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ANTIANGIOGENESIS THERAPY
PEDIATRIC ONCOLOGY IN THE 21st CENTURY, PART I1
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ANTIANGIOGENESIS THERAPY Current and Future Agents Mark W. Kieran, MD, PhD, and Amy Billett, MD
Approaches to cancer therapy for most tumors in adults and children have changed little in 50 years: Surgery, radiation, and chemotherapy are standard for many solid tumors. When the concept of angiogenesis in cancer biology first was introduced in the 1970s, there was little recognition of the therapeutic potential of attacking a tumor’s blood supply. Advances in understanding the molecular processes that regulate tumor blood supply and novel agents that can interfere with them have generated a great deal of scientific interest and excitement. This article reviews the current understanding of angiogenesis and its role in cancer, then discusses new therapeutic options in animals and humans, with a focus on pediatric tumors and the potential for treating them. The interested reader can find many excellent reviews of angiogenesis, antiangiogenesis, and pediatric oncology.* ANGIOGENESIS
Angiogenesis is the development of blood vessels. Although the term is differentiated from vasculogenesis (formation of the primitive venous plexus), the two often are used interchangeably. There is evidence that these processes can be regulated or inhibited differentially, and some of the newer generation of inhibitors are capable of interfering *References 12, 14, 15, 36, 46, 57, 87, 90, 100, 112, 115.
From Pediatric Medical Neuro-Oncology (MK) and the Department of Pediatric Oncology (AB), Dana-Farber Cancer Institute; Oncology Inpatient Unit, Children’s Hospital (AB); and Harvard Medical School (MK, AB), Boston, Massachusetts
HEMATOLOGY/ONCOLOGY CLINICS OF NORTH AMERICA
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specifically with one process or the other. From a practical viewpoint, however, the inhibition of either process leads to a halt in the process of developing, maintaining, or expanding a blood supply and may have similar clinical outcomes. Although a complete review of vasculogenesis and angiogenesis (which together are referred to as neovascularization) is beyond the scope of this article, a simplified model of neovascularization is presented in Fig. 1 and has been reviewed by Yancopoulos et al.ll', 112 The proliferation of vascular endothelial cell precursors into a vascular network (primary capillary plexus) is induced by ligand-mediated signal transduction through receptor tyrosine kinases on these cells.26, 85 The best-characterized ligand of this sort is vascular endothelial growth factor (VEGF), which binds a receptor (ftk-1/KDR/VEGFR-2, a positive activator of vasculogenesisz6).Many other ligand-receptor combinations have been identified that may act in concert with VEGF to induce and maintain vascul~genesis.~~~ 26 Secondary factors, especially angiopoietin-1 (ang-1), then act on the primitive capillary plexus to induce sprouting and branching (angiogenesis), which leads to a more mature and stable vascular system.22Angiopoietin-2 (ang-2) is the antagonist of ang-1, and it is the balance of these two molecules23in the presence of VEGF that determines whether vessel proliferation (high VEGF, balanced ang-1 and ang-2), maturation (high VEGF and ang-1, lower ang-2), or regression (low VEGF and ang-1, high ang-2) will proceed.49,lo7 Although these three factors play a central role in regulating vessel formation or destruction, numerous other elements play into this system to provide the exquisite regulation typical of crucial biologic proce~ses."~ The reader is referred to detailed reviews on the origins and interplay of these different factors.'O, 14, 31, 51, 84 A great deal of understanding of the role of angiogenic factors and their receptors has been obtained from the analysis of knock-out mice, from which the genes for these different molecules have been removed.'l2 VEGFR-2 (also called KDR/flk-l) null mice fail to develop a normal vasculature and have decreased endothelial cells.94By contrast, VEGFR1 (also calledflt-2) null mice have increased numbers of endothelial cells, suggesting that this receptor may act as a negative regulator of VEGF.39 VEGFR-3 (also called flt-3) null mice have relatively normal vasculature but have abnormal lymphatic development.102A similar pattern arises when the ligands are manipulated. VEGF null mice appear similar to VEGFR-2 null mice.'6, 32 VEGF heterozygotes have abnormal vascular formation and are embryonically lethal, suggesting the crucial role this molecule has.16,32 When mice have completed their early development, the effects of VEGF depletion become much less signifi~ant.~~ Using similar strategies, the role of the angiopoietins and their receptors has been elucidated. Mice lacking ang-1 or the angiopoietin receptor Tie-2 develop a vasculature, although the vasculature fails to undergo normal lol Over expression of ang-2, mimicking the effects obrern~deling.~~, served with null mutations in ang-1 or Tie-2, also cause vascular defect~.~~
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Figure 1. Neovascularization. A, Endothelial cell precursors respond to the interaction of ligands and receptor interactions inducing endothelial cell proliferation giving rise to the formation of the primitive venous plexus. In the context of other factors and their receptors, such as angiopoietin-1, remodeling of this network takes place. The coexpression of epherins will determine whether the vessel develops as an arteriole or vein. 6,In the continued presence of vascular endothelial growth factor (VEGF) and angiopoietin-1, vessel maturation and stabilization occur. C,If angiopoietin-1 levels decrease and angiopoietin-2 levels increase, then vessel destabilization occurs. This zone of vessel destabilization allows the vessel to grow further (by adding back angiopoietin-I), or regress. 0,Loss of VEGF signalling results in vessel death.
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ANGIOGENESIS AND TUMOR BIOLOGY
The association of an expanding blood supply as a crucial feature in cancer biology was established almost 100 years ago.45Some tumor grading systems specifically assess neovascularization,6° according lesions a higher grade when present (e.g., glioblastoma multiforme versus anaplastic astrocytoma). In other cases, increasing neovascularization correlates with poorer prognosis.41, In 1971, Folkman et a135,38 proposed a new approach to treating malignant tumors. Instead of directing highly toxic therapy at tumor cells, Folkman suggested attacking the blood supply of these lesions. The theoretic advantages of attacking the blood supply are (1) tumor vasculature is less mature and more sensitive to inhibition than normal vessels and (2) although tumors develop resistance rapidly, tumor-induced vasculature has not in animal experiment^.^ This latter aspect is derived from the principle that normal cells, of any lineage, remain under normal cellular control and lack the mechanisms necessary to generate mutations. Animal data have shown the role of angiogenesis in cancer biology. A few examples are as follows: Avascular tumors remain localized and small (<2 to 3 mm).37 Localized tumors that obtain a blood supply grow and disseminate.44 Many factors (e.g., VEGF, basic fibroblast growth factor [bFGF]) that 95 stimulate neovascularization stimulate tumor Inhibitors of angiogenesis (TNP470, anti-VEGF, endostatin, angiostatin) can reverse neovascularization and cause regression of tumor growth. These agents have no direct inhibitory effects on tumor cells.52,59. 74,76 Agents that interfere with endothelial cell surface receptor signaling can inhibit neovascularization and cause regression of tumor 93 growth.72, At least three naturally occurring inhibitors of neovascularization, specific for endothelial cells, that are derived from the cleavage of native proteins have been identified: angiostatin (from pla~minogen~~), endostatin (from collagen XVII176),and a fragment of antithrombin III.77 These naturally occurring molecules, which normally are present in humans, provide the negative signals required to balance input from stimulatory signals of neovascularization. In addition to the role that angiogenesis plays in the progression to clinically evident disease, neovascularization may be a crucial process in the development of metastases.'*,68, 115 Although tumors that metastasize need to generate a blood supply at their new site, their initial ability to gain access to the blood system may be related in part to the development of their angiogenic potential.4l For some tumors, microvessel density and levels of VEGF are independent prognostic indicators of the risk of developing metastases and overall prognosis.41,lo9
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DEVELOPMENT OF ANTlANGlOGENlC AGENTS Agents that interfere with neovascularization can be grouped loosely into a few categories, based on their presumed mechanism of inhibition. A limited number of these agents are provided as examples. Molecules That Interfere With Signals of Neovascularization
In addition to direct inhibitors of bFGF and VEGF (anti-bFGF and anti-VEGF antibodies), inhibition of their targets (anti-flk-1) and inhibition of transmission of the appropriate signal transduction cascade (SU5416, SU6668) are potentially effective means of turning off neovascularization.48,71,88.93.99
Alteration of Endothelial Shape Endothelial cells must form specific junctions that require certain shape constraints. Agents that interfere with endothelial cell shape can prevent vessel formation, even when all of the appropriate signals are present (squalamine).l,56 Inhibition of Metalloproteinases Metalloproteinases are enzymes that appear to play a crucial role in modulating local tissue and vessel matrix as well as the supply of angiogenic stimulators, which, in a simplistic sense, can alter the environment in which neovascularization occurs. Examples of compounds in this class include marimastat, AG-3340, COL-3, AE-941 (Neovastat), and BMS-27529.3, 28, 89,110. 113 Other Mechanisms of Inhibition Many other components involved in neovascularization have been identified, which provide additional targets for antiangiogenic therapy, including the integrins30and cyclooxygenase inhibitor^.^^ Unknown Mechanisms Many agents are being tested that have shown the ability to inhibit neovascularization, although the specific molecular mechanism remains obscure. Whether compounds in this class will fall into one of the aforementioned categories or lead to new categories is unknown. Exam-
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ples include thalid~mide?~ endostatin,76angi~statin;~ and antiangiogenic antithrombin III.77 PRECLINICAL DATA FOR ANTlANGlOGENlC THERAPY
Many agents have been tested in vitro that show antiangiogenic activity. Many of these agents when tested in animal models have resulted in regression or cure.74,76, Although these results have been confirmed in many independent studies, the mechanism by which this occurs remains obscure for many agents. With the development of specific molecular targets, such as VEGF or its receptor Flk-1, investigators can begin to interpret the animal experiments delineating antiangiogenic responses. To understand how these agents work, it is necessary to understand how they were identified. The methods used to identify inhibitors of angiogenesis (which also were used to identify many of the stimulators) have included many appro ache^.^^
