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Keywords:

  • angiogenesis;
  • antiangiogenics;
  • clinical trials;
  • hypoxia;
  • therapeutic target

Angiogenesis

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Angiogenesis is a multistep process in which new blood vessels grow from existing vessels. It occurs as part of the normal menstrual cycle in the ovary and endometrium and it occurs in pathophysiological conditions such as wound healing, proliferative retinopathy, rheumatoid arthritis, paediatric haemangiomas and within tumours. It has been proposed that a tumour is growth limited to 1–2 mm2 by the diffusion of nutrients unless it recruits additional blood vessels and, hence, an ‘angiogenic switch’ is a critical step in tumorigenesis ( Folkman, 1990).

The sequential steps required for angiogenesis are extracellular matrix remodelling, endothelial cell migration and proliferation, and capillary differentiation and anastomosis ( Risau, 1997). This process can be triggered by the secretion of angiogenic factors, either directly by the tumour cells or by accessory cells in the tumour stroma (Fig 1). Maturation and stabilization of these new vessels occurs through the recruitment of pericytes and involves platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor (TGF-β), vascular endothelial growth factor (VEGF) and angiopoietins ( Hirschi & D'Amore, 1996; Darland & D'Amore, 1999).

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Figure 1. Role of tumour cells and tumour-associated macrophages in angiogenesis.

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A large number of angiogenic factors have been identified (Table I) by looking for the development of new vessels in a variety of in vivo assays: chorioallantoic membrane assay (CAM) ( Folkman, 1974), rabbit corneal implant assay ( Gimbrone et al, 1974 ), hamster cheek pouch assay and rodent subcutaneous sponge model ( Andrade et al, 1987 ). Their effects on the different component steps of angiogenesis have then been dissected out using a variety of in vitro models: thymidine uptake or cell counts to study endothelial cell proliferation, migration in the Boyden chamber or in wound healing models, and tube formation in three-dimensional substrates such as collagen or matrigel. More recently, gene knockout data and xenograft studies have expanded the understanding of the role of individual cytokines. Further complexity exists with one endothelial cell receptor, tyrosine kinase, that contains immunoglobulin-like loops and EGF homology domains (Tie-2), mediating either pro- or antiangiogenic activity depending on the ligand binding, angiopoietin 1 (Ang-1) ( Davis et al, 1996 ; Suri et al, 1996 ) or angiopoietin 2 (Ang-2) ( Maisonpierre et al, 1997 ). Ang-1 activates the Tie-2 receptor and is associated with developing blood vessels, its absence leading to defects in vessel remodelling. Ang-2 antagonizes the actions of Ang-1 and plays a role in the destabilization of existing vessels dependent on local concentrations of angiogenic cytokines.

The phenomenon of concomitant tumour resistance, the ability of a large primary tumour to hold smaller tumours in check, led to the discovery of thrombospondin ( Good et al, 1990 ), an inhibitor of angiogenesis. Many more endogenous inhibitors of angiogenesis have now been identified, many of which are fragments of larger proteins ( Sage, 1997), themselves devoid of any antiangiogenic activity (Table II). These include angiostatin, which is a cleavage product of plasminogen ( O'Reilly et al, 1994 ), and endostatin, a cleavage product of type XVIII collagen ( O'Reilly et al, 1997 ). Most recently described is vasostatin ( Pike et al, 1998 ). It has become apparent that angiogenesis is the result of a complex dynamic balance of positive and negative regulators, and over the time-course of any tumour's development the relative importance of several different angiogenic factors and inhibitors will fluctuate.

The role of hypoxia in the regulation of tumour angiogenesis

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Hypoxia occurs both early in tumour growth and throughout expansion and it can regulate the expression of a diverse group of genes: erythropoietin, VEGF, glycolytic pathway enzymes, transferrin, haem oxygenase and inducible nitric oxide ( Guillemin & Krasnow, 1997). Regulation is controlled by the hypoxic stabilization of a transcriptional complex, termed hypoxia inducible factor 1 (HIF-1), binding to a consensus region of the genes, termed the hypoxia response element (HRE) ( Semenza & Wang, 1992). HIF-1 consists of a heterodimer of two proteins, HIF-1α and HIF-1β, and is targeted for rapid proteasomal degradation under normoxic conditions ( Salceda & Caro, 1997). Alternative dimerization partners for HIF-1β, structurally homologous to HIF-1α, have been identified: endothelial PAS domain protein 1 (EPAS-1) ( Tian et al, 1997 ), recently termed HIF-2α ( Wenger & Gassmann, 1997) and HIF-3α ( Gu et al, 1998 ). HIF-2α can transactivate a similar group of genes as HIF-1α ( Wiesener et al, 1998 ). Inhibition of this pathway has a major effect in xenograft studies, reducing both tumour size and vessel density ( Maxwell et al, 1997 ). Studies of HIF-1α knock-outs and deficient embryonic stem cells have affirmed its essential role in solid tumour vascular formation and embryonic vascularization ( Carmeliet et al, 1998 ; Ryan et al, 1998 ).

