Transworld Research Network 37/661 (2), Fort P.O., Trivandrum-695 023, Kerala, India

Angiogenesis: Basic Science and Clinical Applications, 2007: 377-417 ISBN: 978-81-7895-302-1 Editors: M. E. Maragoudakis and E. Papadimitriou

Angiogenesis-based : Principles and practice for disease 23 prevention and intervention

William W. Li, Michelle Hutnik and Vincent W. Li The Angiogenesis Foundation, 124 Mount Auburn Street, Suites 200N Cambridge, MA 02138, USA

Abstract Angiogenesis, the growth of new capillary blood vessels, has been identified as an organizing principle in medicine. More than 70 different diseases are angiogenesis-dependent, and for many of these, efficacious treatments possess the ability to either inhibit new blood vessel growth (anti-angiogenesis) to suppress disease progression or, alternatively, to stimulate neovascularization (therapeutic angiogenesis) to promote improved local perfusion and the delivery of paracrine, survival factors to injured or ischemic tissues. This chapter provides the first comprehensive overview of the principles and practice of angiogenesis- based medicine across different disciplines, and

describes the definitive clinical data supporting the first

Correspondence/Reprint request: Dr. William W. Li, The Angiogenesis Foundation, 124 Mount Auburn Street, Suites 200N Cambridge, MA 02138, USA. E-mail: [email protected] 378 William W. Li et al. generation of treatments for disease intervention and prevention. Future directions of development for angiogenesis-based therapies are outlined.