Inhibition of endothelial cell proliferation in vitro: Endothelial cells are grown in culture. Drugs to be tested are added in differing concentrations to assess their ability to inhibit endothelial proliferation. These compounds are tested in culture with other cell types to ensure their inhibition is specific for endothelial cells. Inhibition of endothelial cell migration in vitro: Similar to the first assay, this one looks at compounds that interfere with the ability of endothelial cells to move, a crucial feature in neovascularization. Inhibition of endothelial tube formation in vitro: Endothelial cells form three-dimensional tubes that can be replicated in vitro. The addition of drugs to these cultures measures the ability of different agents to disrupt this process. Inhibition of bFGF or VEGF pellet-induced corneal neovascularization in vivo: Small pellets containing slow-release bFGF or VEGF are implanted into the cornea of animals. The animals develop intense neovascularization toward the pellets. Animals are treated systemically with agents, and the ability to reduce or inhibit the neovascularization is measured. Inhibition of chorioallantoic membrane neovascularization in vivo: The chorioallantoic membrane of developing chick embryos is highly vascularized. Small disks containing inhibitors are placed on the membrane, and the zone of inhibition of neovascularization is measured. Decreased in serum or urine bFGF or VEGF levels: Although many angiogenic stimulators have been identified, only a few are easily quantifiable. The two major ones, bFGF and VEGF, can be measured in serum, urine, and cerebrospinal fluid and in tumor specimens. As animals are treated with different inhibitors, decreases in the amount of these molecules can be assessed. Decrease in microvessel density counts of tumor specimens: By examining tissue directly before and after treatment with an agent, a direct
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measure of the number of vessels within the tumor can be determined. Inhibition of one tumor by another growing on an animal: This technique, which has led to the discovery of many of the naturally occurring inhibitors of angiogenesis, is the most difficult to conceptualize. This model is based on a phenomenon recognized by surgeons for more than a century. In a small percentage of cases in which a surgeon removes a localized tumor in a patient without evidence of metastatic disease, within weeks or months of the operation, the patient has widespread metastases. This sudden spread was attributed to the manipulation of the tumor at the time of the operation and extrusion of tumor cells into the vasculature. An alternative explanation, proposed by Folkman in the 1970~:~was that the primary tumor, in addition to secreting factors to stimulate its own blood supply, secreted inhibitors into the circulation. Such inhibitors potentially could prevent metastases from competing for nutrients. If a surgeon removes an apparent localized tumor secreting these inhibitors, the existing microscopic metastases become free to expand. The animal model thought to mimic this clinical phenomenon involves injecting many different clones of a tumor on both flanks of an animal. With most pairs of clones, tumors grow equally on both sides. Rarely, one side grows, while the other remains small (because of inhibitors of angiogenesis produced by the larger tumor mass). If the growing lesion is resected, the smaller side grows rapidly as the inhibitor is removed. By purifying the activity from blood or urine from animals with asymmetric tumor growth, these inhibitors can be isolated. This technique has led to the discovery of many biologically active inhibitors of tumor neovascularization in vivo.74, 76. 77 The preclinical data examining the effect of numerous antiangiogenic agents are extensive, and the reader is referred to reviews and primary articles in the literature.s,48, 67, 83 Some findings from animal studies deserve specific mention because they may affect the design and interpretation of human studies. The first of these findings is the issue of tumor dormancy.44Angiogenesis inhibitors work against dividing endothelial cells, which are most abundant within the small, immature capillaries of the tumor bed. Because the original malignant cells arose from an organ or tissue with a fully mature vasculature, even if all of the tumor-induced neovascularization could be eliminated, tumor cells supplied directly by the original mature vessel could survive. As such, even if completely successful, antiangiogenic therapy likely would control, not eradicate, all of the disease. A small tumor focus that cannot induce neovascularization, even if malignant, can remain in the body indefinitely. Although the cells within the focus are actively proliferating, each new cell is pushed beyond the diffusion capacity of oxygen and nutrients and dies. These lesions are referred to as dormant.47, 50,75
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A second important factor to consider is that to treat the microscopic residual lesion, antiangiogenic therapy likely would require the addition of traditional chemotherapy, radiation therapy, or both. In preclinical studies, the effects of antiangiogenic agents in combination with other therapies would be predicted to be additive in response. Although this has been true for some systems, a surprising result has been observed in others. In these cases, a synergistic instead of additive response was observed when antiangiogenic agents were given with chemotherapy or 114 Although the mechanisms for this response are unradiation.", 92, clear, a possible explanation is inhibition of the paracrine loops that are fundamental to tumor development.22, In addition to producing vasculogenic factors to stimulate the expanding blood supply, the tumor is stimulated by the secretion of factors by the expanding blood supply. For this reason, inhibiting both may account for greater activity than would be expected by inhibiting either alone. Another area of consideration in the use of antiangiogenic agents is the target and what defines an antiangiogenic compound. Most chemotherapy is directed at dividing cells. In addition to tumor cells, normal cells of the hematopoietic system, gastrointestinal tract and hair frequently also are affected. To maintain its growing volume, tumors continually must stimulate endothelial cell turnover so that neovascularization can progress. As such, these dividing cells in the neovasculature may be targeted by traditional drugs. In this sense, many antimitotic agents may act as antiangiogenic compounds. The schedule of most chemotherapy regimens may prevent successful antiangiogenic therapy, however. Only the proportion of endothelial cells in cycle on days of treatment are likely to be killed. Angiogenesis continues between chemotherapy treatments, while normal cell recovery occurs. The suggestion is that long-term administration of low-dose cytotoxic agents could provide a more antiangiogenic effective schedule.l'< This hypothesis could explain responses to low-dose long-term oral etoposide despite progressive disease on high-dose intermittent intravenous therapy2 This potential mechanism is the subject of investigation within the laboratory of one of the authors (MWK). An important concern in the use of angiogenesis inhibitors is the possibility that they will inhibit many of the normal processes within the body that rely on neovascularization. Although this situation was observed with some of the early antiangiogenesis inhibitors,'jl many newer agents appear much more specific? This specificity likely resides in the complex organization of positive and negative signals that regulate neovascularization and a drug's ability to alter one component without upsetting the entire process. Endothelial cells within a tumor should not become resistant to antiangiogenic the rap^.^ As would be predicted in Folkman's original hypothesis, because the endothelial cells making up the tumor's blood supply are inherently normal, they should continue to obey normal signals to proliferate, arrest, or undergo apoptosis. Development of resistance to antiangiogenic agents should thus be unlikely. In numerous
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model systems, with a variety of tumors and agents, this situation has been observed c~nsistently.~ The animal data suggest that antiangiogenic therapy, if successful, is likely to cause tumors to regress to their in situ size, but requires adjunctive therapy if a cure is to be achieved. In contrast to the tumor cells themselves, resistance would not be expected, even in patients who stop and start treatments.
POTENTIAL CHALLENGES TO ANTlANGlOGENlC THERAPY Many theoretic concerns have been proposed to predict the failure of antiangiogenic drugs to treat tumors effe~tively.~~ Examples include the following:
Large tumors have mature vessels that would be insensitive to antiangiogenic therapy. Although large vessels may be present, the capillary bed where oxygen and nutrient exchange occur is the site of neovascularization and the target of these agents.36Because tumors are in a continuous phase of growth, they maintain their capillaries in an immature state so that they can continue to expand with the tumor.35 Some tumors, such as leukemia, grow independent of vessels and would be unaflected by antiangiogenic agents. Data showing the potent expression of inducers of neovascularization and extensive angiogenesis within the bone marrow spaces in leukemia directly refute this ~ t a t e m e n t .In ~ ~animal leukemia models, response to therapy is correlated directly to a decrease in microvessel density. One study using traditional chemotherapy in an antiangiogenic schedule showed that leukemia was highly angiogenic, that low-dose chemotherapy could attack effectively the proliferating endothelial cells within the tumor but not the tumor itself (the tumor cells were made resistant to the drugs), and that tumor cell apoptosis always was preceded by endothelial cell loss.ll, Whether low, long-term dosing of chemotherapy in the treatment of human disease, such as maintenance therapy in acute lymphoblastic leukemia, works by targeting endothelial cells is unknown. Tumors can be divided into angiogenic (red or bloody) and nonangiogenic (white, pale) groups, and antiangiogenic agents would work only against the former. All tissue needs a blood supply to grow. Tumors with the fewest vessels may be the most rapidly responsive to antiangiogenic therapy because, with fewer redundant vessels, damage to any one may cause extensive tumor cell apoptosis. By contrast, lesions with extensive neovascularization have such an enormous redundancy in the number of vessels feeding any one area that even if half the vessels in that area were lost, there could be sufficient remaining vessels to keep the tumor alive and perhaps growing.