Role of oncogenes and tumour-suppressor genes in angiogenesis

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Activation of oncogenes and inactivation of tumour-suppressor genes provides a mechanism by which tumours may switch on and sustain angiogenesis, by gain and loss of function, by increasing angiogenic factors and reducing endogenous inhibitors respectively. Inhibition of overexpressed proto-oncogenes or mutant oncogenes could thus provide a therapeutic target ( Kerbel et al, 1998 ).

The role of tumour-suppressor genes in angiogenesis was first established for p53. Wild-type p53 is a positive regulator of thrombospondin expression, an endogenous inhibitor of angiogenesis; inactivation of p53 by mutation or deletional events results in loss of thrombospondin expression ( Dameron et al, 1994 ). Recently, loss of the von Hippel-Lindau (VHL) tumour-suppressor gene has been shown to interfere with the proteasomal degradation of HIFs, leading to high normoxic expression ( Maxwell et al, 1999 ). This provides a mechanism for the previously reported up-regulation of VEGF within tumours with VHL mutations ( Siemeister et al, 1996 ).

Expression of a variety of oncogenes has been reported to be associated with up-regulation of angiogenic factors. Mutant ras is associated with up-regulated VEGF ( Rak et al, 1995 ). In addition to VEGF, H-ras has been shown to stimulate angiogenesis by up-regulation of matrix metalloproteinase (MMP) activity and down-regulation of tissue inhibitor of MMP ( Arbiser et al, 1997 ). The v-src oncogene has also been found to increase expression of VEGF and HIF-1α under both normoxia and hypoxia ( Jiang et al, 1997 ).

Angiogenesis as a therapeutic target

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Understanding the sequential processes involved has facilitated both the development of targeting strategies for new agents and the screening of potential therapeutic agents. Recently, there have been a number of very exciting publications of inhibition of angiogenesis in a range of animal models by both gene therapy ( Goldman et al, 1998 ; Lin et al, 1998 ) and the new agents endostatin and angiostatin ( Boehm et al, 1997 ), providing further proof of the principle that tumours are dependent on angiogenesis. The majority of antiangiogenic agents are still at an early stage of development in phase I, II or III clinical trials.

Difficulties in designing clinical trials for antiangiogenic factors are related to means of selecting and stratifying patients to different treatment arms and the lack of objective assays for assessing response. As the majority of agents may have no direct anti-tumour effect, they will need to be used in conjunction with conventional chemotherapy or after standard treatment, when tumour burden is low, to induce further tumour regression or as maintenance treatment. Recently, four different antiangiogenic factors were tested at three distinct stages of disease progression in a transgenic mouse model of pancreatic islet cell carcinogenesis and they produced distinct efficacy profiles ( Bergers et al, 1999 ). Even endostatin, one of the most widely investigated agents, was not the most effective at each stage. Thus, different drugs may need to be targeted to specific stages of cancer to be most efficacious. Methods of assessing angiogenesis will be briefly discussed and the evidence for the importance of angiogenesis in haemopoietic conditions before reviewing the data on agents currently in or awaiting clinical trial.

Methods of assessing tumour angiogenesis in tissue sections

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

A number of different methods, based upon that first described by Weidner et al (1991 ), have been used in studies of a wide range of solid and haemopoietic tumour types. Tumour blood vessels, within the section, are highlighted using tinctorial stains, or the immunohistochemical detection of endothelial cell-expressed antigens such as CD31 or von Willebrand factor, and the microvessel density (MVD) is then assessed in the most vascular region. Practical considerations, such as the choice of endothelial-specific antibody and methods of assessing the tumour vascularity, have been recently reviewed ( Fox & Harris, 1997). Increased MVD as a measure of angiogenesis has been found to be a powerful prognostic tool in many solid tumour types: breast ( Horak et al, 1992 ), colon, lung ( Giatromanolaki et al, 1996 ), bladder ( Dickinson et al, 1994 ) and head and neck. The majority of studies have also found an association between high MVD and lymph node metastasis and a significant reduction in relapse-free and overall survival ( Fox et al, 1996 ; Zetter, 1998).