Introduction Angiogenesis-based medicine is a new field of medical therapy based on the control of new blood vessel growth, or angiogenesis. More than 70 different diseases affecting one billion people worldwide have been identified as angiogenesis- dependent [1]. These include a wide-spectrum of health disorders that are malignant, infectious, inflammatory, immune-mediated, and ischemic in nature [2]. Because angiogenesis, or new blood vessel growth, is a “common denominator” in these conditions, the control of angiogenesis is a unifying approach to disease therapy and for maintaining health. Judah Folkman’s pioneering work in tumor angiogenesis established the field of angiogenesis research [3]. Since the early 1970s, an enormous body of angiogenesis research has elucidated the growth control mechanisms of the microcirculation, yielding new knowledge of the critical role played by new blood vessel growth in physiological and pathological conditions. The normal formation of organs and tissues during fetal development relies upon physiological angiogenesis, as well as the closely related processes of vasculogenesis (de novo formation of blood vessels) and arteriogenesis (modification of existing blood vessels into large arterioles or arteries). The terms ‘angiogenesis,’ ‘neovascularization,’ ‘neo-angiogenesis,’ and ‘vascularization’ are often used interchangeably. In the adult, angiogenesis is precisely regulated so that new vessel growth is suppressed by endogenous angiogenesis inhibitors counterbalancing the presence of local stimulators. The female reproduction cycle reflects periodic, controlled bursts of angiogenesis during corpus luteum formation, endometrial regeneration, and placentation. During pregnancy, a number of angiogenic signals are simultaneously produced by both mother and fetus, and these are required for normal gestation. In both males and females, injury provokes angiogenesis and vasculogenesis for wound granulation, wound repair, and tissue regeneration [4]. In numerous pathologies, normal control mechanisms for angiogenesis are dysregulated, so that neovascularization occurs either excessively or insufficiently. Either situation can lead to disease progression and loss of normal organ function. Excessive new blood vessel growth (pathological angiogenesis) provides perfusion and delivers survival factors to diseased cells. Abnormal vessels may also be hemorrhagic and exhibit hyperpermeability, directly causing tissue disruption or destruction. Insufficient angiogenesis occurs in many cardiovascular conditions, and the inability to generate the requisite compensatory microcirculatory response to hypoxia or ischemia may result in infarction, ulceration, debilitation and death. ‘Angiogenesis-based Medicine’ is a term coined by the nonprofit Angiogenesis Foundation in 1994 to describe medical interventions that either inhibit new vessel growth in circumstances where it is undesirable, or that stimulate new vessel growth to restore perfusion and tissue repair. This chapter introduces contemporary concepts of angiogenesis-based medicine, describes currently available treatments in clinical use, and outlines future directions for this new field that is shaping 21st century medical practice. Angiogenesis-based medicine 379 Principles of angiogenesis therapeutic control Therapeutic interventions that inhibit or stimulate angiogenesis rely upon agents that modulate one or more steps in the cascade of events taking place during new blood vessel growth. Endothelial cells (EC) comprise the internal lining of blood vessels, while the outer cells are mural cells, known as pericytes (PC) and smooth muscle cells (SMC). Angiogenesis inhibitory agents target, suppress or disrupt the action or function of these cells during new blood vessel growth. Angiogenesis stimulatory agents, by contrast, not only must foster activation of these cells, but must direct their coordinated assembly into mature functional blood vessels (For reviews see [2, 5-7]). Modern drug therapies that activate or inhibit angiogenesis are directed against one or more discrete cellular or molecular steps identified in neovascularization: 1) the production, transport, and binding of various angiogenic factors; 2) the binding of these factors to specific EC receptors; 3) activation of intracellular EC signal transduction pathways; 4) EC proliferation and migration; 5) EC sprouting and tube formation; 6) stabilization and maturation of newly formed vessels through the recruitment of PCs and SMCs; and 7) mobilization and homing of bone marrow-derived vascular stem cells, known as endothelial progenitor cells (EPCs). Most diseases with pathological angiogenesis exhibit the increased production of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), basic (FGF2), platelet-derived growth factor (PDGF), and other cytokines. Anti-angiogenic therapies developed to treat tumor angiogenesis and ocular neovascularization are designed to neutralize such factors, or interfere with their signaling pathways within endothelial cells or pericytes. Because normal endothelial cells are quiescent, anti-angiogenic agents do not generally have adverse effects on healthy blood vessels. Pathological vessels, by contrast, are architecturally heterogeneous, exhibit hyperpermeability, and do not support uniform blood flow. Inhibitors of angiogenesis may selectively ‘prune’ away these vulnerable, immature vessels, leaving a remaining population of vessels that possess a more mature phenotype and that are more capable of perfusion [8]. This pruning effect has been termed vascular “normalization” by angiogenesis inhibitors, despite the fact that diseased vasculature never becomes truly normal [9]. Because angiogenic vessels support the proliferation of diseased cells, anti- angiogenic therapies often result in cytostatic effects, i.e., lack of disease progression (stabilized disease). In disease conditions where the new vessels themselves directly constitute the pathology, as in age-related macular degeneration, hemangiomas, or rheumatoid arthritis, anti-angiogenic therapies must regress new vessels and prevent their re-growth in order to be clinically successful. Diseases associated with ischemia are often characterized by a compensatory angiogenic response that is insufficient to maintain tissue viability and function. In such conditions, the therapeutic goal is to stimulate angiogenesis to improve perfusion, deliver survival factors to sites of tissue repair, mobilize regenerative stem cell populations, and ultimately, restore form and function to the tissue. Broadly, therapeutic angiogenesis modalities involve the delivery of stimulatory factors, genes, or cells to target sites, with the goal of amplifying angiogenesis, adult vasculogenesis, and arteriogenesis in a clinically meaningful fashion [10-12]. Because physiological angiogenesis requires not only stimulation but also resolution and 380 William W. Li et al. termination of angiogenesis when a vascular bed has been adequately formed, the design of therapeutic angiogenesis strategies is a complex undertaking that is influenced by host factors, such as endogeneous positive and negative angiogenic stimuli present in the local tissues of individual patients. A number of other host factors, such as diabetes, aging, hyperlipidemia, smoking, and even concomitant medications, have been shown to influence angiogenesis and these may negatively impact on the success of therapeutic angiogenesis. Presently, successful therapeutic angiogenesis approaches rely upon the delivery of recombinant growth factor proteins or hyperoxia, the application of tissue engineered skin constructs or negative pressure, and the use of controlled injury or cellular strain in target tissues. Delivery and mobilization of autologous bone marrow- derived stem cells to ischemic tissue also has been successfully performed in individual patients and small clinical studies with positive clinical outcomes [13]. The basic principle of angiogenesis-based therapies is to restore the body’s homeostatic control of angiogenesis, as it exists in the healthy state. The ‘switch’ for angiogenesis in health is constantly maintained in the off position through a balance of co-existing positive and negative factors. Restoration of this balance should be well- tolerated with minimal or no systemic toxicity. Because the long-term therapeutic control is required for many chronic diseases, safety of angiogenesis modulating interventions is as paramount as efficacy. The following sections describe currently approved and available angiogenesis-based therapies, according to their disease indication and categorization as angiogenesis inhibitors or stimulators.

