therapeutic objectives that are more appropriate would be increases in survival time and/or time to disease progression.6,180 Assessment of efficacy of cytostatic inhibitors needs to avoid the pitfall of requiring tumor shrinkage in order to proceed with clinical development.180 Classic cytotoxic chemotherapeutic trials seek to define maximal tolerable doses in phase I trials; by contrast, the optimal biological dose is more relevant to trials of therapeutic angiosuppression.6,180

Drug Resistance: A major problem with current cytotoxic chemotherapy is drug resistance, in part because the malignant cells are genetically unstable and heterogeneous. By contrast, the target population of antiangiogenic drugs consists of a stable, diploid population of endothelial cells. In long-term preclinical studies, drug resistance to angiosuppressive molecules is not observed.67,68

Combination Therapy: Although phase I/II studies evaluate individual agents (monotherapy), angiosuppression could prove to be more effective given in combination with standard therapy, eg, chemotherapy,47,51,94,96,108,150,181,182 radiation,76-78 surgery, or other antiangiogenic compounds.22,183

Staging of Therapy: Just as the selection of current cytotoxic chemotherapy is predicated on the specific pathology, anatomic site, and stage of the disease (eg, the TNM classification), it is likely that antiangiogenesis protocols will be site- and stage-specific. In an elegant model of multistage carcinogenesis, Bergers et al22 recently showed distinct efficacy profiles of four inhibitors of angiogenesis. Three distinct stages of disease progression were defined: the angiogenic switch in premalignant lesions, intervention during the rapid expansion of small tumors, and regression of large end-stage cancers.22 Newer anticancer therapies are typically introduced for the latter group, but with antiangiogenesis compounds, therapeutic intervention at the earlier stages of disease progression or as a chemopreventive agent may prove fruitful.

As there is currently no drug approved by the Food and Drug Administration for antiangiogenesis, there are many opportunities for drug discovery and development. It is presumed that certain compounds will be more effective for specific types of tumors. We proposed that brain tumors would be the most vulnerable to antagonists of angiogenesis, based on their high degree of microscopic angiogenesis (Fig 9).184

Fig 9. — Hierarchy of endothelial dependency based on quantitative measurement of angiogenesis in a variety of solid tumors. Based upon this model, brain tumors, which show the highest degree of vascularity, should be the most susceptible to antiangiogenesis treatment. From Brem et al.184

 Conclusions

The field of angiogenesis research, once the domain of a few laboratories, has enjoyed spectacular growth since the early work of its pioneers. The NCI has identified angiogenesis research as one of the top cancer research priorities. The 35 biological activators, 18 endogenous inhibitors, and 35 pharmacologic antagonists provide at least 88 reasons for cautious optimism: "It is no longer a question 'if' angiosuppression will work, but rather 'when,' 'what indications,' 'which compound,' 'how much, how fast,' 'what route,' 'what risk,' and 'how long.' Much work remains, but the final chapter of the angiogenesis story should be its most challenging and rewarding."164

Several excellent online resources are available to stay current with the rapidly emerging field of angiogenesis inhibitors. The NCI updates information on clinical trials of antiangiogenic therapy on the Angiogenesis Main Page of the NCI’s cancer trials Web site at http://cancertrials.nci.nih.gov, including a fact sheet on "Angiogenesis Inhibitors in Cancer Research." The information also can be obtained by telephone (Cancer Information Service of the NCI at 1-800-4-CANCER.). The PDQ site can be searched via the Internet or the National Institutes of Health Web search engine at http://search.info.nih.gov.

For details of the Penicillamine/Copper Reduction trial or other angiogenesis inhibitors related to brain tumors, recommended Web sites include (1) the NCI’s comprehensive cancer data base, PDQ, accessible via CancerNet (http://cancernet.nci.nih.gov/), (2) the Al Musella Foundation (http://www.virtualtrials.com/), (3) the NABTT home page (http://www.nabtt.org.), and (4) the Neurooncology Program at Moffitt Cancer Center’s home page (http://www.moffitt.usf.edu.).

References

1. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285:1182-1186.

2. Marshall E. The power of the front page of the New York Times. Science. 1998;280:996-997.

3. Harris AL. Are angiostatin and endostatin cures for cancer? Lancet. 1998;351:1598-1599.

4. O’Reilly MS, Holmgren L, Chen C, et al. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med. 1996;2:689-692.

5. O’Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997;88:277-285.

6. Brem S. Angiogenesis antagonists: current clinical trials. Angiogenesis. 1998;2:9-20.

7. Folkman J. What is the evidence that tumors are angiogenesis dependent? J Natl Cancer Inst. 1990;82:4-6.

8. The Antiangiogenic Therapy Self-Help Book for Cancer Patients. 3rd ed. Angiogenesis Foundation, Inc: Cambridge, Massachusetts; 1999.

9. Folkman J, Klagsbrun M. Angiogenic factors. Science. 1987;235:442-447.

10. Brem S, Brem H, Folkman J, et al. Prolonged tumor dormancy by prevention of neovascularization in the vitreous. Cancer Res. 1976;36:2807-2812.

11. Holmgren L, O’Reilly MS, Folkman J. Dormancy of micrometastases: balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med. 1995;1:149-153.

12. Cao Y, O’Reilly MS, Marshall B, et al. Expression of angiostatin cDNA in a murine fibrosarcoma suppresses primary tumor growth and produces long-term dormancy of metastases. J Clin Invest. 1998;101:1055-1063.

13. Holmgren L, Jackson G, Arbiser J. p53 induces angiogenesis-restricted dormancy in a mouse fibrosarcoma. Oncogene. 1998;17:819-824.

14. Folkman J. Anti-angiogenesis: new concept for the therapy of solid tumors. Ann Surg. 1972;175:409-416.

15. Bikfalvi A, Han ZC. Angiogenic factors are hematopoietic growth factors and vice versa. Leukemia. 1994;8:523-529.

16. Bellamy WT, Richter L, Frutiger Y, et al. Expression of vascular endothelial growth factor and its receptors in hematopoietic malignancies. Cancer Res. 1999;59:728-733.

17. Rajkumar SV, Fonseca R, Witzig TE, et al. Bone marrow angiogenesis in patients achieving complete response after stem cell transplantation for multiple myeloma. Leukemia. 1999;13:469-472.