Other markers of angiogenesis

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

As surrogate markers of angiogenesis, measurement of a number of angiogenic factors and/or their receptors is now possible in tumour tissue or patient body fluids; such factors include VEGF, bFGF, FLT1 and urokinase receptor. It is not clear, however, whether these markers will rise and fall in proportion to microvessel density or extent of tumour vascular mass. Up-regulation of several angiogenic factors and their receptors, at the mRNA and protein level, within tissue from different tumour types has shown a relationship to clinicopathological parameters, including survival ( Anandappa et al, 1994 ; Brown et al, 1995 ; Moghaddam et al, 1995 ; Takahashi et al, 1995 ; Ferrara & Davis-Smyth, 1997; Relf et al, 1997 ). The measurement of serum VEGF and bFGF has found them to be elevated in many cancer patients ( Dirix et al, 1997 ) and a raised serum VEGF to be related to poor outcome in some patient case series, including one of non-Hodgkin's lymphoma (NHL) ( Salven et al, 1997 ). Although serum VEGF is the most widely studied marker, recent studies have shown that the major source of serum VEGF is platelets ( Banks et al, 1998 ). Thus, plasma VEGF may be a better marker, although platelets may have a key role in delivering VEGF locally to tumours ( Salgado et al, 1999 ). The role of in vivo techniques in assessing tumour angiogenesis are currently being evaluated: positron emission spectroscopy (PET) to measure tumour perfusion rates, prebiopsy infusion of pimonidazole as a hypoxic marker ( Raleigh et al, 1998 ) and the use of an Eppendorf oximeter to obtain tumour oxygen measurements. The last technique has been found to be of prognostic use in cervical cancer ( Hockel et al, 1998 ).

Evidence for increased angiogenesis in haematological conditions

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

With the exception of multiple myeloma (MM) and NHL, relatively few studies have been published relating angiogenesis to clinicopathological data. However, the report of increased MVD in diagnostic bone marrow trephines in a series of cases of childhood acute lymphocytic leukaemia (ALL) ( Perez-Atayde et al, 1997 ) has kindled interest in the area. Increased MVD appears to be a common finding in haemopoietic malignancies: acute myeloid leukaemia (AML) ( Shami et al, 1998 ), chronic myeloid leukaemia (CML) and myelodysplastic syndrome (MDS) ( Aguayo et al, 1998 ), chronic lymphocytic leukaemia ( Kini et al, 1998 ) and hairy cell leukaemia (HCL) ( Kini et al, 1999 ).

An increased urinary bFGF was found in all of the subset of ALL patients tested, which fell with response to treatment ( Perez-Atayde et al, 1997 ). AML leukaemic blasts have been found to express both VEGF protein and its receptors ( Fiedler et al, 1997 ). Additional elevated serum concentrations of hepatocyte growth factor (HGF) have been found in a subset of cases of AML associated with increased early mortality ( Hjorth-Hansen et al, 1999 ). Both T- and B-lymphoblastic leukaemia and Burkitt's lymphoma cell lines have been shown to produce both angiogenic factors VEGF and bFGF and matrix metalloproteinases ( Vacca et al, 1998 ), and high mRNA expression of VEGF and its receptors have been reported in a wide range of haemopoietic malignancies ( Bellamy et al, 1999 ).

In a study of MM and monoclonal gammopathy of uncertain significance (MGUS), MVD correlated with labelling index and disease activity ( Vacca et al, 1994 ; Munshi et al, 1998 ). The expression of adhesion molecules ( Vacca et al, 1995a ) and the number of infiltrating mast cells ( Vacca et al, 1999 ) also correlate with MVD, suggesting that mast cells may be an important source of angiogenic actors. A recent study has shown dynamic magnetic resonance imaging provides an alternative non-invasive method for quantifying angiogenesis in MM patients, and could be used serially to monitor response to treatment ( Moehler et al, 1998 ). High serum levels of HGF in myeloma patients and HGF production in primary myeloma cell cultures and expression of its receptor c-Met has been reported ( Borset et al, 1996 , 1999). Increased angiogenesis has also been reported in an animal model recently described for studying the biology of MM ( Yaccoby et al, 1998 ). A recent small study has compared the MVD in bone marrow trephines, before and after autologous stem cell transplantation, in patients with MM achieving both complete and partial responses ( Rajkumar et al, 1999 ). A high pretransplant MVD was found with a non-significant reduction after transplantation; it was suggested a continuing high MVD may reflect residual disease. To assess the prognostic value of bone marrow angiogenesis, it is currently being examined prospectively in two Eastern Cooperative Oncology Group (ECOG) randomized trials for MM.