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CLINICAL TRIALS OF ANTlANGlOGENlC AGENTS As with the development of any new field, novel biologic agents, including angiogenesis inhibitors, have forced investigators to reassess the methods used to evaluate and approve such agents.17Formal phase I, 11, and I11 testing has been well suited to classic cytotoxic chemotherapeutic agents in which toxicity is evaluated, while efficacy (as measured by shrinkage) is determined. Antiangiogenic agents come in two major types, chemical and natural. The chemical agents are likely to have many toxicities that would allow determination of a maximal tolerated dose. This determination may be not be possible or appropriate with many of the naturally occurring antiangiogenic inhibitors, such as angiostatin13 and endostatin.zOThey are molecules that have been evolving for the last 200 million years and are present in all humans. Administering these agents at physiologic doses may not cause significant toxicity, and the endpoint of phase I trials becomes less well defined. This situation is complicated further by the difficulties in assessing the efficacy of these agents. Tumors generate excessive amounts of neovascularization.z5If an antiangiogenic agent were given that killed 50% of the tumor vessels, the remaining 50% might be sufficient to allow the tumor to continue to grow. Such an agent would not show activity in a classic phase I1 trial, which measures tumor response. Even when sufficient damage to the tumor's blood supply has been achieved, alteration in fluid flux between the vascular bed and tumor occurs, leading to significant edema, which may lead to further apparent tumor growth.y,z4 Only when there is sufficient tumor cell death to make the lesions smaller would the potential of the therapy be recognized. Time to response may be delayed. Because endothelial cells have a slower turnover rate than tumor cells, longer periods of treatment may be required before response can be assessed. Although many approaches are being considered to overcome these difficulties, none has been successful yet in predicting drug response. That fact underscores the need for new assays or techniques to address these issues more dire~tly.6.57.80~116 There are more than 30 drugs in clinical trials in adults1z,46, 87, lo6 at present. Many of the agents being tested and their specific mechanism of action, disease focus, and sponsor are available on the National Cancer Institute website (www.cancertrials.nci.nih.gov). This section highlights some of the published reports of recent years made about these agents as they progress through clinical trials, recognizing that few published reports are available on the outcomes of the early clinical triais.43 Most clinical data are available for agents that already were commercially available for other indications. Thalidomide, a drug with many effects, including inhibition of neovascularization, has generated a great deal of interest. Response rates (partial response and stable disease) of 50% in recurrent high-grade astro~ytomas~~ and 40% in Kaposi's sarcoma33,64 and renal cancerszyhave been reported. Although responses
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to thalidomide in multiple myeloma may result from some of the immunologic effects of the compound,96the responses noted for astrocytomas, Kaposi's sarcoma, and renal cancers speak to its activity, despite the lack of a molecular basis for this effect. Another agent with many different activities, including inhibition of bFGF-induced neovascularization, is interferon-a, which has been available for some time and has shown activity in selected tumors, such as carcinoid tum0rs.2~Although thalidomide and interferon have been available longest, their relatively nonspecific antiangiogenic activity and numerous other, predominantly immunologic effects complicate their use and interpretation in the way they act. Thalidomide and interferon remain easily available agents, however, and are being tested in numerous clinical trials. Of the initial specific antiangiogenic agents to enter human clinical trials, marimastat and other similar compounds that function by altering 55, 73, 80* 86* 98, lo4, metalloproteinase inhibitors3 are the furthest al0ng,5~, and some have entered phase I11 studies. SU-5416 and SU-6668 are small molecule inhibitors that have generated a great deal of interest based on their preclinical and early clinical evaluations in adults.4O. 70 Antibodydirected inhibition of VEGF has been CMlOl is an endotoxinderived molecule that is picked up by cell surface receptors on the immature vasculature. A phase I trial has been completed, and phase I1 studies are under way.lo8Many other agents, such as IM-862,lo5for which no clear mechanism of action is known, are being tested. Numerous other antiangiogenic agents under investigation have been reported at meetings.30,56, 58, 71, 82, 88, 89, 99 In contrast to the approximately 30 clinical trials of antiangiogenic agents in adults, few have been or are being performed in children. The reasons for this disparity are complex and likely involve multiple forces. Potential contributing factors include the low prevalence of cancer in children, which provides little incentive for companies to invest in expensive clinical trials in this population; the concern that inhibition of neovascularization would cause growth arrest or end-organ damage in growing children; an increase in the number of small companies developing these agents that lack the resources to fund multiple studies; and societal concerns about exposing children to the risks of untested agents. To date, three antiangiogenic drugs have been tested in clinical trials in pediatric oncology patients. The first study used a fumagillin analogue called TNP-470. Originally identified in Folkman's laboratory, the drug was found to inhibit neovascularization in vitro as well as in animal A three-center study (Dana-Farber Cancer Institute, MemorialSloan Kettering, and Baylor University) consisted of a phase I dose escalation in pediatric cancer patients with relapsed or recurrent disease for which no curative therapy remained. The results of this study are awaiting publication. Adult trials with this compound were stopped after little activity was identified.97With a reanalysis of the animal experiments, a different dosing schema has been developed in animals that suggests an increase in the response rate. An adult phase I1 trial
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using this new dosing schedule is in de~elopment.~ No further pediatric studies have been planned for this drug at present. The second antiangiogenic agent to undergo pediatric testing is thalidomide. This compound, which was developed as a safe and nonlethal sedative in the 1950s, was discovered to cause dysmelia in fetuses if exposure occurred between the first and second month of gestation. In 1994, DAmato et alZ1discovered thalidomide’s inhibitory effects on bFGF-induced neovascularization. Further studies have determined that the teratogenicity of thalidomide likely is due to its antiangiogenic properties. Many compounds with minor modifications to thalidomide have been developed. Some compounds maintain the sedative effects but lack the antiangiogenic properties and teratogenicity. Others lack the sedative effects but are antiangiogenic. These molecules also are teratogenic. Many pediatric clinical trials have been proposed for thalidomide, especially in pediatric patients with central nervous system tumors, in part based on the activity of this compound in adult highgrade glial tumors34and multiple myeloma.54, One study, combining thalidomide and radiation therapy for newly diagnosed patients with diffuse pontine gliomas, has been completed through Children’s Hospital, Boston, and the Dana-Farber Cancer Institute as well as National Children’s Hospital, but a report on the findings is awaiting sufficient follow-up. At least five additional trials combining thalidomide with radiation or other chemotherapy (or both) are under way. A pediatric phase I trial of SU-5416 is being conducted through the Pediatric Brain Tumor Consortium. As noted earlier, this VEGF small molecule inhibitor acts by interfering with the phosphorylation site of the VEGF receptor Flk-l.48This trial opened in fall 2000 and is expected to conclude in early 2001. One other phase I trial, due to open in fall 2000 in pediatric centers across Canada and the United States through the Children’s Oncology Group, will study the compound squalamine in combination with carboplatin. In addition to the agents noted here, a few other pediatric antiangiogenic clinical trials have been initiated at the Dana-Farber Cancer Institute and elsewhere. These trials include the use of multiagent low-dose long-term chemotherapy as well as methods that attempt to up-regulate the production of naturally occurring angiogenesis inhibitors in the blood. FUTURE DIRECTIONS