18. Brem SS, Gullino PM, Medina D. Angiogenesis: a marker for neoplastic transformation of mammary papillary hyperplasia. Science. 1977;195:880-882.

19. Brem SS, Jensen HM, Gullino PM. Angiogenesis as a marker of preneoplastic lesions of the human breast. Cancer. 1978;41:239-244.

20. Volpert OV, Dameron KM, Bouck N. Sequential development of an angiogenic phenotype by human fibroblasts progressing to tumorigenicity. Oncogene. 1997;27:1495-1502.

21. Kerbel RS, Viloria-Petit A, Okada F, et al. Establishing a link between oncogenes and tumor angiogenesis. Mol Med. 1998;4:286-295.

22. Bergers G, Javaherian K, Lo KM, et al. Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science. 1999;284:808-812.

23. Matsubara T, Saura R, Hirohata K, et al. Inhibition of human endothelial cell proliferation in vitro and neovascularization in vivo by D-penicillamine. J Clin Invest. 1989;83:158-167.

24. Brem S, Zagzag D, Tsanaclis AM, et al. Inhibition of angiogenesis and tumor growth in the brain. Suppression of endothelial cell turnover by penicillamine and the depletion of copper, an angiogenic cofactor. Am J Pathol. 1990;137:1121-1142.

25. Brem S, Tsanaclis AM, Zagzag D. Anticopper treatment inhibits pseudopodial protrusion and the invasive spread of 9L gliosarcoma cells in the rat brain. Neurosurgery. 1990;26:391-396.

26. Kohn EC, Liotta LA. Molecular insights into cancer invasion: strategies for prevention and intervention. Cancer Res. 1995;55:1856-1862.

27. Fan TPD, Brem S. Angiosuppression. In: Waring MJ, Ponder B, eds. The Search for New Anti-Cancer Drugs, Cancer Biology Series. Vol. 3. Boston, Mass: Kluwer; 1992:183-228.

28. Benjamin LE, Golijanin D, Itin A, et al. Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal. J Clin Invest. 1999;103:159-165.

29. Brem S, Tsanaclis AM, Gately S, et al. Immunolocalization of basic fibroblast growth factor to the microvasculature of human brain tumors. Cancer. 1992;70:2673-2680.

30. Schmidt NO, Westphal M, Hagel C, et al. Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis. Int J Cancer. 1999;84:10-18.

31. Suri C, McCLain J, Thurston G, et al. Increased vascularization in mice overexpressing angiopoietin-1. Science. 1998;282:468-471.

32. Mikkelsen T. Cytostatic agents in the management of malignant gliomas. Cancer Control. 1998;5:150-162.

33. Maisonpierre PC, Suri C, Jones PF, et al. Angiopoietin-2, a natural antagonist fore Tie2 that disrupts in vivo angiogenesis. Science. 1997;277:55-60.

34. Moses MA, Wiederschain D, Wu I, et al. Troponin I is present in human cartilage and inhibits angiogenesis. Proc Natl Acad Sci U S A. 1999;96:2645-2650.

35. Grant SW, Kyshtoobayeva AS, Kurosaki T, et al. Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and metastatic progression in melanoma. Cancer Detect Prev. 1998;22:185-194.

36. Van Meir EG, Polverini PJ, Chazin VR, et al. Release of an inhibitor of angiogenesis upon induction of wild type p53 expression in glioblastoma cells. Nat Genet. 1994;8:171-176.

37. Liu J, Razani B, Tang S, et al. Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1. J Biol Chem. 1999;274:15781-15785.

38. Gross JL, Moscatelli D, Rifkin DB. Increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro. Proc Natl Acad Sci U S A. 1983;80:2623-2627.

39. Gross JL, Hertel D, Herblin WF, et al. Inhibition of basic fibroblast growth factor-induced angiogenesis and glioma tumor growth in vivo in copper depleted rats. Proc Annu Meet Am Assoc Cancer Res. 1991;32:338. Abstract.

40. Takano S, Gately S, Neville ME, et al. Suramin, an anticancer and angiosuppressive agent, inhibits endothelial cell binding of basic fibroblast growth factor, migration, proliferation, and induction of urokinase-type plasminogen activator. Cancer Res. 1994;54:2654-2660.

41. Herblin WF, Brem S, Fan TP, et al. Recent advances in angiogenesis inhibitors. Exp Opin Ther Patients. 1994;4:641-654.

42. Takano S, Gately S, Jiang JB, et al. A diaminoanthraquinone inhibitor of angiogenesis. J Pharmacol Exp Ther. 1994;271:1027-1033.

43. Landau BJ, Kwaan HC, Verrusio EN, et al. Elevated levels of urokinase-type plasminogen activator and plasminogen activator inhibitor type-1 in malignant human brain tumors. Cancer Res. 1994;54:1105-1108.

44. Rasmussen HS, McCann PP. Matrix metalloproteinase inhibition as a novel anticancer strategy: A review with special focus on batimastat and marimastat. Pharmacol Ther. 1997;75:69-75.

45. Steward WP. Marimastat (BB2516): current status of development. Cancer Chemother Pharmacol. 1999;43(suppl):S56-S60.

46. Rosemurgy A, Harris J, Langleben A, et al. Marimastat in patients with advanced pancreatic cancer: a dose-finding study. Am J Clin Oncol. 1999;22:247-252.

47. Tolcher A, Rowinsky EK, Rizzo J, et al. A phase I and pharmacokinetic study of the oral matrix metalloproteinase inhibitor Bay 12-9566 in combination with paclitaxel and carboplatin. Proc Annu Meet Am Soc Clin Oncol. 1999:617. Abstract.

48. Goel R, Hirte H, Major P, et al. Clinical pharmacology of the metalloproteinase (MMP) and angiogenesis inhibitor Bayer 12-9566 in cancer patients. Proc Annu Meet Am Soc Clin Oncol. 1999:616. Abstract.

49. Shalinsky DR. Broad antiangiogenic and antitumor activities of AG3340, a novel matrix metalloprotease (MMP) inhibitor, in preclinical human tumor models. IBC Fifth Annual Conference: Angiogenesis: Novel Therapeutic Development, Boston, March 1999.