In NHL, increased angiogenesis has been found to be associated with tumour progression ( Vacca et al, 1995b ; Ribatti et al, 1996 ). Both macrophage ( Ribatti et al, 1999 ) and mast cell numbers ( Ribatti et al, 1998 ) in NHL are also positively correlated with MVD.

The development of haemopoiesis is closely related to that of angiogenesis, with evidence that a number of the endothelial cell receptors, Flt-1 and Tie-2, are essential for the development of normal haemopoiesis ( Shalaby et al, 1995 ; Takakura et al, 1998 ).

Antiangiogenic agents

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

The broad mechanisms by which these agents are thought to work are listed in Table III. Antiangiogenic agents, both those currently in or awaiting clinical trial and those already licensed that have antiangiogenic activity in addition to other therapeutic actions, will be discussed within this framework along with newly identified therapeutic targets. Tables IV and V give overviews of the agents in phase II and III trials. Further details may be found in a recent review ( Zhang & Harris, 1998). The potential of gene therapy to deliver antiangiogenic agents will not be explored, but has recently been reviewed ( Kong & Crystal, 1998). Information on on-going trials may be obtained from the NCI, who provide information on new clinical trials of antiangiogenic agents at a web site http://207.121.187.155/nci_cancer_ trials/zones/pressinfo/angio/.

Table III.  Mechanisms of action of antiangiogenic factors. Thumbnail image of
Table IV.  Agents in phase III trials. Thumbnail image of
Table V.  Agents in phase II trials. Thumbnail image of

Interference with angiogenic stimulators

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Interferon a

Interferon-α inhibits the production of bFGF, and has been used successfully in the treatment of life-threatening airway haemangiomas in infants ( Ezekowitz et al, 1991 ; Ohlms et al, 1994 ) and in the therapy of a recurrent giant cell tumour of the mandible ( Kaban et al, 1999 ). Interferon alpha-2a, 3 million units/m2 was given by daily s.c. injection for between 2 and 31 months. It has also shown some activity in phase II trials of metastatic renal cell carcinoma in combination with interleukin 2 or 13-cis-retinoic acid ( Motzer et al, 1995 ). It is currently in phase II/III trials.

Thalidomide

Thalidomide has been shown to inhibit bFGF-induced angiogenesis in both the CAM and rabbit cornea assay ( D'Amato et al, 1994 ). It would seem likely that this antiangiogenic activity accounts for its teratogenic effect in developing limb buds. The antiangiogenic activity of thalidomide is mediated by an epoxide metabolite, generated in vivo in the liver. Neither the parental thalidomide nor its hydrolysis degradation product is active ( Kenyon et al, 1997 ). It is currently undergoing phase II trials in patients with solid tumours, including breast, brain and prostate cancer ( Figg et al, 1999 ), AIDS-related Kaposi's sarcoma (AIDS-KS) and other angiogenic disorders such as diabetic retinopathy and age-related macular degeneration. A recent abstract reported thalidomide to have marked anti-tumour activity when used in refractory MM ( Singhal et al, 1998 ) and proposed that it be incorporated into combination chemotherapy trials. Its oral bioavailability and the existing clinical experience of its use make it a potentially useful adjuvant therapy.

Suramin

Suramin is a polysulphonated naphthylurea that has been used for the treatment of protozoal and helminthic infections since the 1920s. It has multiple actions, including inhibiting mitochondrial function and reducing DNA polymerase activity. Recently, it has been shown to have an antiangiogenic effect by inhibiting the binding of a number of angiogenic factors: bFGF ( Braddock et al, 1994 ), EGF, insulin-like growth factor (IGF), interleukin 12 (IL-12), PDGF, TGF-α and TGF-B ( Boylan et al, 1998 ). Suramin is a difficult drug to administer because it has a narrow therapeutic window and a long and variable half-life (44–54 d). It has been used with some success in the treatment of hormone-refractory prostate cancer ( Kehinde et al, 1995 ), and is continuing in various phase I/II trials in this condition. Local instillation has also been reported to be successful in treating superficial transitional cell carcinoma of the bladder ( Walther et al, 1996 ) and peritoneal mesothelioma ( Westermann et al, 1997 ).