The field of vascular biology and angiogenesis has been transformed from one with little relevance to the treatment of cancer to its current status as a field that has generated enormous energy and effort in identifying possible new targets and agents. Although antiangiogenic agents have not cured patients yet, their remarkable efficacy in animal studies is cause for some optimism. As investigators continue to seek better antiangiogenic agents for testing in pediatric patients, results from
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many phase I11 adult studies will soon be available. They should help guide in the choice of agents to be considered. References 1. Akhter S, et al: Squalamine, a novel cationic steroid, specifically inhibits the brushborder N a + / H + exchanger isoform NHE3. Am J Physiol276C136-144, 1999 2. Ashley DM, et a1 Response of recurrent medulloblastoma to low-dose oral etoposide. J Clin Onc01 14:1922-1927, 1996 3. Belotti D, Paganoni P, Giavazzi R MMP inhibitors: Experimental and clinical studies. Int J Biol Markers 14232-238, 1999 4. Berger AC, et al: The angiogenesis inhibitor, endostatin, does not affect murine cutaneous wound healing. J Surg Res 91%-31,2000 5. Bhargava P, et al: A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer. Clin Cancer Res 5:1989-1995, 1999 6. Bhujwalla ZM, Artemov D, Glockner J: Tumor angiogenesis, vascularization, and contrast-enhanced magnetic resonance imaging. Top M a p Reson Imaging 10:92103, 1999 7. Boehm T, et al: Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404-407, 1997 8. Boehm Viswanathan T Is angiogenesis inhibition the Holy Grail of cancer therapy? Curr Opin Oncol 12:89-94, 2000 9. Brem SS, et a 1 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 1371121-1142, 1990 10. Browder T, Folkman J, Pirie Shepherd S The hemostatic system as a regulator of angiogenesis. J Biol Chem 275:1521-1524, 2000 11. Browder T, et al: Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878-1886, 2000 12. Brower V Tumor angiogenesis-new drugs on the block. Nat Biotechnol 17963-968, 1999 13. Cao Y: Therapeutic potentials of angiostatin in the treatment of cancer. Haematologica 84:643-650, 1999 14. Carmeliet P:Mechanisms of angiogenesis and arteriogenesis. Nat Med 6389-395,2000 15. Carmeliet P, Jain R Angiogenesis in cancer and other diseases. Nature 407249-257, 2000 16. Carmeliet P, et al: Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380435439, 1996 17. Carter SK. Clinical strategy for the development of angiogenesis inhibitors. Oncologist ~ ( S U P 1):51-54, P~ 2000 18. Chambers AF, et a1 Clinical targets for anti-metastasis therapy. Adv Cancer Res 79:91-121, 2000 19. Cherrington JM, Strawn LM, Shawver L K New paradigms for the treatment of cancer: The role of anti-angiogenesis agents. Adv Cancer Res 79:l-38, 2000 20. Cirri L, et a1 Endostatin: A promising drug for antiangiogenic therapy. Int J Biol Markers 14263-267, 1999 21. DAmato RJ, et a 1 Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A 91:40824085, 1994 22. Dankbar B, et al: Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma. Blood 95:2630-2636, 2000 23. Davis S, Yancopoulos GD: The angiopoietins: Yin and yang in angiogenesis. Curr Top Microbiol Immunol237173-185, 1999 24. Del Maestro RF, Megyesi JF, Farrell C L Mechanisms of tumor-associated edema: A review. Can J Neurol Sci 17177-183, 1990 25. Denekamp J: Vascular attack as a therapeutic strategy for cancer. Cancer Metastasis Rev 9:267-282, 1990
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26. Desai SB, Libutti SK Tumor angiogenesis and endothelial cell modulatory factors. J Immunother 22:186-211, 1999 27. Dirix LY, et al: Long-term results of continuous treatment with recombinant interferon-alpha in patients with metastatic carcinoid tumors-an antiangiogenic effect? Anticancer Drugs 7175-181, 1996 28. Drummond AH, et a1 Preclinical and clinical studies of MMP inhibitors in cancer. Ann N Y Acad Sci 878:22&235, 1999 29. Eisen T, et a1 Continuous low dose thalidomide: A phase I1 study in advanced melanoma, renal cell, ovarian and breast cancer. Br J Cancer 82812-817,2000 30. Eskens F, et al: Phase I and pharmacologic study of EMD 121974, an a n b 3 and a n b 5 integrin inhibitor that perturbs tumor angiogenesis in patients with solid tumors. ASCO, 2000, poster 801 31. Ferrara N, Alitalo K: Clinical applications of angiogenic growth factors and their inhibitors. Nat Med 53359-1364, 1999 32. Ferrara N, et a1 Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439442,1996 33. Fife K, et al: Activity of thalidomide in AIDS-related Kaposi’s sarcoma and correlation with HHV8 titre. Int J STD AIDS 9:751-755, 1998 34. Fine HA, et al: Phase I1 trial of the antiangiogenic agent thalidomide in patients with recurrent high-grade gliomas. J Clin Oncol 18708-715,2000 35. Folkman J: Tumor angiogenesis: Therapeutic implications. N Engl J Med 285:11821186,1971 36. Folkman J: Tumor angiogenesis. In Holland JF, et a1 (eds): Cancer Medicine, ed 5. Toronto, BC Decker, 2000, pp 132-152 37. Folkman J, Hochberg M: Self-regulation of growth in three dimensions. J Exp Med 138:745-753, 1973 38. Folkman J, et al: Isolation of a tumor factor responsible or angiogenesis. J Exp Med 133:275-288, 1971 39. Fong GH, et al: Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature 37666-70, 1995 40. Fong TA, 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 59:99-106, 1999 41. Gasparini G: Prognostic value of vascular endothelial growth factor in breast cancer. Oncologist 5(suppl 1):3744, 2000 42. Gerber HP, et a1 VEGF is required for growth and survival in neonatal mice. Development 126:1149-1159, 1999 43. Giavazzi R, Giulia T: Angiogenesis and angiogenesis inhibitors in cancer. Forum (Genova) 9:261-272, 1999 44. Gimbrone MA Jr, et a1 Tumor dormancy in vivo by prevention of neovascularization. J Exp Med 136:261-276, 1972 45. Goldman E: The growth of malignant disease in man and the lower animals with special reference to the vascular system. Lancet 21236-1240, 1907 46. Hagedom M, Bikfalvi A Target molecules for anti-angiogenic therapy: From basic research to clinical trials. Crit Rev Oncol Hematol 34:89-110, 2000 47. Hahnfeldt P, et al: Tumor development under angiogenic signaling: A dynamical theory of tumor growth, treatment response, and postvascular dormancy. Cancer Res 59:4770-4775, 1999 48. Hamby JM, Showalter HD: Small molecule inhibitors of tumor-promoted angiogenesis, including protein tyrosine kinase inhibitors. Pharmacol Ther 82:169-193, 1999 49. Holash J, Wiegand SJ, Yancopoulos G D New model of tumor angiogenesis: Dynamic balance between vessel regression and growth mediated by angiopoietins and VEGF. Oncogene 18:5356-5362, 1999 50. Holmgren L, et al: Dormancy of micrometastases: Balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nat Med 1:149-153, 1995 51. Ilan N, Madri JA: New paradigms of signaling in the vasculature: Ephrins and metalloproteases. Curr Opin Biotechnol 10:536-540, 1999
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52. Ingber D, et al: Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature 348:555-557, 1990 53. Jones L, et a 1 The matrix metalloproteinases and their inhibitors in the treatment of pancreatic cancer. Ann N Y Acad Sci 880288-307, 1999 54. Juliusson G, et a1 Frequent good partial remissions fromthalidomide including best response ever in patients with advanced refractory and relapsed myeloma. Br J Haematol 109239-96, 2000 55. Kahari VM, Saarialho Kere U Matrix metalloproteinases and their inhibitors in tumour growth and invasion. Ann Med 3133445,1999 56. Kalidas M, et a1 A phase I and pharmacokinetic (PK) study of the angiogenesis inhibitor squalamine lactate (MSI-1256F). ASCO, 2000, poster 698 57. Kerbel RS: Tumor angiogenesis: Past, present and the near future. Carcinogenesis 21:505-515,2000 58. Keshet E, Ben Sasson S A Anticancer drug targets: Approaching angiogenesis. J Clin Invest 104:1497-1501,1999 59. Kim KJ, et a1 Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature 3628414344, 1993 60. Kleihues P, Cavenee WK: Pathology and Genetics of Tumours of the Nervous System. Lyon, France, WHO, 2000 61. Klein SA, et al: Angiogenesis inhibitor TNP-470 inhibits murine cutaneous wound healing. J Surg Res 82:268-274,1999 62. Klement G, et al: Continuous low-dose therapy with vinblastine and VEGF receptor2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105:R15-24,2000 63. 