50. Price A, Shi Q, Morris D, et al. Marked inhibition of tumor growth in a malignant glioma tumor model by a novel synthetic matrix metalloproteinase inhibitor AG3340. Clin Cancer Res. 1999;5:845-854.

51. D’Olimpio J, Hande K, Collier M, et al. Phase I study of the matrix metalloprotease inhibitor AG3340 in combination with paclitaxel and carboplatin for the treatment of patients with advanced solid tumors. Proc Annu Meet Am Soc Clin Oncol. 1999:615. Abstract.

52. Nelson NJ. Inhibitors of angiogenesis enter phase III testing. J Natl Cancer Inst. 1998;90:960-963.

53. Collier M, Shepherd F, Ahmann FR, et al. A novel approach to studying the efficacy of AG3340, a selective inhibitor of matrix metalloproteases (MMPs). Proc Annu Meet Am Soc Clin Oncol. 1999. Abstract 1861.

54. Rivičre M. An update of clinical trials in oncology and dermatology. IBC Fifth Annual Conference: Angiogenesis: Novel Therapeutic Development. Boston, March 1999.

55. Evans WK, Latreille J, Batist G, et al. AE-941, an inhibitor of angiogenesis: rationale for development in combination with induction chemotherapy/radiotherapy in patients with non small cell lung cancer (NSCLC). Proc Annu Meet Am Soc Clin Oncol. 1999:1938. Abstract.

56. Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature. 1990;348:555-559

57. Brem H, Ehrlich P, Tsakayannis D, et al. Delay of wound healing by the angiogenesis inhibitor TNP-470. Surg Forum. 1997;48: 714-716.

58. Figg WD, Pluda JM, Lush RM, et al. The pharmacokinetics of TNP-470, a new angiogenesis inhibitor. Pharmacotherapy. 1997;17;91-97.

59. Kudelka AP, Levy T, Verschraegen CF, et al. A phase I study of TNP-470 administered to patients with advanced squamous cell cancer of the cervix. Clin Cancer Res. 1997;3:1501-1505.

60. Dezube BJ, Von Roenn JH, Holden-Wiltse J, et al. Fumagillin analog in the treatment of Kaposi’s sarcoma: a phase I AIDS Clinical Trial Group study. Clinical Trial Group No. 215 Team. J Clin Oncol. 1998;16:1444-1449.

61. Sills AK Jr, Williams JI, Tyler BM, et al. Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vasculature. Cancer Res. 1998;58:2784-2792.

62. Patnaik A, Rowinsky E, Hammond L, et al. A phase I and pharmacokinetic (PK) study of the unique angiogenesis inhibitor, squalamine lactate (MSI-1256F). Proc Annu Meet Am Soc Clin Oncol. 1999. Abstract 622.

63. Bhargava P, Trocky N, Marshall J, et al. A phase I safety, tolerance and pharmacokinetic study of rising dose, rising duration continuous infusion of MSI-1256F (squalamine lactate) in patients with advanced cancer. Proc Annu Meet Am Soc Clin Oncol. 1999:623. Abstract.

64. Iyer S, Chaplin DJ, Rosenthal DS, et al. Induction of apoptosis in proliferating human endothelial cells by the tumor-specific antiangiogenesis agent combretastatin A-4. Cancer Res. 1998;58:4510-4514.

65. Li L, Rojiani A, Siemann DW. Targeting the tumor vasculature with combretastatin A-4 disodium phosphate: effects on radiation therapy. Int J Radiat Oncol Biol Phys. 1998;42:899-903.

66. Boehm T, O’Reilly MS, Keough K, et al. Zinc-binding of endostatin is essential for its antiangiogenic activity. Biochem Biophys Res Commun. 1998;252:190-194.

67. Kerbel RS. A cancer therapy resistant to resistance. Nature. 1997;390:335-336.

68. Boehm T, Folkman J, Browder T, et al. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature. 1997;390:404-7.

69. Sim BK, O’Reilly MS, Liang H, et al. A recombinant human angiostatin protein inhibits experimental primary and metastatic cancer. Cancer Res. 1997;57:1329-1334.

70. Gately S, Twardowski P, Stack MS, et al. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin. Proc Natl Acad Sci U S A. 1997;94:10868-10872.

71. Moser TL, Stack MS, Asplin I, et al. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci U S A. 1999;96:2811-2816.

72. Lucas R, Holmgren L, Garcia I, et al. Multiple forms of angiostatin induce apoptosis in endothelial cells. Blood. 1998;92:4730-4741.

73. Claesson-Welch L, Welsh M, Ito N, et al. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD. Proc Natl Acad Sci U S A. 1998;95:5579-5583.

74. Kirsch M, Strasser J, Allende R, et al. Angiostatin suppresses malignant glioma growth in vivo. Cancer Res. 1998;58:4654-4659.

75. Stack MS, Gately S, Bafetti LM, et al. Angiostatin inhibits endothelial and melanoma cellular invasion by blocking matrix-enhanced plasminogen activation. Biochem J. 1999;340:77-84.

76. Teicher BA, Dupuis N, Kusomoto T, et al. Antiangiogenic agents can increase tumor oxygenation and response to radiation therapy. Radiat Oncol Invest. 1995;2:269-276.

77. Mauceri HJ, Hanna NN, Beckett MA, et al. Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature. 1998;394:287-291.

78. Gorski DH, Mauceri HJ, Salloum RM, et al. Potentiation of the antitumor effect of ionizing radiation by brief concomitant exposures to angiostatin. Cancer Res. 1998;58:5686-5689.

79. Tanaka T, Cao Y, Folkman J, et al. Viral vector-targeted antiangiogenic gene therapy utilizing an angiostatin complementary DNA. Cancer Res. 1998;58:3362-3369.

80. Yoshida D, Ikeda Y, Nakazawa S. Suppression of 9L gliosarcoma growth by copper depletion with copper-deficient diet and D-penicillamine. J Neurooncol. 1993;17:91-97.

81. Gately S, Takano S, Engelhard H, et al. D-penicillamine, a novel antiglioma agent, selectively suppresses endothelial cell growth and migration. Proc Annu Meet Am Assoc Cancer Res. 1993;34:2235. Abstract.