Monoclonal antibodies to angiogenic stimulators

In animal xenograft experiments, monoclonal blocking antibodies to VEGF have reduced tumour growth and angiogenesis in prostate ( Borgstrom et al, 1998 ), colon and glioblastoma ( Kim et al, 1993 ) tumours. Humanized anti-VEGF IgG monoclonal antibodies are currently in phase II/III trials against lung, breast, prostate, colorectal and renal cancers.

Fragmin and other heparin derivatives: (ISMS)3 analogues and pentosan polysulphate

In addition to its other actions, fragmin has antiangiogenic activity and potential indications in the treatment of solid tumours. A specific region of heparin, IdoA[2-OSO3]α1-4aManR[6-OSO3](ISMS)3, binds to bFGF and blocks its interactions with its receptors. (ISMS)3 analogues GM-306 and GM-1474 have been shown to inhibit bFGF proliferation of some tumour cell lines. The synthetic heparinoid pentosan polysulphate sodium (PPS) that has been used as an anticoagulant also has antiangiogenic activity. It is currently in phase I and II trials of advanced cancer and AIDS-KS, with disease stabilization observed in a small subgroup. Although s.c. administration has been mainly used, an oral preparation appears safe and is continuing in trial ( Marshall et al, 1997 ).

Interference with angiogenic receptors

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Angiogenic receptor inhibitors: SU5416, lefluonomide (SU101), SU006668 and PTK 787/ZK22584

SU5416 is a synthetic low molecular weight (MW) inhibitor of the Flk-1/KDR tyrosine kinase receptor expressed in endothelial cells, which prevents VEGF-induced angiogenesis. It is given i.v., and in a phase I trial of patients with end-stage cancer disease stabilization was seen in various solid tumour types. Phase I/II trials are open for enrolment in both the UK and USA in combination with either paclitaxel or 5-FU/leucovorin in advanced malignancies and colorectal cancer, respectively, or as a single agent for AIDS-KS.

Lefluonomide (SU101) is another synthetic receptor tyrosine kinase inhibitor that blocks platelet-derived growth factor (PDGF) receptor signalling. In subsets of brain, prostate, ovarian and non-small-cell lung tumours, the PDGF receptor is thought to be the driving oncogene and trials, using i.v. administration, are under way in these conditions. A phase III trial is under way in the treatment of first relapse glioblastoma multiforme, the condition in which most experience has been gained. Earlier trials have shown partial response or disease stabilization in 41% of glioma patients for up to 94 weeks. It has also been used as an immunosuppressant drug with efficacy in advanced rheumatoid arthritis ( Silva & Morris, 1997).

SU006668 inhibits the signalling of the VEGF receptor Flk-1/KDR, the PDGF receptor and the fibroblast growth factor 1 receptor. Phase I trials are under way in solid tumours with both an oral and i.v. formulation.

PTK787/ZK22584 blocks VEGF receptor signalling and is to enter phase I trials in patients with advanced cancer in Germany and in the UK. In the USA, a phase I trial against glioblastoma and AIDS-KS and a phase I/II trial against von Hippel-Lindau disease have been started.

Soluble receptor fragments

A variant of the VEGF receptor Flt-1 exists as a soluble truncated form, sFLT-1. Its binding affinity for its ligand VEGF is maintained and, hence, it can compete with the wild-type full-length receptor for VEGF. Its normal biological function is unclear. In animal experiments, expression of sFLT-1 is associated with inhibition of tumour growth, metastasis and mortality rate ( Goldman et al, 1998 ). This negative regulator of VEGF has not reached the stage of therapeutic trials, but might provide a target for gene therapy.

Dominant negative receptors

In animal models of many tumour types, suppression of tumour growth has been observed by the retroviral expression of a dominant negative Flk-1/KDR mutant ( Millauer et al, 1996 ). This mutant is a truncated Flk-1/KDR receptor, lacking the kinase domain, which associates preferentially with the wild-type receptor. Ligand binding to these dimers fails to produce any tyrosine kinase activity. They may also be possible targets for gene therapy.

Carboxyamidotriazole

Carboxyamidotriazole (CAI) is a synthetic low MW inhibitor of non-excitable calcium channels that inhibits the tyrosine kinases associated with angiogenic factor receptors. It has additional cytostatic properties related to its interference of calcium influx. Phase I trials of an oral formulation found it to be well tolerated, with disease stabilization observed in some patients with refractory solid tumours ( Kohn et al, 1996 ). Phase II/III trials are under way against ovarian, non-small-cell lung and renal cancers.