Angiogenesis based therapies Anti-angiogenic therapies Anti-angiogenic therapies encompass a spectrum of interventions that inhibit new blood vessel growth in pathological tissues. Presently, anti-angiogenic therapies are in clinical use for four major indications: 1) cancer (solid tumors, hematological malignancies); 2) ophthalmic conditions (age-related macular degeneration); 3) skin disorders (actinic keratosis, non-melanoma skin cancers); and 4) vascular tumors (hemangiomas, giant cell tumors) in children. This section briefly describes the role of angiogenesis in each disease condition and the specific anti-angiogenic therapies that have been developed. The therapies and their dosing regimens are listed in Table 1.

Oncology i). Solid tumors All solid tumors are dependent upon angiogenesis [14]. Anti-angiogenic therapy for cancer is originally based on a large body of experimental evidence showing that inhibiting angiogenesis in animal bearing tumors can slow, halt, or regress tumor size. Unlike chemotherapy and radiation, anti-angiogenic therapy does not directly kill tumor cells, but instead targets the vasculature compartment supporting tumor growth. This cytostatic effect represents a paradigm shift for cancer treatment, because tumor shrinkage does not always accurately reflect the clinical benefit of angiogenesis inhibition. Important benefits such as improved survival and stabilization of disease, with good quality of life, are also seen. Angiogenesis-based medicine 381

Table 1. Approved Anti-angiogenic Therapies.

Generic Name Indication Date Dosing Schedule (Brand Name: Approved Company) (Country) Cancer Treatments Metastatic Colorectal 2/2004 (USA) Bevacizumab 5mg/kg IV (Avastin; Cancer every 2 weeks + ) 5-FU and Leucovorin or FOLFOX or FOLFIRI Bevacizumab 7.5 mg/kg IV every 3 weeks + CapeOX Metastatic NSCLC 10/2006 (USA) Bevacizumab 15 mg/kg First Line every 3 weeks + paclitaxel and carboplatin Bortezomib Multiple Myeloma – 5/2003 (USA) 3- to 5-second bolus 1.3 (Velcade: second line mg/m2 i.v. twice weekly Millennium) for 2 weeks followed by a 10-day rest period (21 day cycle) Mantle Cell 12/2006 1.3 mg/m2 i.v 3- to 5- Lymphoma – second (USA) second bolus. twice line weekly for 2 weeks followed by a 10-day rest period (21 day cycle) Cetuximab Metastatic Colorectal 2/2004 400 mg/m2 initial (Erbitux: ImClone, Cancer – second line (USA) loading dose BMS) 250 mg/m2 weekly Locally Advanced or 3/2006 400 mg/m2 initial Metastatic Head and (USA) loading dose Neck Cancer 250 mg/m2 weekly with or without radiation (Endostar: rh- NCSLC First and 9/2005 (China) rhEndostatin 7.5 mg/m2 Second Line days 1 to 14 in 3 week Simcere cycle + vinorelbine + Pharmaceuticals) cisplatin Erlotinib Locally Advanced or 11/2004 150 mg/day (Tarceva: Metastatic Non-Small (USA) Genentech/OSI/ Cell Lung Cancer – Roche) second line Pancreatic cancer – 11/2005 100 mg/day + first line (USA) gemcitabine Lenalidomide Multiple Myeloma - 6/2006 (USA) 25 mg/day capsule on (Revlimid: Celgene) second line Days 1 to 21 of repeated 28-day cycles + dexamethasone 382 William W. Li et al.