82. Rak J, Mitsuhashi Y, Bayko L, et al. Mutant ras oncogenes upregulate VEGF/VPF expression: implications of induction and inhibition of tumor angiogenesis. Cancer Res. 1995;55:4575-4580.

83. Arbiser JL, Moses MA, Fernandez CA, et al. Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc Natl Acad Sci U S A. 1997;94:861-866.

84. Okada F, Rak JW, Croix BS, et al. Impact of oncogenes in tumor angiogenesis: mutant K-ras up-regulation of vascular endothelial growth factor/vascular permeability factor is necessary, but not sufficient for tumorigenicity of human colorectal carcinoma cells. Proc Natl Acad Sci U S A. 1998;95:3609-3614.

85. Shi YP, Ferrara N. Oncogenic ras fails to restore an in vivo tumorigenic phenotype in embryonic stem cells lacking vascular endothelial growth factor (VEGF). Biochem Biophys Res Commun. 1999;254:480-483.

86. Brem S, Duckworth E, Obadia M, et al. Inhibitors of protein prenylation inhibit glioma and endothelial cell proliferation. Proc Annu Meet Am Assoc Cancer Res. 1999;40:462. Abstract.

87. Britten CD, Rowinsky E, Yao SL, et al. The farnesyl protein transferase (FPTase) inhibitor L-778,123 in patients with solid cancers. Proc Annu Meet Am Soc Clin Oncol. 1999:597. Abstract.

88. Eskens F, Awada A, Verweij J, et al. Phase 1 and pharmacologic study of continuous daily oral SCH 66336, a novel farnesyl transferase inhibitor, in patients with solid tumors. Proc Annu Meet Am Soc Clin Oncol. 1999. Abstract 600.

89. Adjei AA, Erlichman C, Davis JN, et al. A phase I and pharmacologic study of the farnesyl protein transferase (FPT) inhibitor SCH 66336 in patients with locally advanced or metastatic cancer. Proc Annu Meet Am Soc Clin Oncol. 1999:598. Abstract.

90. Hurwitz HI, Colvin OM, Petros WP, et al. Phase I and pharmacokinetic study of SCH66336, a novel FPTI, using a 2-week on, 2-week off schedule. Proc Annu Meet Am Soc Clin Oncol. 1999:599. Abstract.

91. Hudes GR, Schol J, Baab J, et al. Phase I clinical and pharmacokinetic trail of the farnesyltransferase inhibitor R115777 on a 21-day dosing schedule. Proc Annu Meet Am Soc Clin Oncol. 1999:601. Abstract.

92. Ferrara N. Role of vascular endothelial growth factor in regulation of angiogenesis. In: Teicher BA, ed. Antiangiogenic Agents in Cancer Therapy. Totowa, NJ: Humana Press Inc; 1999:119-142.

93. Gordon MS, Talpaz M, Margolin K, et al. Phase I trial of recombinant humanized monoclonal anti-vascular endothelial growth factor (anti-VEGF MAB) in patients with metastatic cancer. Proc Annu Meet Am Soc Clin Oncol. 1999:1678. Abstract.

94. Margolin K, Gordon MS, Talpaz M, et al. Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: pharmacologic and longterm safety data. Proc Annu Meet Am Soc Clin Oncol. 1999:1678. Abstract.

95. D’Amato RJ, Loughnan MS, Flynn E, et al. Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A. 1994;91:4082-4085.

96. Glass J, Gruber ML, Nirenberg A. Phase I/II study of carboplatin and thalidomide in recurrent glioblastoma multiforme. Proc Annu Meet Am Soc Clin Oncol. 1999:551. Abstract.

97. Baidas SM, Isaacs C, Crawford J, et al. A phase II evaluation of thalidomide in patients with metastatic breast cancer. Proc Annu Meet Am Soc Clin Oncol. 1999:475. Abstract.

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

99. Rosen L, Mulay M, Mayers A, et al. Phase I dose-escalating trial of SU5416, a novel angiogenesis inhibitor in patients with advanced malignancies. Proc Annu Meet Am Soc Clin Oncol. 1999:618. Abstract.

100. Cropp G, Rosen L, Mulay M, et al. Pharmacokinetics and pharmacodynamics of SU5416 in a phase I, dose escalating trial in patients with advanced malignancies. Proc Annu Meet Am Soc Clin Oncol. 1999:619. Abstract.

101. Sandberg JA. Pharmacology and toxicology of an antiangiogenic ribozyme. IBC Fifth Annual Conference: Angiogenesis: novel therapeutic development. Boston; March 1999.

102. Shawver LK, Strawn IM, Fong TAT, et al. SU6668 is a potent, broad spectrum angiogenesis inhibitor that exhibits antitumor properties. Proc Annu Meet Am Assoc Cancer Res. 1999;40:4777. Abstract.

103. Folkman J. Successful treatment of an angiogenic disease. N Engl J Med. 1989;320:1211-1212.

104. Folkman J. Seminars in Medicine of the Beth Israel Hospital, Boston. Clinical applications of research on angiogenesis. N Engl J Med. 1995;333:1757-1763.

105. Clifford JL, Miano JM, Lippman SM. Retinoids and interferons as antiangiogenic cancer drugs. In: Teicher BA, ed. Antiangiogenic Agents in Cancer Therapy. Totowa, NJ: Humana Press Inc; 1999:355-370.

106. Kaban LB, Mulliken JB, Ezekowitz RA, et al. Antiangiogenic therapy of a recurrent giant cell tumor of the mandible with interferon alfa-2a. Pediatrics. 1999;103:1145-1149.

107. Benjamin RS, Patel SR, Gutterman JU, et al. Interferon a2b as anti-angiogenesis therapy of giant cell tumors of bone: implications for the study of newer angiogenesis-inhibitors. Proc Annu Meet Am Soc Clin Oncol. 1999:2114. Abstract.

108. Teicher BA. Potentiation of cytotoxic therapies. In: Teicher BA, ed. Antiangiogenic Agents in Cancer Therapy. Totowa, NJ: Humana Press Inc; 1999:277-316.

109. Grossman SA, Brem S, Lesser G, et al. Efficacy, toxicity, and pharmacology of suramin in adults with recurrent high grade astrocytomas. Proc Soc Neurooncol. In press.

