Anti-Angiogenesis Drug Discovery and Development

Angiogenesis is a physiological process associated with development and repair of tissues. In embryonic stage, vasculogenesis occurs by de novo synthesis of a network of primitive blood vessels from precursors of endothelial cells called angioblasts which proliferate and coalesce to form the primary capillary plexus. The primary capillary plexus serves as a scaffold for further angiogenesis. It involves remodeling by sprouting and branching of preexisting vessels. In adults, angiogenesis occurs during ovarian cycles and in physiological processes like wound healing and tissue repair. Tumor induced angiogenesis is a pathological condition wherein angiogenesis is up regulated due to aberrant deployment of normal angiogenic machinery. In small tumors, the cells receive nutrition initially by passive diffusion. However, as the tumor grows in size, within the confinement of tumor the availability of nutrients is limited due to increasing competition between rapidly proliferating cells and the diffusion of nutrients is impeded by high interstitial pressure. In order to overcome this nutrient deprivation and for growth, invasion and subsequent metastasis, the tumor cells induce formation of new blood vessels from preexisting ones. This enables the survival of the tumor cells in a hostile microenvironment. Neo-angiogenesis is a complex process involving an extensive interplay between cells, soluble factors and extracellular matrix components. A critical equilibrium is regulated by anti and proangiogenic factors and the balance is shifted in favor of angiogenesis by hypoxia or inflammation. In tumor associated angiogenesis, the cancerous cells secrete or stimulate the secretion of various pro-angiogenic factors including Angiogenin, Vascular endothelial growth factor (VEGF), Fibroblast growth factor (FGF) and Transforming growth factor-β (TGF-β). The stimulation for neo-angiogenesis is called an angiogenic switch. The principal stimulation is thought to be oxygen deprivation possibly assisted by inflammation, oncogenic mutation, mechanical stress etc. VEGF is the most specific angiogenic factor for endothelial cells. VEGF binds to its receptors inducing signaling pathways that in turn bring about endothelial cell proliferation, differentiation, migration, increased vascular permeability and release of endothelial cell precursors from the bone marrow. Sequentially, angiogenesis involves the degradation of basement by proteases, migration of endothelial cells (EC) into interstitial spaces and sprouting, ECs proliferation at the migrating tip and lumen formation, generation of new basement membrane with the recruitment of pericytes, formation of anastomoses and finally blood flow. Targeting angiogenesis for treatment of cancer has been an appealing concept among researchers for over three decades and recently many angiogenic inhibitors have moved from preclinical to clinical trials. Most of angiogenesis inhibitors have been found to be cytostatic rather than cytocidal. Hence, anti-angiogenic therapy is useful when administered in combination with conventional chemotherapeutic agents. Today there are more than 30 anti-angiogenic drugs in use showing considerable disease response. The development of anti-angiogenic drugs involves identification of new targets in the angiogenic pathway as well as identification and management of a new range of toxicities.

Culture techniques using matrix structures have been improved for in vitro studies of angiogenesis. Collagen gel culture was used for studying the biological process of angiogenesis. During angiogenesis, electron microscopic and immunohistochemical studies were performed. DNA micro-array gene expression was also conducted. Capillary tubes in the collagen gel were positive for Tnf-α, Nrp-1 and CD133. To test anti-angiogenic drugs, the collagen gel culture was applied. Thalidomide induced the inhibition of cell migration and suppression of Tnf-α. Thalidomide-induced inhibition of angiogenesis involves apoptosis. Cell migration was inhibited by lovastatin. Lovastatin caused the capillary tube degradation. The collagen gel culture provides a useful method for assaying anti-angiogenic effect of drugs.