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 42899-903, 1998 64. Little RF, et al: Activity of thalidomide in AIDS-related Kaposi's sarcoma. J Clin Oncol 182593-2602, 2000 65. Maisonpierre PC, et a1 Angiopoietin-2, a natural antagonist for Tie2 that disrupts in vivo angiogenesis. Science 2775540, 1997 66. Malone C, Kerin MJ: Angiogenesis-related markers and their potential clinical significance. Int J Biol Markers 14:3-7, 1999 67. Masferrer JL, Koki A, Seibert K COX-2 inhibitors: A new class of antiangiogenic agents. Ann N Y Acad Sci 88994-86, 1999 68. McDonnell CO, et al: Tumour micrometastases: The influence of angiogenesis. Eur J Surg Oncol 26305-15, 2000 69. McMahon G: VEGF receptor signaling in tumor angiogenesis. Oncologist 5(suppl 1):3-10, 2000 70. Mendel DB, et al: Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenesis agent. Anticancer Drug Des 15:29-41, 2000 71. Miles S, et al: A multicenter dose-escalating study of SU5416 in AIDS-related Kaposi's sarcoma. ASCO, 2000, poster 683 72. Millauer 8, et al: Glioblastoma growth inhibited in vivo by a dominant-negative Flk1 mutant. Nature 367576579, 1994 73. Nemunaitis J, et al: Combined analysis of studies of the effects of the matrix metalloproteinase inhibitor marimastat on serum tumor markers in advanced cancer: Selection of a biologically active and tolerable dose for longer-term studies. Clin Cancer Res 4:1101-1109, 1998 74. OReilly MS, et al: Angiostatim A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315-328, 1994 75. OReilly MS, et al: Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Med 2:689-692, 1996 76. OReilly MS, et al: Endostatim An endogenous inhibitor of angiogenesis and tumor growth. Cell 88:277-285, 1997 77. OReilly MS, et al: Antiangiogenic activity of the cleaved conformation of the serpin antithrombin. Science 285:1926-1928,1999
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78. Perez Atayde AR, et al: Spectrum of tumor angiogenesis in the bone marrow of children with acute lymphoblastic leukemia. Am J Pathol, 150:815-821, 1997 79. Plate KH, et al: Up-regulation of vascular endothelial growth factor and its cognate receptors in a rat glioma model of tumor angiogenesis. Cancer Res 53:5822-5827,1993 80. Primrose JN, et al: Marimastat in recurrent colorectal cancer: Exploratory evaluation of biological activity by measurement of carcinoembryonic antigen. Br J Cancer 79:509-514, 1999 81. Rak J, Filmus J, Kerbel RS Reciprocal paracrine interactions between tumour cells and endothelial cells: The 'angiogenesis progression' hypothesis. Eur J Cancer 32A:24362450, 1996 82. Remick SC, et al: A phase I pharmacokinetic (PK) study of single dose intravenous (iv) Combretatstatin A4 prodrug (CaA4) in patients (pts) with advanced cancer. ASCO, 2000, poster 697 83. Ribatti D, Vacca A Models for studying angiogenesis in vivo. Int J Biol Markers 14:207-213, 1999 84. Richard DE, Berra E, Pouyssegur J: Angiogenesis: How a tumor adapts to hypoxia. Biochem Biophys Res Commun 266:71&722, 1999 85. Risau W, Flamme I: Vasculogenesis. Annu Rev Cell Dev Biol 11:73-91, 1995 86. Rosemurgy A, et al: Marimastat in patients with advanced pancreatic cancer: A dosefinding "studv., Am T Clin Oncol22247-252. 1999 87. Rosen L: Antiangiogenic strategies and agents in clinical trials. Oncologist 5(suppl 1):20-27, 2000 88. Rosen PJ, et al: A phase 1/11 study of SU5416 in combination with 5-FU/leucovorin in patients with metastatic colorectal cancer. ASCO, 2000, poster 5D 89. Rowinsky EK, et al: Protracted daily treatment with COL-3, an oral tetracycline analog, matrix metalloproteinase (MMP) inhibitor; is feasible: A phase I, pharmacokinetic, and biologic study. ASCO, 2000, poster 700 90. Rubin JB, Kieran MW: Innovative therapies for pediatric brain tumors. Curr Opin Pediatr 11:3946, 1999 91. Sat0 TN, et al: Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation. Nature 37670-74, 1995 92. Schiller J, Bittner G: Potentiation of platinum antitumor effects in human lung tumor xenografts by the angiogenesis inhibitor squalamine: Effects on tumor neovascularization. Clin Cancer Res 5:4287-4294, 1999 93 Schlaeppi JM, Wood JM: Targeting vascular endothelial growth factor (VEGF) for anti-tumor therapy, by anti-VEGF neutralizing monoclonal antibodies or by VEGF receptor tyrosine-kinase inhibitors. Cancer Metastasis Rev 18:473-481, 1999 94. Shalaby F, et al: Failure of blood-island formation and vasculogenesis in Flk-ldeficient mice. Nature 376:62-66, 1995 95. Shing Y, et al: Heparin affinity: Purification of a tumor-derived capillary endothelial cell growth factor. Science 223:1296-1299, 1984 96. Singhal S, et al: Antitumor activity of thalidomide in refractory multiple myeloma. N Engl J Med 341:1565-1571, 1999 97. Stadler WM, et al: Multi-institutional study of the angiogenesis inhibitor TNP-470 in metastatic renal carcinoma. J Clin Oncol 172541-2545, 1999 98. Steward WP: Marimastat (882516):Current status of development. Cancer Chemother Pharmaco143(suppl):S5640, 1999 99. Stopeck A: Results of a phase I dose-escalating study of the antiangiogenic agent, SU5416, in patients with advanced malignancies. ASCO, 2000, poster 802 100. Suda T, Takakura N, Oike Y Hematopoiesis and angiogenesis. Int J Hematol 71:99107, 2000 101. Suri C, et a1 Requisite role of angiopoietin-1, a ligand for the TIE2 receptor, during embryonic angiogenesis. Cell 871171-1180, 1996 102. Taipale J, et al: Vascular endothelial growth factor receptor-3. Curr Top Microbiol Immunol 237:85-96, 1999 103. Teicher BA, et al: Potential of the aminosterol, squalamine in combination therapy in the rat 13,762 mammary carcinoma and the murine Lewis lung carcinoma. Anticancer Res 18:256-273. 1998
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104. Tiemey GM, et al: A pilot study of the safety and effects of the matrix metalloprotainase inhibitor marimastat in gastric cancer. Eur J Cancer 35(4):563-568, 1998 105. Tidpule A, et al: Results of a randomized study of IM862 nasal solution in the treatment of AIDS-related Kaposi's sarcoma. J Clin Oncol 18:71&723,2000 106. Twardowski P, Gradishar WJ: Clinical trials of antiangiogenic agents. Curr Opin Oncol 93584-589, 1997 107. Veikkola T, et al: Regulation of angiogenesis via vascular endothelial growth factor receptors. Cancer Res 60:203-212, 2000 108. Wamil BD, et al: Soluble E-selectin in cancer patients as a marker of the therapeutic efficacy of CM101, a tumor-inhibiting anti-neovascularization agent, evaluated in phase I clinical trial. J Cancer Res Clin Oncol 123:173-179, 1997 109. Weidner N, et al: Tumor angiogenesis and metastasis-correlation in invasive breast carcinoma. N Engl J Med 324:1-8, 1991 110. Wojtowicz Praga S, et al: Phase I trial of Marimastat, a novel matrix metalloproteinase inhibitor, administered orally to patients with advanced lung cancer. J Clin Oncol 16:2150-2156, 1998 111. Yancopoulos GD, Klagsbrun M, Folkman J: Vasculogenesis, angiogenesis, and growth factors: Ephrins enter the fray at the border. Cell 93:661-664, 1998 112. Yancopoulos GD, et al: Vascular-specific growth factors and blood vessel formation. Nature 407242-248, 2000 113. Yip D, et al: Matrix metalloproteinase inhibitors: Applications in oncology. Invest New Drugs 17387-399, 1999 114. Yokoyama Y, et al: Synergy between angiostatin and endostatin: Inhibition of ovarian cancer growth. Cancer Res 60:219(r2196,2000 115. Zetter B R Angiogenesis and tumor metastasis. Annu Rev Med 49:407424, 1998 116. Ziche M, et al: Biological parameters for the choice of antiangiogenic therapy and efficacy monitoring. Int J Biol Markers 14214-217, 1999
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ANTIANGIOGENESIS THERAPY Current and Future Agents Mark W. Kieran, MD, PhD, and Amy Billett, MD
Approaches to cancer therapy for most tumors in adults and children have changed little in 50 years: Surgery, radiation, and chemotherapy are standard for many solid tumors. When the concept of angiogenesis in cancer biology first was introduced in the 1970s, there was little recognition of the therapeutic potential of attacking a tumor’s blood supply. Advances in understanding the molecular processes that regulate tumor blood supply and novel agents that can interfere with them have generated a great deal of scientific interest and excitement. This article reviews the current understanding of angiogenesis and its role in cancer, then discusses new therapeutic options in animals and humans, with a focus on pediatric tumors and the potential for treating them. The interested reader can find many excellent reviews of angiogenesis, antiangiogenesis, and pediatric oncology.* ANGIOGENESIS
Angiogenesis is the development of blood vessels. Although the term is differentiated from vasculogenesis (formation of the primitive venous plexus), the two often are used interchangeably. There is evidence that these processes can be regulated or inhibited differentially, and some of the newer generation of inhibitors are capable of interfering *References 12, 14, 15, 36, 46, 57, 87, 90, 100, 112, 115.