Platelet factor 4

Platelet factor 4 (PF-4) is an endogenous inhibitor of angiogenesis ( Maione et al, 1990 ), which is stored in platelet α granules. PF-4 interacts with heparin-binding angiogenic factors bFGF and VEGF165, inhibiting their receptor binding ability. It also appears to disrupt VEGF receptor-mediated transduction by an additional, as yet unknown, mechanism ( Camussi et al, 1995 ). Several phase I and II trials with recombinant protein have been undertaken in AIDS-KS, high-grade glioma, renal and colon cancer. PF-4 appears to be non-toxic, but activity was limited to intralesional use in AIDS-KS and the short half-life of the current form has limited its systemic uses.

Interference with the extracellular matrix

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Antibodies/peptides blocking integrins: vitaxin, PEX and EMD 121974

Blocking the αvβ3 integrin receptor can induce endothelial cell (EC) apoptosis in dividing or remodelling ECs, leading to inhibition of angiogenesis and shrinkage of tumours ( Brooks et al, 1994 ; Cheresh, 1998). Vitaxin is an IgG1 humanized version of the mouse anti-αvβ3 monoclonal antibody LM-609, produced in a eukaryotic expression system. Phase I trials have shown it to be well tolerated, and phase II trials are under way in AIDS-KS, advanced cancer and other diseases in which there is evidence of a role for vitronectin, i.e. arthritis, psoriasis and inflammatory diseases.

PEX is the C-terminal haemopexin-like domain of the matrix metalloproteinase 2 (MMP-2). It prevents binding of MMP-2 to the integrin receptor and blocks cell-surface collagenolytic activity ( Brooks et al, 1998 ). A naturally occurring form can be identified, suggesting it may be an endogenous inhibitor of MMP-2. Its potential as an antiangiogenic agent is being assessed.

EMD 121974 is a small peptide that blocks integrins and will be entering phase I/II trials for AIDS-KS, brain tumours and solid tumours later this year.

Interference with the control of angiogenesis

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Modulators of the hypoxia response

As hypoxia is a driving force for angiogenesis and it is found to some extent within all solid tumours, the hypoxia response pathways have become therapeutic targets. To be able to target tumour cells with bioreductive drugs, on the basis of their hypoxic status, would help to overcome the observed resistance of hypoxic cells to conventional radiation and chemotherapy. Hypoxic induction of modified promoters have been used in vitro to increase expression of a bacterial cytosine deaminase, promoting conversion of the pro-drug 5-fluorocytosine to 5-fluorouracil, resulting in increased cell death ( Dachs et al, 1997 ). Hypoxia-regulated gene therapy with activation of cyclophosphamide will enter a clinical trial this year (Oxford Biomedica).

Oncogene inhibitors

Therapeutic targeting of oncogenes and their downstream signalling pathways is at an early stage of development. Farnesyltransferase inhibitors which block the activity of H-ras and antibodies against the product of the erb-B2 oncogene, epidermal growth factor (heregulin), suppress angiogenesis and tumour proliferation in xenograft experiments ( Ciardiello et al, 1996 ; Cox & Der, 1997). All these agents are in phase I/II trials or are already marketed.

Interference with proteolysis

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Matrixmetalloproteinases (MMPs)

Degradation of the extracellular matrix is essential for EC migration and tumour invasion and metastasis. MMPs are an important group of degradative proteases ( Jones & Walker, 1997), comprising over 18 enzymes. Six synthetic MMP inhibitors and one naturally occurring MMP inhibitor are currently in clinical trial. The most advanced of these are Marimastat, Bay 12-9566 and AG 3340, which are in phase III trials in pancreatic, non-small-cell lung, ovarian, breast and prostate cancers.

Marimastat chelates zinc at the active site of MMPs and is a broad inhibitor of MMPs. It has good oral bioavailability and has shown favourable biological responses with significant reductions in serum levels of tumour antigens ( Gore et al, 1996 ), but, to date, not tumour regression. Its main toxicity is reversible musculoskeletal pain.

Bay 12-9566 is an orally active non-peptide biphenyl compound that inhibits particularly MMP-2 and MMP-9. It is better tolerated than marimastat, but with little evidence yet of disease activity. Interestingly, neither marimastat nor Bay 12-9566 appear to affect wound healing, as assessed in patients requiring surgery while on the drug.

AG 3340/Agouron is currently being assessed in combination with paclitaxel/carboplatin in non-small-cell lung cancer and with mitoxantrone/prednisolone in hormone refractory prostate cancer.