Table 1. Continued

Deletion 5q 12/2005 (USA) 10 mg/day capsule on Myelodysplastic Days 1 to 21 of repeated Syndrome 28-day cycles Sorafenib Advanced Renal Cell 12/2005 (USA) 400 mg twice per day (Nexavar: Carcinoma – second Bayer/Onyx) line

Sunitinib Malate Advanced Renal Cell 1/2006 (USA) 50 mg once daily for 4 (Sutent: Pfizer) Carcinoma - first line weeks, followed by 2 weeks off treatment Gastrointestinal 1/2006 (USA) 50 mg once daily for 4 Stromal Tumor weeks, followed by 2 weeks off treatment ENL 6/1998 (USA) 100 to 400 mg/day (Thalomid: Celgene) Multiple Myeloma 12/2003 200 mg/day + (Australia) dexamethasone

Ophthalmic Treatments Pegaptanib Wet Age Related 12/2004 (USA) Once every six weeks (Macugen: Macular 0.3 mg Pfizer/OSI) Degeneration (90 μL) intravitreal injection Wet Age Related 7/2006 (USA) Once a month 0.5 mg (Lucentis: Macular (0.05 mL) intravitreal Genentech) Degeneration injection Dermatologic Treatments Aldara External Genital 5/1997 5% cream applied 3 (Imiquimod; Warts (USA) times per week for a Graceway maximum of 16 weeks Pharmaceuticals) Actinic Keratosis 3/2004 5% cream applied 2 (USA) times per week for 16 weeks Superficial Basal Cell 7/2004 5% cream applied 5 Carcinoma (USA) times per week for 6 weeks

Overview of approaches Three major groupings of anti-angiogenic agents are used for cancer therapy: 1) biologic agents; 2) small molecule drugs; and 3) off-label use of older drugs with anti- angiogenic activity. The biologic agents include monoclonal antibodies directed against the angiogenic factors VEGF (bevacizumab, Avastin) and epidermal growth factor (EGF) Angiogenesis-based medicine 383

(erlotinib, Tarceva; cetuximab, Erbitux), and modified endostatin (rhEndostatin, Endostar), a recombinant protein based on an endogenous . Small molecule anti-angiogenic agents are multi-kinase inhibitors (sorafenib, Nexavar; sunitinib, Sutent), anti-cytokine drugs (thalidomide, Thalomid; lenalidomide, Revlimid), and a proteosome inhibitor (bortezomib, Velcade). The drugs used off-label are administered singly or as ‘cocktails’ of several drugs, or they are administered over long periods of time at low doses in a ‘metronomic’ schedule that induces optimal biological activity. These include: Celexocib (Celebrex), doxycyline (Doryx), (Nolvadex), cyclophosphamide (Cytoxan), and interferon (Roferon), among others.

Colorectal cancer Colorectal cancer (CRC) is the third most common cancer worldwide, with one million new cases diagnosed annually. CRC ranks 4th among all cancer deaths [15]. Numerous studies have established VEGF and its receptors as key regulators of tumor angiogenesis. Their expression correlates with vascularity, metastasis, and clinical outcome in patients with colorectal cancer [16, 17]. Bevacizumab. The US Food and Drug Administration (FDA) approved the first specific anti-angiogenic drug, bevacizumab (Avastin) in patients with metastatic CRC based upon the results of a Phase 3 clinical trial enrolling 813 patients who had received no previous therapy for metastatic disease. Patients were randomized to receive IFL (irinotecan [Camptosar], 5-fluorouracil, and leucovorin) with either bevacizumab (BV) or a placebo [18]. The primary study endpoint was overall survival. Adding bevacizumab to IFL increased median overall survival to 20.3 months vs. 15.6 months, for IFL alone (P<0.001). The objective response rate was 44.8 percent vs. 34.8 percent (P=0.004), and median progression-free survival was 10.6 months vs. 6.2 months (P<0.001), for BV + IFL vs. IFL alone, respectively. The main adverse event of bevacizumab was grade 3 hypertension (11%). Additional clinical studies of bevacizumab combined with other chemotherapy regimens, including platinum-based protocols, are underway. Bevacizumab is a key component of first line therapy in metastatic CRC [19, 20]. Cetuximab. The cetuximab (Erbitux) is FDA-approved as a second line therapy for patients with CRC. Cetuximab is an inhibitor of the epidermal growth factor receptor (EGFR), which is upregulated in 60 to 80% of colorectal tumors [21, 22]. The EGFR signaling pathway regulates cell proliferation, apoptosis, angiogenesis, and metastatic spread [21]. Cetuximab’s approval was based upon the results of a Phase 3 clinical trial of 329 CRC patients whose disease had progressed. Patients were randomized to receive either cetuximab and irinotecan, or cetuximab alone. Cetuximab plus irinotecan increased median survival by 1.7 months [22]. Approximately 80% of patients had skin reactions to cetuximab and developed an acne-like rash. Notably, patients with skin reactions had higher tumor response rates than those without skin reactions.