From Pediatric Medical Neuro-Oncology (MK) and the Department of Pediatric Oncology (AB), Dana-Farber Cancer Institute; Oncology Inpatient Unit, Children’s Hospital (AB); and Harvard Medical School (MK, AB), Boston, Massachusetts
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specifically with one process or the other. From a practical viewpoint, however, the inhibition of either process leads to a halt in the process of developing, maintaining, or expanding a blood supply and may have similar clinical outcomes. Although a complete review of vasculogenesis and angiogenesis (which together are referred to as neovascularization) is beyond the scope of this article, a simplified model of neovascularization is presented in Fig. 1 and has been reviewed by Yancopoulos et al.ll', 112 The proliferation of vascular endothelial cell precursors into a vascular network (primary capillary plexus) is induced by ligand-mediated signal transduction through receptor tyrosine kinases on these cells.26, 85 The best-characterized ligand of this sort is vascular endothelial growth factor (VEGF), which binds a receptor (ftk-1/KDR/VEGFR-2, a positive activator of vasculogenesisz6).Many other ligand-receptor combinations have been identified that may act in concert with VEGF to induce and maintain vascul~genesis.~~~ 26 Secondary factors, especially angiopoietin-1 (ang-1), then act on the primitive capillary plexus to induce sprouting and branching (angiogenesis), which leads to a more mature and stable vascular system.22Angiopoietin-2 (ang-2) is the antagonist of ang-1, and it is the balance of these two molecules23in the presence of VEGF that determines whether vessel proliferation (high VEGF, balanced ang-1 and ang-2), maturation (high VEGF and ang-1, lower ang-2), or regression (low VEGF and ang-1, high ang-2) will proceed.49,lo7 Although these three factors play a central role in regulating vessel formation or destruction, numerous other elements play into this system to provide the exquisite regulation typical of crucial biologic proce~ses."~ The reader is referred to detailed reviews on the origins and interplay of these different factors.'O, 14, 31, 51, 84 A great deal of understanding of the role of angiogenic factors and their receptors has been obtained from the analysis of knock-out mice, from which the genes for these different molecules have been removed.'l2 VEGFR-2 (also called KDR/flk-l) null mice fail to develop a normal vasculature and have decreased endothelial cells.94By contrast, VEGFR1 (also calledflt-2) null mice have increased numbers of endothelial cells, suggesting that this receptor may act as a negative regulator of VEGF.39 VEGFR-3 (also called flt-3) null mice have relatively normal vasculature but have abnormal lymphatic development.102A similar pattern arises when the ligands are manipulated. VEGF null mice appear similar to VEGFR-2 null mice.'6, 32 VEGF heterozygotes have abnormal vascular formation and are embryonically lethal, suggesting the crucial role this molecule has.16,32 When mice have completed their early development, the effects of VEGF depletion become much less signifi~ant.~~ Using similar strategies, the role of the angiopoietins and their receptors has been elucidated. Mice lacking ang-1 or the angiopoietin receptor Tie-2 develop a vasculature, although the vasculature fails to undergo normal lol Over expression of ang-2, mimicking the effects obrern~deling.~~, served with null mutations in ang-1 or Tie-2, also cause vascular defect~.~~
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Proliferation and Formation of primitive
with loss of Ang-1 and gain
of -9-2 Of VEGF and vessel LOSS
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Figure 1. Neovascularization. A, Endothelial cell precursors respond to the interaction of ligands and receptor interactions inducing endothelial cell proliferation giving rise to the formation of the primitive venous plexus. In the context of other factors and their receptors, such as angiopoietin-1, remodeling of this network takes place. The coexpression of epherins will determine whether the vessel develops as an arteriole or vein. 6,In the continued presence of vascular endothelial growth factor (VEGF) and angiopoietin-1, vessel maturation and stabilization occur. C,If angiopoietin-1 levels decrease and angiopoietin-2 levels increase, then vessel destabilization occurs. This zone of vessel destabilization allows the vessel to grow further (by adding back angiopoietin-I), or regress. 0,Loss of VEGF signalling results in vessel death.
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ANGIOGENESIS AND TUMOR BIOLOGY
The association of an expanding blood supply as a crucial feature in cancer biology was established almost 100 years ago.45Some tumor grading systems specifically assess neovascularization,6° according lesions a higher grade when present (e.g., glioblastoma multiforme versus anaplastic astrocytoma). In other cases, increasing neovascularization correlates with poorer prognosis.41, In 1971, Folkman et a135,38 proposed a new approach to treating malignant tumors. Instead of directing highly toxic therapy at tumor cells, Folkman suggested attacking the blood supply of these lesions. The theoretic advantages of attacking the blood supply are (1) tumor vasculature is less mature and more sensitive to inhibition than normal vessels and (2) although tumors develop resistance rapidly, tumor-induced vasculature has not in animal experiment^.^ This latter aspect is derived from the principle that normal cells, of any lineage, remain under normal cellular control and lack the mechanisms necessary to generate mutations. Animal data have shown the role of angiogenesis in cancer biology. A few examples are as follows: Avascular tumors remain localized and small (<2 to 3 mm).37 Localized tumors that obtain a blood supply grow and disseminate.44 Many factors (e.g., VEGF, basic fibroblast growth factor [bFGF]) that 95 stimulate neovascularization stimulate tumor Inhibitors of angiogenesis (TNP470, anti-VEGF, endostatin, angiostatin) can reverse neovascularization and cause regression of tumor growth. These agents have no direct inhibitory effects on tumor cells.52,59. 74,76 Agents that interfere with endothelial cell surface receptor signaling can inhibit neovascularization and cause regression of tumor 93 growth.72, At least three naturally occurring inhibitors of neovascularization, specific for endothelial cells, that are derived from the cleavage of native proteins have been identified: angiostatin (from pla~minogen~~), endostatin (from collagen XVII176),and a fragment of antithrombin III.77 These naturally occurring molecules, which normally are present in humans, provide the negative signals required to balance input from stimulatory signals of neovascularization. In addition to the role that angiogenesis plays in the progression to clinically evident disease, neovascularization may be a crucial process in the development of metastases.'*,68, 115 Although tumors that metastasize need to generate a blood supply at their new site, their initial ability to gain access to the blood system may be related in part to the development of their angiogenic potential.4l For some tumors, microvessel density and levels of VEGF are independent prognostic indicators of the risk of developing metastases and overall prognosis.41,lo9
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DEVELOPMENT OF ANTlANGlOGENlC AGENTS Agents that interfere with neovascularization can be grouped loosely into a few categories, based on their presumed mechanism of inhibition. A limited number of these agents are provided as examples. Molecules That Interfere With Signals of Neovascularization
In addition to direct inhibitors of bFGF and VEGF (anti-bFGF and anti-VEGF antibodies), inhibition of their targets (anti-flk-1) and inhibition of transmission of the appropriate signal transduction cascade (SU5416, SU6668) are potentially effective means of turning off neovascularization.48,71,88.93.99
Alteration of Endothelial Shape Endothelial cells must form specific junctions that require certain shape constraints. Agents that interfere with endothelial cell shape can prevent vessel formation, even when all of the appropriate signals are present (squalamine).l,56 Inhibition of Metalloproteinases Metalloproteinases are enzymes that appear to play a crucial role in modulating local tissue and vessel matrix as well as the supply of angiogenic stimulators, which, in a simplistic sense, can alter the environment in which neovascularization occurs. Examples of compounds in this class include marimastat, AG-3340, COL-3, AE-941 (Neovastat), and BMS-27529.3, 28, 89,110. 113 Other Mechanisms of Inhibition Many other components involved in neovascularization have been identified, which provide additional targets for antiangiogenic therapy, including the integrins30and cyclooxygenase inhibitor^.^^ Unknown Mechanisms Many agents are being tested that have shown the ability to inhibit neovascularization, although the specific molecular mechanism remains obscure. Whether compounds in this class will fall into one of the aforementioned categories or lead to new categories is unknown. Exam-
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ples include thalid~mide?~ endostatin,76angi~statin;~ and antiangiogenic antithrombin III.77 PRECLINICAL DATA FOR ANTlANGlOGENlC THERAPY
Many agents have been tested in vitro that show antiangiogenic activity. Many of these agents when tested in animal models have resulted in regression or cure.74,76, Although these results have been confirmed in many independent studies, the mechanism by which this occurs remains obscure for many agents. With the development of specific molecular targets, such as VEGF or its receptor Flk-1, investigators can begin to interpret the animal experiments delineating antiangiogenic responses. To understand how these agents work, it is necessary to understand how they were identified. The methods used to identify inhibitors of angiogenesis (which also were used to identify many of the stimulators) have included many appro ache^.^^