110. Cheresh DA. Death to a blood vessel, death to a tumor. Nat Med. 1998;4:395-396.

111. Petitclerc E, Stromblad S, von Schalscha TL, et al. Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival. Cancer Res. 1999;59:2724-2730

112. Gasparini G, Brooks PC, Biganzoli E, et al. Vascular integrin alpha(v)beta3: a new prognostic indicator in breast cancer. Clin Cancer Res. 1998;4:2625-2634.

113. Sipkins DA, Cheresh DA, Kazemi MR, et al. Detection of tumor angiogenesis in vivo by alphaVbeta3-targeted magnetic resonance imaging. Nat Med. 1998:4:623-626.

114. Cheresh DA. Integrins signaling during angiogenesis: therapeutic implications. IBC Fifth Annual Conference: Angiogenesis: Novel Therapeutic Development. Boston; March 1999.

115. Brem S, Wotoczek-Obadia MC. Regulation of angiogenesis by copper reduction and penicillamine: antagonism of cytokine and growth factor activity. AACR Special Conference: Angiogenesis and Cancer Research. Orlando, Fla; January 24-28, 1998. Abstract A-16.

116. McAuslan B, Reilly W. Endothelial cell phagokinesis in response to specific metal ions. Exp Cell Res. 1980;130:147-157.

117. Hu GF. Copper stimulates proliferation of human endothelial cells under culture. J Cell Biochem. 1998;69:326-335.

118. Gullino P. Considerations of the mechanisms of the angiogenic response. Anticancer Res. 1986;6:153-158.

119. Ziche M, Jones J, Gullino PM. Role of prostaglandin E1 and copper in angiogenesis. J Natl Cancer Inst. 1982;69:475-482.

120. Linder M, Hazegh-Azam M. Copper biochemistry and molecular biology. Am J Clin Nutr. 1996;63:797S-811S.

121. Apelgot S, Coppey J, Fromentin A, et al. Altered distribution of copper (64Cu) in tumor-bearing mice and rats. Anticancer Res. 1986;6:159-164.

122. Ungar-Waron H, Gluckman A, Spira E, et al. Ceruloplasmin as a marker of neoplastic activity in rabbits bearing the VX-2 carcinoma. Cancer Res. 1978:38:1296-1299.

123. Senra VA, Lopez SJ, Quintela SD. Serum ceruloplasmin as a diagnostic marker of cancer. Cancer Lett. 1997;121:139-145.

124. Coates RJ, Weiss NS, Daling JR, et al. Cancer risk in relation to serum copper levels. Cancer Res. 1989;49:4353-4356.

125. Gupta SK, Shukla VK, Vaidya MP, et al. Serum and tissue trace elements in colorectal cancer. J Surg Oncol. 1993;52:172-175.

126. Gupta S, Shukla VK, Vaidya MP, et al. Serum trace elements and Cu/Zn ratio in breast cancer patients. J Surg Oncol. 1991;46:178-181.

127. Diez M, Cerdan FJ, Arroyo M, et al. Use of copper/zinc ratio in the diagnosis of lung cancer. Cancer. 1989;63:726-730.

128. Turecky L, Kalina P, Uhlikova E. Serum ceruloplasmin and copper levels in patients with primary brain tumors. Klin Wochenschr. 1984;62:187-189.

129. Yoshida D, Ikeda Y, Nakazawa S. Quantitative analysis of copper, zinc and copper/zinc ration in selected human brain tumors. J Neurooncol. 1993;16:109-115.

130. Merajver SD, Irani J, vanGolen K. Copper depletion as an anti-angiogenic strategy in HER2-neu transgenic mice. AACR Special Conference: Angiogenesis and Cancer Research. Orlando, Fla; January 24-28, 1998.

131. Merajver S. Protocol 97-08. Online: http://www.cancermed.umich.edu/cgi-bin/protocol?9708_701; http://www.cancer.med.umich.edu/news/angio.htm; http://www.healthyliving.com/ 9811110503h.html.

132. Volpert OV, Ward WF, Lingen MW, et al. Captopril inhibits angiogenesis and slows the growth of experimental tumors in rats. J Clin Invest. 1996;98:671-679.

133. Brem S, Gately S, Takano S, et al. Captopril selectively inhibits endothelial proliferation in situ in the rat C6 glioma. Proc Annu Meet Am Assoc Cancer Res. 1994;35:398. Abstract.

134. Kotsaki-Kovatsi VP, Koehler-Samouilidis G, Kovatsis A, et al. Fluctuation of zinc, copper, magnesium and calcium concentrations in guinea pig tissues after administration of captopril (SQ 14225). J Trace Elem Med Biol. 1997;11:32-36.

135. Mueller SA, Driscoll WJ, Mueller GP. Captopril inhibits peptidylgycine-alpha-hydroxylating monoxygenase: implications for therapeutic effects. Pharmacology. 1999;58:270-280.

136. Prontera C, Mariani B, Rossi C, et al. Inhibition of gelatinase A (MMP-2) by batimastat and captopril reduces tumor growth and lung metastases in mice bearing Lewis lung carcinoma. Int J Cancer. 1999;81:761-766.

137. Kohn EC, Reed E, Sarosy G, et al. Clinical investigation of a cytostatic calcium influx inhibitor in patients with refractory cancers. Cancer Res. 1996;56:569-573.

138. Kohn EC, Figg WD, Sarosy GA, et al. Phase I trial of a micronized formulation carboxyamidotriazole in patients with refractory solid tumors: pharmacokinetics, clinical outcome, and comparison of formulations. J Clin Oncol. 1997;15:1985-1993.

139. Stiles JD, Ostrow PT, Balos LL, et al. Correlation of endothelin-1 and transforming growth factor beta 1 with malignancy and vascularity in human gliomas. J Neuropathol Exp Neurol. 1997;56:435-439.

140. Nelson JB, Hedican SP, George DJ, et al. Identification of endothelin-1 in the pathophysiology of metastatic adenocarcinoma of the prostate. Nat Med. 1995;1:944-949.

141. Bagnato A, Salani D, Di Castro V, et al. Expression of endothelin 1 and endothelin A receptor in ovarian carcinoma: evidence for an autocrine role in tumor growth. Cancer Res. 59:720-727.