Neovastat is a naturally occurring MMP inhibitor, derived from shark cartilage extract, with both antiangiogenic and anti-inflammatory activity. No published results are yet available for the phase I/II trials in solid tumours or for the phase III trial under way in Canada.

Three new synthetic MMPs, CG527023A, COL-3 and BMS-275291, are entering phase I trials in the USA.

Urokinase receptor antagonists

Urokinase plasminogen activator receptor antibodies are active at inhibiting tumour growth in mice ( Min et al, 1996 ). Low MW antagonists that block urokinase binding to its receptor are in preclinical studies, and they provide another therapeutic approach to target angiogenesis.

AGM 1470/TNP 470

AGM-1470 is a semisynthetic analogue of fumagillin, a naturally occurring antibiotic of Aspergillus fumigatus fresenius ( Ingber et al, 1990 ). It inhibits the MMP type 2 aminopeptidase metAP-2, and thus inhibits EC proliferation and migration ( Sin et al, 1997 ).

AGM-1470 has been given as either a 4-h i.v. infusion once a week or a 1-h infusion three times a week with similar low toxicity. The major dose-limiting complication observed was neurotoxicity. In female mice, AGM-1470 inhibited endometrial maturation and corpora lutea ( Klauber et al, 1997a ).

Most clinical experience has been obtained in AIDS-KS, in which partial remissions have been reported and a phase III trial is under way. There has also been a report of complete remission in a patient with metastatic cervical cancer, after 22 months of treatment, for at least 8 months after discontinuation of treatment ( Kudelka et al, 1998 ). Phase II trials are under way in adults with a variety of advanced cancers: glioblastoma multiforme, pancreatic adenocarcinoma, renal cell cancer and advanced cervical cancer. A phase I trial in paediatric solid tumours, lymphomas and acute leukaemia has recently opened in the USA.

Direct attack on the endothelial cell (including vascular targeting)

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

CM 101

CM 101 is a group B haemolytic streptococcus polysaccharide exotoxin that binds selectively to developing blood vessels. By activating complement c3, it initiates an acute cytokine inflammatory response (IL-1, IL-8 and TNFα), which results in vessel disruption and tumour necrosis ( Hellerqvist et al, 1993 ). In phase I trials, anti-tumour activity has been noted in various tumour types, with tumour regression in duodenal adenocarcinoma and AIDS-KS ( Harris, 1997). Dose-limiting toxicities are similar to TNFα and other cytokines therapies: dyspnoea and cardiac dysrhythmias. It would appear that measurement of E-selectin serves as biochemical evidence of EC damage and could be used to monitor therapy ( Wamil et al, 1997 ). Further trials are ongoing in various solid tumours.

Squalamine/MS-1246

Squalamine is an aminosteroid antibiotic first extracted from dogfish shark liver. It inhibits a sodium hydrogen exchanger, NHE3, and has both antiangiogenic and anti-infective activity. Both an oral and an i.v. formulation are under assessment ( Selinsky et al, 1998 ) in phase I trials in combination with carmustine in brain and breast tumours.

Combretastin A4

Combretastin A4 is a tubulin inhibitor that induces apoptosis in proliferating EC cells, causing acute shutdown of vessels. This probably reflects the key role of the cytoskeleton in migration and proliferation of the EC. It is in phase I trials, with a phase II trial due to start in late 1999.

Endostatin and angiostatin

Endostatin is a 20-kDa C-terminal fragment of type XVIII collagen that was first isolated from a murine haemangioendothelioma ( O'Reilly et al, 1997 ). Recombinant endostatin was then produced in baculovirus and Escherichia coli expression systems and was administered to tumour-bearing mice as a s.c. depot of non-refolded protein that induced dramatic tumour regression. Furthermore, repeated cycles of endostatin have been followed by prolonged tumour dormancy and no emergence of drug resistance ( Boehm et al, 1997 ). Producing it in large quantities in a stable active form has proved difficult ( Cohen, 1999), however the appreciation that zinc was required for its antiangiogenic activity has enabled large-scale culture to take place successfully ( Boehm et al, 1998 ). It appears that it induces EC apoptosis ( Dhanabal et al, 1999 ). Endostatin is due to enter a phase 1 solid tumour study, including lymphoma, later this year. Angiostatin, another endogenous inhibitor which was discovered by Folkman's group from mice with primary Lewis lung carcinoma xenografts ( O'Reilly et al, 1994 ), has been shown to bind to a membrane ATPase. Synergy has been observed with combinations of angiostatin and radiation ( Mauceri et al, 1998 ). It is hoped that angiostatin will also enter clinical trials soon.