Inhibition of endothelial cell proliferation in vitro: Endothelial cells are grown in culture. Drugs to be tested are added in differing concentrations to assess their ability to inhibit endothelial proliferation. These compounds are tested in culture with other cell types to ensure their inhibition is specific for endothelial cells. Inhibition of endothelial cell migration in vitro: Similar to the first assay, this one looks at compounds that interfere with the ability of endothelial cells to move, a crucial feature in neovascularization. Inhibition of endothelial tube formation in vitro: Endothelial cells form three-dimensional tubes that can be replicated in vitro. The addition of drugs to these cultures measures the ability of different agents to disrupt this process. Inhibition of bFGF or VEGF pellet-induced corneal neovascularization in vivo: Small pellets containing slow-release bFGF or VEGF are implanted into the cornea of animals. The animals develop intense neovascularization toward the pellets. Animals are treated systemically with agents, and the ability to reduce or inhibit the neovascularization is measured. Inhibition of chorioallantoic membrane neovascularization in vivo: The chorioallantoic membrane of developing chick embryos is highly vascularized. Small disks containing inhibitors are placed on the membrane, and the zone of inhibition of neovascularization is measured. Decreased in serum or urine bFGF or VEGF levels: Although many angiogenic stimulators have been identified, only a few are easily quantifiable. The two major ones, bFGF and VEGF, can be measured in serum, urine, and cerebrospinal fluid and in tumor specimens. As animals are treated with different inhibitors, decreases in the amount of these molecules can be assessed. Decrease in microvessel density counts of tumor specimens: By examining tissue directly before and after treatment with an agent, a direct
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measure of the number of vessels within the tumor can be determined. Inhibition of one tumor by another growing on an animal: This technique, which has led to the discovery of many of the naturally occurring inhibitors of angiogenesis, is the most difficult to conceptualize. This model is based on a phenomenon recognized by surgeons for more than a century. In a small percentage of cases in which a surgeon removes a localized tumor in a patient without evidence of metastatic disease, within weeks or months of the operation, the patient has widespread metastases. This sudden spread was attributed to the manipulation of the tumor at the time of the operation and extrusion of tumor cells into the vasculature. An alternative explanation, proposed by Folkman in the 1970~:~was that the primary tumor, in addition to secreting factors to stimulate its own blood supply, secreted inhibitors into the circulation. Such inhibitors potentially could prevent metastases from competing for nutrients. If a surgeon removes an apparent localized tumor secreting these inhibitors, the existing microscopic metastases become free to expand. The animal model thought to mimic this clinical phenomenon involves injecting many different clones of a tumor on both flanks of an animal. With most pairs of clones, tumors grow equally on both sides. Rarely, one side grows, while the other remains small (because of inhibitors of angiogenesis produced by the larger tumor mass). If the growing lesion is resected, the smaller side grows rapidly as the inhibitor is removed. By purifying the activity from blood or urine from animals with asymmetric tumor growth, these inhibitors can be isolated. This technique has led to the discovery of many biologically active inhibitors of tumor neovascularization in vivo.74, 76. 77 The preclinical data examining the effect of numerous antiangiogenic agents are extensive, and the reader is referred to reviews and primary articles in the literature.s,48, 67, 83 Some findings from animal studies deserve specific mention because they may affect the design and interpretation of human studies. The first of these findings is the issue of tumor dormancy.44Angiogenesis inhibitors work against dividing endothelial cells, which are most abundant within the small, immature capillaries of the tumor bed. Because the original malignant cells arose from an organ or tissue with a fully mature vasculature, even if all of the tumor-induced neovascularization could be eliminated, tumor cells supplied directly by the original mature vessel could survive. As such, even if completely successful, antiangiogenic therapy likely would control, not eradicate, all of the disease. A small tumor focus that cannot induce neovascularization, even if malignant, can remain in the body indefinitely. Although the cells within the focus are actively proliferating, each new cell is pushed beyond the diffusion capacity of oxygen and nutrients and dies. These lesions are referred to as dormant.47, 50,75
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A second important factor to consider is that to treat the microscopic residual lesion, antiangiogenic therapy likely would require the addition of traditional chemotherapy, radiation therapy, or both. In preclinical studies, the effects of antiangiogenic agents in combination with other therapies would be predicted to be additive in response. Although this has been true for some systems, a surprising result has been observed in others. In these cases, a synergistic instead of additive response was observed when antiangiogenic agents were given with chemotherapy or 114 Although the mechanisms for this response are unradiation.", 92, clear, a possible explanation is inhibition of the paracrine loops that are fundamental to tumor development.22, In addition to producing vasculogenic factors to stimulate the expanding blood supply, the tumor is stimulated by the secretion of factors by the expanding blood supply. For this reason, inhibiting both may account for greater activity than would be expected by inhibiting either alone. Another area of consideration in the use of antiangiogenic agents is the target and what defines an antiangiogenic compound. Most chemotherapy is directed at dividing cells. In addition to tumor cells, normal cells of the hematopoietic system, gastrointestinal tract and hair frequently also are affected. To maintain its growing volume, tumors continually must stimulate endothelial cell turnover so that neovascularization can progress. As such, these dividing cells in the neovasculature may be targeted by traditional drugs. In this sense, many antimitotic agents may act as antiangiogenic compounds. The schedule of most chemotherapy regimens may prevent successful antiangiogenic therapy, however. Only the proportion of endothelial cells in cycle on days of treatment are likely to be killed. Angiogenesis continues between chemotherapy treatments, while normal cell recovery occurs. The suggestion is that long-term administration of low-dose cytotoxic agents could provide a more antiangiogenic effective schedule.l'< This hypothesis could explain responses to low-dose long-term oral etoposide despite progressive disease on high-dose intermittent intravenous therapy2 This potential mechanism is the subject of investigation within the laboratory of one of the authors (MWK). An important concern in the use of angiogenesis inhibitors is the possibility that they will inhibit many of the normal processes within the body that rely on neovascularization. Although this situation was observed with some of the early antiangiogenesis inhibitors,'jl many newer agents appear much more specific? This specificity likely resides in the complex organization of positive and negative signals that regulate neovascularization and a drug's ability to alter one component without upsetting the entire process. Endothelial cells within a tumor should not become resistant to antiangiogenic the rap^.^ As would be predicted in Folkman's original hypothesis, because the endothelial cells making up the tumor's blood supply are inherently normal, they should continue to obey normal signals to proliferate, arrest, or undergo apoptosis. Development of resistance to antiangiogenic agents should thus be unlikely. In numerous
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model systems, with a variety of tumors and agents, this situation has been observed c~nsistently.~ The animal data suggest that antiangiogenic therapy, if successful, is likely to cause tumors to regress to their in situ size, but requires adjunctive therapy if a cure is to be achieved. In contrast to the tumor cells themselves, resistance would not be expected, even in patients who stop and start treatments.
POTENTIAL CHALLENGES TO ANTlANGlOGENlC THERAPY Many theoretic concerns have been proposed to predict the failure of antiangiogenic drugs to treat tumors effe~tively.~~ Examples include the following:
Large tumors have mature vessels that would be insensitive to antiangiogenic therapy. Although large vessels may be present, the capillary bed where oxygen and nutrient exchange occur is the site of neovascularization and the target of these agents.36Because tumors are in a continuous phase of growth, they maintain their capillaries in an immature state so that they can continue to expand with the tumor.35 Some tumors, such as leukemia, grow independent of vessels and would be unaflected by antiangiogenic agents. Data showing the potent expression of inducers of neovascularization and extensive angiogenesis within the bone marrow spaces in leukemia directly refute this ~ t a t e m e n t .In ~ ~animal leukemia models, response to therapy is correlated directly to a decrease in microvessel density. One study using traditional chemotherapy in an antiangiogenic schedule showed that leukemia was highly angiogenic, that low-dose chemotherapy could attack effectively the proliferating endothelial cells within the tumor but not the tumor itself (the tumor cells were made resistant to the drugs), and that tumor cell apoptosis always was preceded by endothelial cell loss.ll, Whether low, long-term dosing of chemotherapy in the treatment of human disease, such as maintenance therapy in acute lymphoblastic leukemia, works by targeting endothelial cells is unknown. Tumors can be divided into angiogenic (red or bloody) and nonangiogenic (white, pale) groups, and antiangiogenic agents would work only against the former. All tissue needs a blood supply to grow. Tumors with the fewest vessels may be the most rapidly responsive to antiangiogenic therapy because, with fewer redundant vessels, damage to any one may cause extensive tumor cell apoptosis. By contrast, lesions with extensive neovascularization have such an enormous redundancy in the number of vessels feeding any one area that even if half the vessels in that area were lost, there could be sufficient remaining vessels to keep the tumor alive and perhaps growing.