Non-small cell lung cancer Worldwide, lung cancer is the leading cause of death by cancer and accounts for one third of all cancer deaths [15]. EGFR, an angiogenic marker, is overexpressed in about 60% of all non-small cell lung cancer (NSCLC) cases and correlates with poor prognosis [23]. VEGF is another angiogenic factor correlated to NSCLC. 384 William W. Li et al.

Erlotinib. Erlotinib (Tarceva) is a small molecule EGFR inhibitor and is FDA- approved for the treatment of patients with locally advanced or metastatic NSCLC after failure of at least one prior chemotherapy regimen [24]. In a Phase 3 trial of 731 NSCLC patients who had prior chemotherapy, erlotinib improved survival by 42%, from 4.7 months (placebo group) to 6.7 months. The tumor response rate increased from 0.7% to 8.9%, compared to placebo. Bevacizumab. VEGF signaling plays an important role in the pathology of NSCLC. Poor outcomes are associated with patients whose tumors express VEGF or VEGF receptors.[25] Bevacizumab has been FDA-approved for first line treatment of NSCLC, based upon the results from a Phase 3 clinical trial enrolling 878 patients with recurrent or advanced NSCLC [26]. Patients were randomized to receive paclitaxel and carboplatin alone or paclitaxel and carboplatin plus bevacizumab. The addition of bevacizumab to the chemotherapy combination increased median survival from 10.3 to 12.3 months. rhEndostatin. In China, where 600,000 people die annually from lung cancer, recombinant human endostatin (Endostar) has been approved for the treatment of NSCLC [27]. A randomized, double-blinded, multicenter Phase 3 trial of vinorelbine and cisplatin plus rhEndostatin or placebo was conducted in 486 Chinese patients with advanced NSCLC who had received no previous therapy [28]. The overall response rate for the group receiving rhEndostatin was 35.4% vs. 19.5% in the placebo group. Median time-to-progression was 6.3 months vs. 3.6 months, in the rhEndostatin and placebo group, respectively.

Breast cancer Breast cancer is the leading worldwide cause of cancer death in women [15]. In invasive disease, high intratumoral microvessel density (MVD) is associated with increased VEGF expression, greater metastatic potential, and shorter relapse-free and overall survival [29, 30]. Bevacizumab. Treatment of breast cancer with bevacizumab, in combination with chemotherapy, has clinical benefit. A phase 3 trial (ECOG-E2100) in 715 patients with metastatic breast cancer compared the results of treatment using paclitaxel alone, or paclitaxel with bevacizumab, as first line treatment. The addition of bevacizumab increased progression free survival from 6.11 months to 11.4 months, and the overall response rate from 14.2% to 28.2%, compared to paclitaxel alone [31]. The specific chemotherapy used, however, appears to influence the benefit of bevacizumab. A Phase 3 trial of capecitabine (Xeloda) with or without bevacizumab in 462 patients with heavily pretreated metastatic breast cancer showed no increase in progression-free survival [32]. Multiple clinical trials of bevacizumab combined with various chemotherapy regimens for breast cancer are being studied in first line, adjuvant, and neoadjuvant settings [33]. Metronomic chemotherapy. A number of clinical studies have been conducted studying the effects of metronomic dosing of chemotherapy drugs in breast cancer. A Phase 2 clinical trial of methotrexate (MTX) and cyclophosphamide (Cytoxan), both administered with metronomic dose-schedules, in patients with previously-treated metastatic breast cancer revealed an overall tumor response rate of 19% [34]. A second Phase 2 study enrolling 55 patients added bevacizumab to metronomic low-dose MTX and cyclophosphamide as second line treatment in metastatic disease. The addition of Angiogenesis-based medicine 385 bevacizumab significantly increased the median time-to-progression from 2 months to 5.5 months [35].