Vascular-targeted drug delivery

Targeting drug delivery to tumour vasculature by exploiting differences in protein expression between new and established vessels provides another therapeutic strategy. Doxorubicin coupled to an αv integrin binding motif has recently been shown to have enhanced efficacy and reduced toxicity, compared with conventional chemotherapy, in a breast cancer xenograft model ( Arap et al, 1998 ). An alternative strategy of targeting toxins to vasculature by creating fusion proteins of diphtheria toxin to VEGF165 or VEGF121 has successfully retarded tumour growth in animals ( Arora et al, 1999 ).

Other

Utilizing differences between new and established vessels has been shown to be an effective anti-tumour strategy in animal models. Immature vessels lacking mural pericytes are very dependent on high local concentrations of VEGF to maintain their stability, removing this has proved an effective anti-tumour strategy ( Benjamin et al, 1999 ).

Other/unknown mechanisms

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Interleukin 12 (IL-12)

IL-12 inhibits angiogenesis by up-regulation of IFNγ and IP-10, a CXC chemokine induced by IFNγ. Animal models suggest natural killer cells (NK) mediate angiogenesis inhibition by IL-12 ( Yao et al, 1999 ) and tumour activity has been inhibited in Burkitt lymphoma models. IL-12 has recently entered a phase I/II trial against AIDS-KS and solid tumours.

Dexrazoxane

Dexrazoxane is the + isomer of razoxane and has recently been marketed as a cardioprotectant for use with doxorubicin therapy. It is known to have DNA topoisomerase II inhibitory effects, but appears to have additional antiangiogenic activity ( Hasinoff et al, 1998 ). It has been used in a variety of combination chemotherapy schedules in solid tumours with some partial responses seen. However, the trial designs have not allowed the role of dexrazoxane to be assessed. These issues and issues about myelosuppression are currently being addressed.

Paclitaxel/taxol

Paclitaxel is an anti-cancer agent first discovered in the Taxus species of yew. It stabilizes microtubules and is licensed for use as second line treatment for AIDS-KS, recurrent ovarian and metastatic brain cancers ( Spencer & Faulds, 1994). It may also have additional antiangiogenic activity ( Klauber et al, 1997b ) and has been, and is, under clinical investigation for various solid tumours, lymphoma and leukaemia. The principle toxicities include dose- and schedule-dependent neutropenia and sensory neuropathy. Premedication with steroids and histamine antagonists has decreased the incidence of major hypersensitivity reactions.

2-Methoxyoestradiol (2-ME)

2-ME is a low MW metabolite of oestrogen and is a naturally occurring angiogenic inhibitor ( Fotsis et al, 1994 ). In animal studies, it appears to have angiostatic and paclitaxel-like effects on microtubules and will be entering a clinical trial.

Cyclosporin

A preliminary report has recently shown cyclosporin to have antiangiogenic activity in a range of assays at low non-cytotoxic doses in addition to its previously characterized immunosuppressive effects on IL-1 and IL-2 production and T-cell responsiveness ( Iurlaro et al, 1998 ). It inhibits both MMP-2 and MMP-9 secretion as well as inhibiting EC function, which may be a consequence of its inhibition of EC respiratory chain enzymes.

Conclusions

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References

Angiogenesis has been confirmed to be a prerequisite for tumour development. More recently, the mechanisms have been appreciated to be of relevance not only to solid tumours but also to haemopoietic malignancies. The huge number of antiangiogenic factors currently undergoing clinical trials and the experimental validation of additional therapeutic targets is very exciting. Over the next 5 years, experience on the role in different stages of tumours, methods of assessing response, optimal scheduling regimens and drug combinations will be gained. It would then seem inevitable that antiangiogenics will make the transition from trials to complementing existing modalities of treatment.

References

  1. Top of page
  2. Angiogenesis
  3. The role of hypoxia in the regulation of tumour angiogenesis
  4. Role of oncogenes and tumour-suppressor genes in angiogenesis
  5. Angiogenesis as a therapeutic target
  6. Methods of assessing tumour angiogenesis in tissue sections
  7. Other markers of angiogenesis
  8. Evidence for increased angiogenesis in haematological conditions
  9. Antiangiogenic agents
  10. Interference with angiogenic stimulators
  11. Interference with angiogenic receptors
  12. Interference with the extracellular matrix
  13. Interference with the control of angiogenesis
  14. Interference with proteolysis
  15. Direct attack on the endothelial cell (including vascular targeting)
  16. Other/unknown mechanisms
  17. Conclusions
  18. References
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