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CLINICAL TRIALS OF ANTlANGlOGENlC AGENTS As with the development of any new field, novel biologic agents, including angiogenesis inhibitors, have forced investigators to reassess the methods used to evaluate and approve such agents.17Formal phase I, 11, and I11 testing has been well suited to classic cytotoxic chemotherapeutic agents in which toxicity is evaluated, while efficacy (as measured by shrinkage) is determined. Antiangiogenic agents come in two major types, chemical and natural. The chemical agents are likely to have many toxicities that would allow determination of a maximal tolerated dose. This determination may be not be possible or appropriate with many of the naturally occurring antiangiogenic inhibitors, such as angiostatin13 and endostatin.zOThey are molecules that have been evolving for the last 200 million years and are present in all humans. Administering these agents at physiologic doses may not cause significant toxicity, and the endpoint of phase I trials becomes less well defined. This situation is complicated further by the difficulties in assessing the efficacy of these agents. Tumors generate excessive amounts of neovascularization.z5If an antiangiogenic agent were given that killed 50% of the tumor vessels, the remaining 50% might be sufficient to allow the tumor to continue to grow. Such an agent would not show activity in a classic phase I1 trial, which measures tumor response. Even when sufficient damage to the tumor's blood supply has been achieved, alteration in fluid flux between the vascular bed and tumor occurs, leading to significant edema, which may lead to further apparent tumor growth.y,z4 Only when there is sufficient tumor cell death to make the lesions smaller would the potential of the therapy be recognized. Time to response may be delayed. Because endothelial cells have a slower turnover rate than tumor cells, longer periods of treatment may be required before response can be assessed. Although many approaches are being considered to overcome these difficulties, none has been successful yet in predicting drug response. That fact underscores the need for new assays or techniques to address these issues more dire~tly.6.57.80~116 There are more than 30 drugs in clinical trials in adults1z,46, 87, lo6 at present. Many of the agents being tested and their specific mechanism of action, disease focus, and sponsor are available on the National Cancer Institute website (www.cancertrials.nci.nih.gov). This section highlights some of the published reports of recent years made about these agents as they progress through clinical trials, recognizing that few published reports are available on the outcomes of the early clinical triais.43 Most clinical data are available for agents that already were commercially available for other indications. Thalidomide, a drug with many effects, including inhibition of neovascularization, has generated a great deal of interest. Response rates (partial response and stable disease) of 50% in recurrent high-grade astro~ytomas~~ and 40% in Kaposi's sarcoma33,64 and renal cancerszyhave been reported. Although responses
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to thalidomide in multiple myeloma may result from some of the immunologic effects of the compound,96the responses noted for astrocytomas, Kaposi's sarcoma, and renal cancers speak to its activity, despite the lack of a molecular basis for this effect. Another agent with many different activities, including inhibition of bFGF-induced neovascularization, is interferon-a, which has been available for some time and has shown activity in selected tumors, such as carcinoid tum0rs.2~Although thalidomide and interferon have been available longest, their relatively nonspecific antiangiogenic activity and numerous other, predominantly immunologic effects complicate their use and interpretation in the way they act. Thalidomide and interferon remain easily available agents, however, and are being tested in numerous clinical trials. Of the initial specific antiangiogenic agents to enter human clinical trials, marimastat and other similar compounds that function by altering 55, 73, 80* 86* 98, lo4, metalloproteinase inhibitors3 are the furthest al0ng,5~, and some have entered phase I11 studies. SU-5416 and SU-6668 are small molecule inhibitors that have generated a great deal of interest based on their preclinical and early clinical evaluations in adults.4O. 70 Antibodydirected inhibition of VEGF has been CMlOl is an endotoxinderived molecule that is picked up by cell surface receptors on the immature vasculature. A phase I trial has been completed, and phase I1 studies are under way.lo8Many other agents, such as IM-862,lo5for which no clear mechanism of action is known, are being tested. Numerous other antiangiogenic agents under investigation have been reported at meetings.30,56, 58, 71, 82, 88, 89, 99 In contrast to the approximately 30 clinical trials of antiangiogenic agents in adults, few have been or are being performed in children. The reasons for this disparity are complex and likely involve multiple forces. Potential contributing factors include the low prevalence of cancer in children, which provides little incentive for companies to invest in expensive clinical trials in this population; the concern that inhibition of neovascularization would cause growth arrest or end-organ damage in growing children; an increase in the number of small companies developing these agents that lack the resources to fund multiple studies; and societal concerns about exposing children to the risks of untested agents. To date, three antiangiogenic drugs have been tested in clinical trials in pediatric oncology patients. The first study used a fumagillin analogue called TNP-470. Originally identified in Folkman's laboratory, the drug was found to inhibit neovascularization in vitro as well as in animal A three-center study (Dana-Farber Cancer Institute, MemorialSloan Kettering, and Baylor University) consisted of a phase I dose escalation in pediatric cancer patients with relapsed or recurrent disease for which no curative therapy remained. The results of this study are awaiting publication. Adult trials with this compound were stopped after little activity was identified.97With a reanalysis of the animal experiments, a different dosing schema has been developed in animals that suggests an increase in the response rate. An adult phase I1 trial
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using this new dosing schedule is in de~elopment.~ No further pediatric studies have been planned for this drug at present. The second antiangiogenic agent to undergo pediatric testing is thalidomide. This compound, which was developed as a safe and nonlethal sedative in the 1950s, was discovered to cause dysmelia in fetuses if exposure occurred between the first and second month of gestation. In 1994, DAmato et alZ1discovered thalidomide’s inhibitory effects on bFGF-induced neovascularization. Further studies have determined that the teratogenicity of thalidomide likely is due to its antiangiogenic properties. Many compounds with minor modifications to thalidomide have been developed. Some compounds maintain the sedative effects but lack the antiangiogenic properties and teratogenicity. Others lack the sedative effects but are antiangiogenic. These molecules also are teratogenic. Many pediatric clinical trials have been proposed for thalidomide, especially in pediatric patients with central nervous system tumors, in part based on the activity of this compound in adult highgrade glial tumors34and multiple myeloma.54, One study, combining thalidomide and radiation therapy for newly diagnosed patients with diffuse pontine gliomas, has been completed through Children’s Hospital, Boston, and the Dana-Farber Cancer Institute as well as National Children’s Hospital, but a report on the findings is awaiting sufficient follow-up. At least five additional trials combining thalidomide with radiation or other chemotherapy (or both) are under way. A pediatric phase I trial of SU-5416 is being conducted through the Pediatric Brain Tumor Consortium. As noted earlier, this VEGF small molecule inhibitor acts by interfering with the phosphorylation site of the VEGF receptor Flk-l.48This trial opened in fall 2000 and is expected to conclude in early 2001. One other phase I trial, due to open in fall 2000 in pediatric centers across Canada and the United States through the Children’s Oncology Group, will study the compound squalamine in combination with carboplatin. In addition to the agents noted here, a few other pediatric antiangiogenic clinical trials have been initiated at the Dana-Farber Cancer Institute and elsewhere. These trials include the use of multiagent low-dose long-term chemotherapy as well as methods that attempt to up-regulate the production of naturally occurring angiogenesis inhibitors in the blood. FUTURE DIRECTIONS
The field of vascular biology and angiogenesis has been transformed from one with little relevance to the treatment of cancer to its current status as a field that has generated enormous energy and effort in identifying possible new targets and agents. Although antiangiogenic agents have not cured patients yet, their remarkable efficacy in animal studies is cause for some optimism. As investigators continue to seek better antiangiogenic agents for testing in pediatric patients, results from
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many phase I11 adult studies will soon be available. They should help guide in the choice of agents to be considered. References 1. Akhter S, et al: Squalamine, a novel cationic steroid, specifically inhibits the brushborder N a + / H + exchanger isoform NHE3. Am J Physiol276C136-144, 1999 2. Ashley DM, et a1 Response of recurrent medulloblastoma to low-dose oral etoposide. J Clin Onc01 14:1922-1927, 1996 3. Belotti D, Paganoni P, Giavazzi R MMP inhibitors: Experimental and clinical studies. Int J Biol Markers 14232-238, 1999 4. Berger AC, et al: The angiogenesis inhibitor, endostatin, does not affect murine cutaneous wound healing. J Surg Res 91%-31,2000 5. Bhargava P, et al: A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer. Clin Cancer Res 5:1989-1995, 1999 6. Bhujwalla ZM, Artemov D, Glockner J: Tumor angiogenesis, vascularization, and contrast-enhanced magnetic resonance imaging. Top M a p Reson Imaging 10:92103, 1999 7. Boehm T, et al: Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390:404-407, 1997 8. Boehm Viswanathan T Is angiogenesis inhibition the Holy Grail of cancer therapy? Curr Opin Oncol 12:89-94, 2000 9. Brem SS, et a 1 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 1371121-1142, 1990 10. Browder T, Folkman J, Pirie Shepherd S The hemostatic system as a regulator of angiogenesis. J Biol Chem 275:1521-1524, 2000 11. Browder T, et al: Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878-1886, 2000 12. Brower V Tumor angiogenesis-new drugs on the block. Nat Biotechnol 17963-968, 1999 13. Cao Y: Therapeutic potentials of angiostatin in the treatment of cancer. Haematologica 84:643-650, 1999 14. Carmeliet P:Mechanisms of angiogenesis and arteriogenesis. Nat Med 6389-395,2000 15. Carmeliet P, Jain R Angiogenesis in cancer and other diseases. Nature 407249-257, 2000 16. Carmeliet P, et al: Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380435439, 1996 17. Carter SK. Clinical strategy for the development of angiogenesis inhibitors. Oncologist ~ ( S U P 1):51-54, P~ 2000 18. Chambers AF, et a1 Clinical targets for anti-metastasis therapy. Adv Cancer Res 79:91-121, 2000 19. Cherrington JM, Strawn LM, Shawver L K New paradigms for the treatment of cancer: The role of anti-angiogenesis agents. Adv Cancer Res 79:l-38, 2000 20. Cirri L, et a1 Endostatin: A promising drug for antiangiogenic therapy. Int J Biol Markers 14263-267, 1999 21. DAmato RJ, et a 1 Thalidomide is an inhibitor of angiogenesis. Proc Natl Acad Sci U S A 91:40824085, 1994 22. Dankbar B, et al: Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interactions in multiple myeloma. Blood 95:2630-2636, 2000 23. Davis S, Yancopoulos GD: The angiopoietins: Yin and yang in angiogenesis. Curr Top Microbiol Immunol237173-185, 1999 24. Del Maestro RF, Megyesi JF, Farrell C L Mechanisms of tumor-associated edema: A review. Can J Neurol Sci 17177-183, 1990 25. Denekamp J: Vascular attack as a therapeutic strategy for cancer. Cancer Metastasis Rev 9:267-282, 1990
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