142. Shankar A, Loizidou M, Aliev G, et al. Raised endothelin 1 levels in patients with colorectal liver metastases. Br J Surg. 1998;85:502-506.

143. Zonnenberg BA, Anbaum B, Kronemeier R, et al. Results of an initial phase I dose-escalation study of the endothelin-A receptor antagonist ABT-627 in patients with hormone refractory prostate cancer. Proc Annu Meet Am Soc Clin Oncol. 1999:626. Abstract.

144. Carducci M, Bowling M, Rogers T, et al. ABT-627, and endothelin-receptor antagonist for refractory adenocarcinomas: phase I and pharmacologic evaluation. Proc Annu Meet Am Soc Clin Oncol. 1999;abst.625.

145. Hellerqvist CG. CM101: An anti-pathoangiogenic agent and its receptor CM2101-areas of application. IBC Fifth Annual Conference: Angiogenesis: Novel Therapeutic Development. Boston; March 1999.

146. DeVore RF, Hellerqvist CG, Wakefield GB, et al. A phase I study of the antineovascularization drug CM101. J Clin Cancer Res. 1997;3:365-372.

147. Voest EE, Kenyon BM, O’Reilly MS, et al. Inhibition of angiogenesis in vivo by interleukin 12. J Natl Cancer Inst. 1995;87:581-586.

148. Coughlin CM, Salhany KE, Wysocka M, et al. Interleukin-12 and interleukin-18 synergistically induce murine tumor regression which involves inhibition of angiogenesis. J Clin Invest. 1998;101:1441-1452.

149. Cavallo F, Di Carlo E, Butera M, et al. Immune events associated with the cure of established tumors and spontaneous metastases by local and systemic interleukin 12. Cancer Res. 1999;59:414-421.

150. Teicher BA, Holden SA, Ara G, et al. Potentiation of cytotoxic cancer therapies by TNP-470 alone and with other anti-angiogenic agents. Int J Cancer. 1994;57:1-6.

151. Lenzi R, Kudelka AP, Vershraegen C, et al. Recombinant human interleukin-12 (RHIL-12) in patients with ovarian and gastrointestinal cancers: evidence of biological activity and lack of significant toxicity with low-dose intraperitoneal (IP) administration. Proc Annu Meet Am Soc Clin Oncol. 1999:1721. Abstract.

152. Tulpule A, Espina BM, Cabriales S, et al. IM862 nasal solution is an active anti-angiogenic agent in the treatment of AIDS-related Kaposi’s sarcoma. Proc Annu Meet Am Soc Clin Oncol. 1999:2065. Abstract.

153. Finch PW, Yee LK, Chen MY, et al. Inhibition of growth factor mitogenicity and growth of tumor cell xenografts by a sulfonated distamycin A derivative. Pharmacology. 1997;55:269-278.

154. Corallini A, Betti M, Rusnati M, et al. Characterization of the effects of two polysulfonated distamycin A derivatives, PNU145156E and PNU153429, on HIV type 1 Tat protein. AIDS Res Hum Retroviruses. 1998;14:1561-1571.

155. De Vries EGE, Groen HJM, Wynendaele W, et al. PNU-145156E-a novel angiogenesis inhibitor-in patients with solid tumors: an update of a phase I and pharmacokinetic study. Proc Annu Meet Am Soc Clin Oncol. 1999:620. Abstract.

156. Fueyo J, Gomez-Manzano C, Yung WK, et al. Targeting in gene therapy for gliomas. Arch Neurol. 1999;56:445-448.

157. Im SA, Gomez-Manzano C, Fueyo J, et al. Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo. Cancer Res. 1999:59:895-900.

158. Bouvet M, Ellis LM, Nishizaki M, et al. Adenovirus-mediated wild-type p53 gene transfer down-regulates vascular endothelial growth factor expression and inhibits angiogenesis in human colon cancer. Cancer Res. 1998;58:2288-2292.

159. Blezinger P, Wang J, Gondo M, et al. Systemic inhibition of tumor growth and tumor metastases by intramuscular administration of the endostatin gene. Nat Biotechnol. 1999;17:343-348

160. Li H, Lu H, Griscelli F, et al. Adenovirus-mediated delivery of a uPA/uPAR antagonist suppresses angiogenesis-dependent tumor growth and dissemination in mice. Gene Ther. 1998;5:1105-1113.

161. Machein MR, Risau W, Plate KH. Antiangiogenic gene therapy in a rat glioma model using a dominant-negative vascular endothelial growth factor receptor 2. Hum Gene Ther. 1999;10:1117-1128.

162. Ke LD, Fueyo J, Chen X, et al. A novel approach to glioma gene therapy: down regulation of the vascular endothelial growth facto in glioma cells using ribozymes. Int J Oncol. 1998;12:1391-1396.

163. Weyerbrock A, Oldfield EH. Gene transfer technologies for malignant gliomas. Curr Opin Oncol. 1999;11:168-173.

164. Brem S. The development of therapeutic angiosuppression: problems and progress. In: Maragoudakis M, ed. Angiogenesis in Health and Disease. New York, NY: Plenum Press. NATO-ASI series; 1992:295-302.

165. Wu JT. Review of circulating tumor markers: from enzyme, carcinoembryonic protein to oncogene and suppressor gene. Ann Clin Lab Sci. 1999;29:106-111.

166. Grant SW, Kyshtoobayeva AS, Kurosaki T, et al. Mutant p53 correlates with reduced expression of thrombospondin-1, increased angiogenesis, and metastatic progression in melanoma. Cancer Detect Prev. 1998;22:185-194.

167. Fox SB, Harris AL. Markers of tumor angiogenesis: clinical applications in prognosis and anti-angiogenic therapy. Invest New Drugs. 1997;15:15-28.

168. Abdulrauf SI, Edvardsen K, Ho KL, et al. Vascular endothelial growth factor expression and vascular density as prognostic markers of survival in patients with low-grade astrocytoma. J Neurosurg. 1998;88:513-520.

169. Weidner N. Tumoural vascularity as a prognostic factor in cancer patients: the evidence continues to grow. J Pathol. 1998;184:119-122.

170. Gasparini G, Harris AL. Prognostic significance of tumor vascularity. In: Teicher BA, ed. Antiangiogenic Agents in Cancer Therapy. Totowa, NJ: Humana Press Inc; 1999:317-340.