Renal cell carcinoma More than 200,000 people worldwide are diagnosed each year with renal cell carcinoma (RCC), and over 100,000 people die annually from the disease [15]. Renal tumors are among the most highly vascularized cancers. Mutations in the Von Hippel- Lindau gene, which lead to overexpression of VEGF and PDGF through a HIF-1α mechanism, are associated with 80% of cases of sporadic RCC [36]. Conventional first line treatment of metastatic RCC includes interferon-alpha, a cytokine therapy that has shown to have immunomodulatory, cytotoxic, and anti-angiogenic affects [37]. More recently, VEGF inhibitors have demonstrated remarkable single agent activity against RCC [36]. The small molecule multi-kinase inhibitors sunitinib (Sutent) and sorafenib (Nexavar) have been approved by the FDA for the treatment of metastatic renal cell carcinoma. Temsirolimus (Torisel), an inhibitor of mTOR (mammalian target of rapamycin), has also shown significant clinical benefit in RCC. Sunitinib. A Phase 3 clinical trial of sunitinib vs. interferon-alpha as first line therapy was conducted in 750 patients with metastatic renal cell carcinoma. Patients receiving sunitinib experienced a median progression-free survival of 11 months vs. 5 months for the interferon-treated group. Sunitinib significantly increased tumor response. compared with interferon. This data confirmed sunitinib’s efficacy in RCC as first line therapy [38]. Sorafenib. Sorafenib was evaluated in a Phase 3 study of 903 patients with metastatic RCC who had failed prior therapies [39]. Treatment with sorafenib increased median progression-free survival to 5.5 months vs. 2.8 months in the placebo group, although tumor response was not significantly increased. Sorafenib is FDA-approved as a second line therapy for RCC. Temsirolimus. Temsirolimus, an inhibitor of mTOR, alone or in combination with interferon-alpha, was evaluated in a Phase 3 study of 626 patients with metastatic RCC who were determined to have a poor outlook [40]. Median overall survival for the temsirolimus alone group was 10.9 months vs. 7.3 months for those treated with interferon-alpha alone. The addition of interferon-alpha to temsirolimus did not provide additional clinical benefit. Bevacizumab. Bevacizumab was investigated in a Phase 2 study of 116 patients with pretreated metastatic RCC. A biweekly dose of bevacizumab (10 mg/kg) increased time to progression from 2.5 months to 4.8 months, compared to a placebo. The probability of being progression-free at 4 months increased with bevacizumab from 20% to 64% [41]. Although bevacizumab is not yet approved for RCC, there is significant off-label use of this agent for the disease.

Gastrointestinal stromal tumor Gastrointestinal stromal tumor (GIST) is a soft tissue sarcoma, originating mainly in connective tissue supporting the stomach and intestines. High MVD in GIST pathology specimens correlated with VEGF overexpression, tumor location (intestine > stomach), tumor size (>/ = 5 cm), and tumor grade (high > intermediate > low grade) [42]. In metastatic GIST, activating mutations of KIT and PDGFRA are found in 85 – 90% and 5% of tumors, respectfully [43, 44]. 386 William W. Li et al.

Imatinib. Imatinib mesylate (Gleevec), which selectively targets KIT and PDGFR kinase activity, was approved by the FDA in 2001 as the first effective treatment for metastatic GIST. The clinical benefit of this drug is limited, however, with 5% of patients exhibiting primary resistance, and 14% developing early resistance to imatinib. Acquired drug resistance to the drug is also observed [45]. Sunitinib. Sunitinib is approved for patients with metastatic GIST, who are either intolerant to imatinib, or whose disease has progressed despite imatinib treatment. A Phase 3 clinical trial was conducted in 312 patients with metastatic GIST after failure and discontinuation of imatinib. Patients were randomized to receive either sunitinib or a placebo. The median time to progression was 27.3 weeks in patients receiving sunitinib vs. 6.4 weeks in patients receiving placebo [46].