Angiogenesis is a physiological phenomenon that establishes the vascular tree during development and maintains the supply of oxygen and nutrients to organs during adult life. A tight balance exists between several pro- or anti-angiogenic actors. The level of these molecules determines de novo angiogenesis or a highly controlled steady-state. Hence, a slight modification of this equilibrium inducing an increase of angiogenesis may provoke pathological angiogenesis in particular favouring aggressiveness of cancers by contributing to tumour growth and metastasis. Disruption of the angiogenic balance is a result of over production of chemokines, in particular the Vascular Endothelial Growth Factor (VEGF) or members of the ELR+CXCL family (ELR for glutamic acid (E), leucine (L) and arginin (R)), which are major proangiogenic factors. Their receptors (VEGFR or CXCR) present at the surface of endothelial and tumour cells are also essential to drive angiogenesis through activation of signalling pathways (RAS/RAF/MEK/ERK and PI3 Kinase/AKT/mTOR). Both signalling pathways drive cell proliferation, survival, and production of angiogenic and inflammatory cytokines. In this context, clear cell renal cell carcinomas (ccRCC) represent a paradigm of deregulated angiogenesis. Thus, ccRCC have been widely used to better understand pathological angiogenesis leading to over vascularization and it will be discussed in this revue the different approaches used by companies to study the implication of angiogenesis in cancers and to develop antibodies or pharmacological inhibitors targeting major receptors or cytokines. Although some therapeutic compounds are used in the clinic, they have given disappointing results particularly on the improvement of global survival. Hence the current challenge is to improve the existing therapeutics or to stratify the patients that will really benefit from them in order to tend to a more personalized therapy.

The hallmarks of cancer include cell growth and metastasis, facilitated by angiogenesis and the remodeling of the extracellular matrix (ECM) by vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), transforming growth factor (TGF-β) and matrixmetalloproteinases (MMPs), which are the predominant factors. These factors are secreted by tumors or the stromal cells in the tumor niche. Oxidative stress and inflammation are the primary causes of the pro-angiogenic factors, including VEGF, MMPs, TGF-β, and IL-8 that collectively activate several signal transduction pathways such as MAP kinase and NF-kB to accentuate ECM remodeling, angiogenesis and cancer metastasis.

Ascorbate (Vitamin C) is a major regulator of the ECM and regulates cancer biology. It inhibits the invasiveness of several cancers such as gastric, oral, pulmonary, fibrosarcoma and melanoma. We have reported ascorbate’s dose-dependent inverse effects on cancer cell growth and the expression MMPs and TGF-β. An extract from P. leucotomos (a fern) in combination with ascorbate simultaneously reduces cancer cell growth as well as the expression of MMP-1 and TGF-β. Further, ascorbate and P. leucotomos, independently and in combination, inhibit the expression of VEGF in a dose dependent manner. A combination of ascorbate and P. leucotomos would benefit as preventive measure; and well as a supplemental regimen for cancer patients.

Angiogenesis is the formation of new blood vessels from the pre-existing ones. It forms the core of the metastatic cascade, since the tumor cells needs nutrition for their growth at distant sites. The work by Folkman et al., has led to a significant understanding of the angiogenesis process and development of novel anti-cancer strategies based on angiogenesis inhibition. In the present chapter we have discussed various pro-angiogenic and anti-angiogenic molecules that are involved in angiogenesis. Further, we have tried to summarise the various molecules both natural and synthetic that inhibit angiogenesis and are being used as novel strategies for the treatment of cancer.

It is more than 30 years since the seminal observations by Folkman of the development of new blood vessels (angiogenesis) in tumors. Ovarian cancer remains the most lethal gynecologic malignancy in the US, and angiogenesis is a particularly important target as VEGF levels are high, manifest as ascites and pleural effusions, and the response rates to single agent bevacizumab, a recombinant humanized monoclonal antibody directed against VEGF, are the highest (16-25%) of any reported in oncology. Antiangiogenics have generally been well tolerated, but are associated with gastrointestinal perforation in 1-2%. New angiogenesis targets are being identified (ANG-2, PDGFR, FGFR, inflammation and the microenvironment), and a plethora of new agents is in clinical development: tyrosine-kinase inhibitors (sunitinib, cediranib, pazopanib), multitargeted agents (XL-184), anti-angiopoietins (trebananib), novel antivascular approaches (VB-111 and ombrabulin). Antiangiogenic therapy appears to impact PFS, but does not impact cure. In subsets of patients, it may improve overall survival (OS), and its use remains costly and controversial. Although approved in Europe, the pathway to approval of bevacizumab for ovarian cancer in the US is currently still unclear. There is a clinical need to define the role of these drugs in ovarian cancer management and to identify robust predictive biomarkers.