171. Ho H, Brem S, Phuphanich S, et al. The uPA receptor: a marker of angiogenesis and malignancy in human gliomas. Proc Soc Neuro-Oncol. 1996:11. Abstract.

172. Li VW, Folkerth RD, Watanabe H, et al. Microvessel count and cerebrospinal fluid basic fibroblast growth factor in children with brain tumours. Lancet. 1994;344:82-86.

173. Brem S. The role of vascular proliferation in the growth of brain tumors. Clin Neurosurg. 1976;23:440-453.

174. Hoffer FA, Taylor GA, Spevak M, et al. Metabolism of tumor regression from angiogenesis inhibition: 31P magnetic resonance spectroscopy. Magn Reson Med. 1989;11:202-208.

175. Dennie J, Mandeville JB, Boxerman JL, et al. NMR imaging of changes in vascular morphology due to tumor angiogenesis. Magn Reson Med. 1998;40:793-799.

176. Lev MH, Hochberg F. Perfusion magnetic resonance imaging to assess brain tumor responses to new therapies. Cancer Control. 1998;5:115-123.

177. Tynninen O, Aronen HJ, Ruhala M, et al. MRI enhancement and microvascular density in gliomas. Correlation with tumor cell proliferation. Invest Radiol. 1999;34:427-434.

178. Zagzag D, Goldenberg M, Brem S. Angiogenesis and blood-brain barrier breakdown modulate CT contrast enhancement: an experimental study in a rabbit brain tumor model. AJR Am J Roentgenol. 1989;153:141-146.

179. Ferrara N, Chen H, Davis-Smyth T, et al. Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nat Med. 1998;4:336-340.

180. Gradishar WJ. End-points for determination of efficacy of antiangiogenic agents in clinical trials. In: Teicher BA, ed. Antiangiogenic Agents in Cancer Therapy. Totowa, NJ: Humana Press Inc; 1999:341-354.

181. Arrieta O, Guevara P, Reyes S, et al. Protamine inhibits angiogenesis and growth of C6 rat glioma, a synergistic effect when combined with carmustine. Eur J Cancer. 1998;34:2101-2106.

182. Sipos EP, Tamargo RJ, Weingart JD, et al. Inhibition of tumor angiogenesis. Ann N Y Acad Sci. 1994;732:263-272.

183. Parangi S, O’Reilly M, Christofori G, et al. Antiangiogenic therapy of transgenic mice impairs de novo tumor growth. Proc Natl Acad Sci U S A. 1996;93:2002-2007.

184. Brem S, Cotran R, Folkman J. Tumor angiogenesis: a quantitative method for histologic grading. J Natl Cancer Inst. 1972;48:347-356.

185. Blois MS. Medicine and the nature of vertical reasoning. N Engl J Med. 1988;318:847-851.


From the Departments of Neurosurgery and Pharmacology of the University of South Florida, and the Neurooncology Program of the H. Lee Moffitt Cancer Center & Research Center, Tampa, FL.

Address reprint requests to Steven Brem, MD, Neurooncology Program, Suite 3136, H. Lee Moffitt Cancer Center & Research Institute, 12902 Magnolia Dr, Tampa, FL 33612-9497.

This publication has been supported by grants from the American Institute of Cancer Research (95B127) and the National Cancer Institute (1 UO1 CA76614-01) to Dr Brem.

http://www.moffitt.usf.edu/pubs/ccj/v6n5/article2.htm

===========================

Lymphedema People Angiogenesis Related Pages:

Angiogenesis

http://www.lymphedemapeople.com/thesite/angiogenesis.htm

Angiogenesis and Cancer

http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer.htm

Angiogenesis and Cancer Control

http://www.lymphedemapeople.com/thesite/angiogenesis_and_cancer_control.htm

Angiogenesis Inhibitors and Cancer

http://www.lymphedemapeople.com/thesite/angiogenesis_inhibitors_and_cancer.htm

 ===========================

Lymphedema People Lymphangiogenesis Related Pages:

The Formation of Lymphatic Vessels and Its Importance in the Setting of Malignancy

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_formation_of_lymphatic_vessels_and_malignancy.htm

Lymphangiogenesis Lymphedema and Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_lymphedema_and_cancer.htm

Lymphangiogenesis and Gastric Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_gastric_cancer.htm

Lymphangiogenesis in Head and Neck Cancer

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_head_and_neck_cancer.htm

Lymphangiogenesis and Kaposi's Sarcoma VEGF-C

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_and_kaposis_sarcoma_vegfc.htm

Lymphangiogenesis in Wound Healing

http://www.lymphedemapeople.com/thesite/lymphangiogenesis_in_wound_healing.htm

A model for gene therapy of human hereditary lymphedema

http://www.lymphedemapeople.com/thesite/a_model_for_gene_therapy_of_human_lymphedema.htm

VEGFR-3 Ligands and Lymphangiogenesis (1)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_1.htm

VEGFR-3 Ligands and Lymphangiogenesis (2)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_2.htm

VEGFR-3 Ligands and Lymphangiogenesis (3)

http://www.lymphedemapeople.com/thesite/vegfr3_ligands_lymphangiogenesis_3.htm

Vascular Endothelial Growth Factor; VEGF

http://www.lymphedemapeople/thesite/vascular_endothelial_growth_factor_VEGF.htm

VEGF-D is the strongest angiogenic and lymphangiogenic effector

http://www.lymphedemapeople.com/thesite/VEGFD_Angiogenic_Lymphangiogenic_Effector.htm

Inhibition of Lymphatic Regeneration by VEGFR3

http://www.lymphedemapeople.com/thesite/Inhibition_of_Lymphatic_Regeneration_by_VEGFR3.htm

VEGFR3 and Metastasis in Prostate Cancer

http://www.lymphedemapeople.com/thesite/VEGFR3_and_Metastasis_in_Prostate_Cancer.htm

===========================

Lymphedema People Genetics, Research, Lymphangiogenesis, Angiogenesis Forum

http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=16

===========================

Join us as we work for lymphedema patients everywehere:

Advocates for Lymphedema

Dedicated to be an advocacy group for lymphedema patients. Working towards education, legal reform, changing insurance practices, promoting research, reaching for a cure.