Primary brain tumors (Glioma) Glioblastoma multiforme (GBM) is the most common primary brain tumor. Prognosis for newly diagnosed GBM is extremely poor, with a median survival of only 8 to 15 months after diagnosis [47]. Patients with recurrent malignant gliomas have an even worse prognosis, with a median survival of 3 to 9 months. Advanced gliomas are highly vascularized. Increased VEGF expression in tumors correlates with poor prognosis. Bevacizumab. A Phase 2 trial was conducted with bevacizumab and irinotecan in 32 adult patients with recurrent grade III and grade IV malignant glioma. In this study, 63% of the patients had a radiographic tumor response. The median overall survival for grade IV glioma patients was 40 weeks and for patients with grade III glioma, the median overall survival had not been reached at this writing, with 16 patients remaining alive after 48 to 64 weeks treatment. Metronomic chemotherapy. A Phase 2 study of metronomic dosing of thalidomide and celexocib alternating with etoposide and cyclophosphamide was conducted in 29 heavily pre-treated patients with recurrent malignant glioma. The results showed 8% of patients had a partial response and 52% had stable disease [48]. The 6 month progression- free survival (PFS6) in patients was 32%, while median progression-free survival was 3.4 months and median survival was 7 months. This metronomic chemotherapy regimen was reported to be well-tolerated, and the results are considered encouraging in a pre-treated population. A feasibility study of the same protocol was conducted in 20 pediatric patients with recurrent or progressive cancer. Published results reported that 25% of the patients were progression free at 123 weeks from initiation of therapy [49]. ii). Hematogenous malignancies Hematogenous cancers are also dependent upon angiogenesis. The degree of bone marrow vascularity in patients correlates with extent of disease and patient outcome in leukemia, multiple myeloma, and myelodysplastic syndromes (MDS). In these conditions, angiogenesis in the bone marrow improves the delivery of paracrine survival factors to cancer cells.

Multiple myeloma Multiple myeloma is the second most common blood cancer, and an estimated 750,000 people worldwide have the disease. Angiogenesis-based medicine 387

Bortezomib. Bortezomib (Velcade) was FDA-approved for the treatment of multiple myeloma that has relapsed after two prior treatments or where resistance has developed following the last treatment. In a Phase 3 trial of 669 patients with multiple myeloma treated with at least one prior therapy, bortezomib treatment was compared to high-dose dexamethasone. Patients receiving bortezomib experienced increased median time to progression (6.2 months vs. 3.5 months), improved overall survival, and increased response rate (38% vs. 18%), compared with dexamethasone [50]. Thalidomide. Thalidomide (Thalomid) was approved by the FDA in 2006 for the treatment of newly diagnosed multiple myeloma, despite years of off-label use for this disease, based on Phase 2 clinical trial data. A Phase 3 clinical trial of 207 patients with newly-diagnosed multiple myeloma was conducted studying thalidomide plus dexamethasone vs. dexamethasone alone [51]. The response rate with was 63% for the thalidomide plus dexamethasone group vs. 41% with dexamethasone alone. Dose-limiting toxicities to thalidomide include fatigue, constipation, and peripheral neuropathy. Lenalidomide. Lenalidomide (Revlimid), a rationally designed thalidomide analog, was FDA-approved for the treatment of multiple myeloma, in combination with dexamethasone, for patients who have received at least one prior therapy. Pooled data from two Phase 3 trials (MM009 and MM010) enrolling a combined total of 692 patients demonstrated an improved overall response (59.2% vs. 22.5%) and increased median time to progression (48.1 vs. 20.1 wks) for patients treated with lenalidomide and dexamethasone, compared with dexamethasone alone [52]. The toxicities of lenalidomide are less severe and less frequent than those of thalidomide. Its major side effects include a three-fold risk (12% vs. 4%) for deep vein thrombosis and pulmonary embolism, compared to placebo plus dexamethasone alone.