Signal Transducer and Activator of Transcription 3 (STAT3) transmits signals from receptors of the IL-6 family of cytokines and from receptors for several growth factors, including vascular endothelial growth factor (VEGF). Transmission occurs directly to the nucleus where STAT3 participates in the expression of numerous genes involved in tumor cell survival, cell cycling, invasion, and angiogenesis. Mechanistic studies have established that upon cytokine or growth factor binding to their respective receptors, STAT3 is recruited to pTyr residues on those receptors via its Src homology 2 (SH2) domain where it becomes phosphorylated on Tyr705. Reciprocal SH2-pTyr705 interactions lead to STAT3 dimerization, followed by its nuclear translocation and a resultant cascade of gene transcription. STAT3 is activated (phosphorylated on Tyr705) in numerous tumor types. Many small molecule inhibitors of STAT3 phosphorylation have been reported. These include natural products such as cryptotanshinone, resveratrol and analogues, quercetin, curcumin and analogues, and 2- methoxystypandrone, as well as synthetic compounds: e.g., Stattic, STA-21, sorafenib, STX-0119, CPA-7, LLL12, PM-73G, sorafenib, and others. For most of these agents, associated with their capacity to inhibit STAT3 phosphorylation is expression of cytotoxicity for tumor cells, raising the issue of whether they also inhibit pathways other than those responsible for STAT3 phosphorylation. Such off-target toxicities could lead to dose-limiting side effects and become a developmental impediment when evaluated in the clinic. However, recent reports on JAK inhibitors, such as Pyridone P6, AZD1480 and ruxolotinib, and SH2-targeted inhibitors suggest that inhibiting STAT3 phosphorylation is not in and of itself cytotoxic to tumor cells. In vivo, administration of AZD1480 or the STAT3 phosphorylation inhibitor, PM-73G, result in reduced tumor volume and microvessel density in human tumor xenografts in mice, suggesting blockade of VEGF signaling. Collectively, these data support the hypothesis that specific, small molecule-mediated inhibition of STAT3 activation may be a viable antitumor treatment strategy by inhibiting interactions between the tumor cells and the stromal compartment. This review will focus on key developments, spanning more than the last decade, that create the foundation for this pursuit.

The process involving new blood vessel sprouting from already existing ones is regulated by a physiological complex mechanism, known as angiogenesis. It plays a key role in wound healing but is also present in pathophysiological conditions such as cancer and cardiovascular disease, which have the highest rates of morbidity and mortality worldwide. It is stimulated mechanically or chemically, with the latter involving several signaling pathways and proteins widely known as growth factors. Anti-angiogenesis has always been an appealing target for cardiovascular related diseases, such as atherosclerosis, with its role still eluding our grasp. In this chapter we focus on the latest trends in antiangiogenic therapy and drug discovery as well as highlight the distinct pathways underlying it. Therapies can range from use of peptides, proteins as well as well-defined chemically synthesized molecules. Latest trends involve gene therapy related approaches, with delivery of anti-angiogenic factors to target areas. Furthermore, toxicity issues arising from the use of anti-angiogenic drugs are discussed and highlighted as many of the drugs employed can cause serious side effects, while others may not achieve maximum therapeutic effect. Anti-angiogenic therapy is a very dynamic field and will continue to evolve and improve in the future. A very interesting addition to the anti-angiogenesis drug arsenal can be achieved with the aim of nanotechnology, a novel but promising scientific field. It is certain that in the future new, more potent drugs will be discovered, posing greater therapeutic potential and lower side effects, providing a much needed boost in this continuously evolving scientific field.