http://health.groups.yahoo.com/group/AdvocatesforLymphedema/

Subscribe: AdvocatesforLymphedema-subscribe@yahoogroups.com

Pat O'Connor

Lymphedema People / Advocates for Lymphedema

===========================

For information about Lymphedema

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema\

For Information about Lymphedema Complications

http://www.lymphedemapeople.com/wiki/doku.php?id=complications_of_lymphedema

For Lymphedema Personal Stories

http://www.lymphedemapeople.com/phpBB2/viewforum.php?f=3

For information about How to Treat a Lymphedema Wound

http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound

For information about Lymphedema Treatment 

http://www.lymphedemapeople.com/wiki/doku.php?id=treatment

For information about Exercises for Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema

For information on Infections Associated with Lymphedema

http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema

For information on Lymphedema in Children

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children

Lymphedema Glossary

http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing

===========================

Lymphedema People - Support Groups

-----------------------------------------------

Children with Lymphedema

The time has come for families, parents, caregivers to have a support group of their own. Support group for parents, families and caregivers of chilren with lymphedema. Sharing information on coping, diagnosis, treatment and prognosis. Sponsored by Lymphedema People.

http://health.groups.yahoo.com/group/childrenwithlymphedema/

Subscribe: childrenwithlymphedema-subscribe@yahoogroups.com

......................

Lipedema Lipodema Lipoedema

No matter how you spell it, this is another very little understood and totally frustrating conditions out there. This will be a support group for those suffering with lipedema/lipodema. A place for information, sharing experiences, exploring treatment options and coping.

Come join, be a part of the family!

http://health.groups.yahoo.com/group/lipedema_lipodema_lipoedema/?yguid=209645515

Subscribe: lipedema_lipodema_lipoedema-subscribe@yahoogroups.com

......................

MEN WITH LYMPHEDEMA

If you are a man with lymphedema; a man with a loved one with lymphedema who you are trying to help and understand come join us and discover what it is to be the master instead of the sufferer of lymphedema.

http://health.groups.yahoo.com/group/menwithlymphedema/

Subscribe: menwithlymphedema-subscribe@yahoogroups.com

......................

All About Lymphangiectasia

Support group for parents, patients, children who suffer from all forms of lymphangiectasia. This condition is caused by dilation of the lymphatics. It can affect the intestinal tract, lungs and other critical body areas.

http://health.groups.yahoo.com/group/allaboutlymphangiectasia/

Subscribe: allaboutlymphangiectasia-subscribe@yahoogroups.com

......................

Lymphatic Disorders Support Group @ Yahoo Groups

While we have a number of support groups for lymphedema... there is nothing out there for other lymphatic disorders. Because we have one of the most comprehensive information sites on all lymphatic disorders, I thought perhaps, it is time that one be offered.

DISCRIPTION

Information and support for rare and unusual disorders affecting the lymph system. Includes lymphangiomas, lymphatic malformations, telangiectasia, hennekam's syndrome, distichiasis, Figueroa
syndrome, ptosis syndrome, plus many more. Extensive database of information available through sister site Lymphedema People.

http://health.groups.yahoo.com/group/lymphaticdisorders/

Subscribe: lymphaticdisorders-subscribe@yahoogroups.com

===========================

Lymphedema People New Wiki Pages

Have you seen our new “Wiki” pages yet?  Listed below are just a sample of the more than 140 pages now listed in our Wiki section. We are also working on hundred more.  Come and take a stroll! 

Lymphedema Glossary 

http://www.lymphedemapeople.com/wiki/doku.php?id=glossary:listing 

Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema 

Arm Lymphedema  

http://www.lymphedemapeople.com/wiki/doku.php?id=arm_lymphedema 

Leg Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=leg_lymphedema 

Acute Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=acute_lymphedema 

The Lymphedema Diet 

http://www.lymphedemapeople.com/wiki/doku.php?id=the_lymphedema_diet 

Exercises for Lymphedema  

http://www.lymphedemapeople.com/wiki/doku.php?id=exercises_for_lymphedema 

Diuretics are not for Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=diuretics_are_not_for_lymphedema 

Lymphedema People Online Support Groups 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_people_online_support_groups 

Lipedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=lipedema 

Treatment 

http://www.lymphedemapeople.com/wiki/doku.php?id=treatment 

Lymphedema and Pain Management 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pain_management 

Manual Lymphatic Drainage (MLD) and Complex Decongestive Therapy (CDT)

http://www.lymphedemapeople.com/wiki/doku.php?id=manual_lymphatic_drainage_mld_complex_decongestive_therapy_cdt 

Infections Associated with Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=infections_associated_with_lymphedema 

How to Treat a Lymphedema Wound 

http://www.lymphedemapeople.com/wiki/doku.php?id=how_to_treat_a_lymphedema_wound 

Fungal Infections Associated with Lymphedema 

http://www.lymphedemapeople.com/wiki/doku.php?id=fungal_infections_associated_with_lymphedema 

Lymphedema in Children 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_in_children 

Lymphoscintigraphy 

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphoscintigraphy 

Magnetic Resonance Imaging 

http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging 

Extraperitoneal para-aortic lymph node dissection (EPLND) 

http://www.lymphedemapeople.com/wiki/doku.php?id=extraperitoneal_para-aortic_lymph_node_dissection_eplnd 

Axillary node biopsy 

http://www.lymphedemapeople.com/wiki/doku.php?id=axillary_node_biopsy

Sentinel Node Biopsy 

http://www.lymphedemapeople.com/wiki/doku.php?id=sentinel_node_biopsy

 Small Needle Biopsy - Fine Needle Aspiration 

http://www.lymphedemapeople.com/wiki/doku.php?id=small_needle_biopsy 

Magnetic Resonance Imaging 

http://www.lymphedemapeople.com/wiki/doku.php?id=magnetic_resonance_imaging 

Lymphedema Gene FOXC2

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_foxc2

 Lymphedema Gene VEGFC

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_vegfc

 Lymphedema Gene SOX18

 http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_gene_sox18

 Lymphedema and Pregnancy

http://www.lymphedemapeople.com/wiki/doku.php?id=lymphedema_and_pregnancy

Home page: Lymphedema People

http://www.lymphedemapeople.com

Page Updated: Dec. 15, 2011