Myelodysplastic syndromes In 2003, over 10,000 people were diagnosed with of myelodysplastic syndrome, a dysfunction of bone marrow function, in the USA alone. Clinically, underproduction of erythrocytes, leukocytes, and platelets lead to a constellation of anemia, infection, or bleeding. The 3-year survival rate of patients with severe MDS is as low as 35%, with mortality associated with infection or hemorrhage [53]. Transformation from MDS to acute myelodysplastic leukemia (AML) occurs in up to 40% of patients, and carries a poor prognosis. Multiple studies have demonstrated VEGF overexpression by immature myeloid cells in the bone marrow of patients with MDS, and this is associated with increase bone marrow vascularity and the presence of neoplastic cells. Anti-angiogenic therapy has been actively explored due to bone marrow angiogenesis and the association of MDS with malignant transformation. Early clinical studies of thalidomide in hematological conditions included a trial of the drug in 34 patients with myelodysplastic syndromes revealing an impressive 56% hematological response with some patients achieving transfusion independence [54]. Lenalidomide. Lenalidomide is FDA-approved for the treatment of patients with transfusion-dependent anemia due to Low-or- Intermediate-1-risk myelodysplastic syndromes associated with a deletion 5q cytogenetic abnormality (del 5q MDS) with or without additional cytogenetic abnormalities. In a clinical trial of lenalidomide in 43 MDS patients with transfusion-dependent or symptomatic anemia, 56% of the patients 388 William W. Li et al. exhibited a therapeutic response [55]. For patients with del 5q MDS, the response rate was 83%, and 75% of those patients achieved a complete cytogenetic remission. In a larger study of 148 patients with del 5q MDS, lenalidomide treatment resulted in a response rate of 76%, with 45% of 85 evaluable patients achieving complete cytogenetic remission [56]. iii). Cancer prevention Prevention of angiogenesis at the earliest stages of tumor growth has been shown to keep tumors at a dormant, microscopic size [57, 58]. Cancer chemoprevention is achievable using angiogenesis inhibitors derived from both natural products as well as synthetic drugs in human tumors implanted into experimental animals. Human data supports this preventative strategy, in familial colon polyposis and breast cancer, with the drugs such as celecoxib (Celebrex), tamoxifen (Nolvadex) and raloxifene (Evista) that have known anti-angiogenic activity [59-61].

Colon polyposis Familial adenomatous polyposis (FAP) is an inherited autosomal dominant condition in which thousands of polyps develop throughout the colon. Left untreated, 100% of patients develop colon or other intestinal cancers [62]. The average age of onset of polyp development is 16 years. The average age for onset of colorectal cancer is 39 years old. Sporadic colon polyposis occurs later (40 – 60 years old) and is associated with a smaller risk of malignant transformation (8% relative risk without polyp removal). Angiogenesis occurs during polyp development and is considered a pathological driver for the transformation into carcinoma. Celexocib. In a Phase 3 randomized trial, 77 patients with FAP with documented colorectal polyps were treated with celecoxib (100 or 400 mg twice daily) or placebo for 6 months [63]. In patients receiving 400 mg celecoxib twice daily, reductions were seen in both polyp number (28% vs. 4.5%) and in the polyp burden (31% vs. 4.9%), compared to placebo-treated patients. A separate Phase 3 study was conducted to evaluate the effects of celecoxib in 2035 patients with spontaneous colon polyposis who had adenomas removed before study entry. Patients were randomized to receive celecoxib twice daily (200 mg or 400 mg) or a placebo [62]. The number of newly detected colorectal adenomas was compared at three years. The cumulative incidence of having a newly detected adenoma was 61% for patients in the placebo group vs. 43% in the 200 mg of celecoxib twice a day group vs. 37.5% in the 400 mg of celecoxib twice a day group [62] Compared to placebo, an increased risk of cardiovascular events was observed in the group receiving celecoxib.

Breast cancer Substantial clinical evidence supporting the preventative effects of drugs with anti- angiogenic activity for breast cancer. Non-steroidal anti-inflammatory drugs. The chemopreventive effects of nonsteroidal anti-inflammatory drugs (NSAIDs) have been established through numerous studies. A case control study of 323 women with breast cancer compared with 649 matched cancer- free controls determined that selective COX-2 inhibitors (celexocib, rofecoxib) produced a 71% reduction in the risk of breast cancer. The same study showed use of regular Angiogenesis-based medicine 389 aspirin (325 mg) reduced the risk by 51%, and ibuprofen or naproxen reduced the risk by 64% [64, 65]. Acetaminophen and low dose aspirin (81 mg) use did not lower breast cancer risk. Tamoxifen. The National Surgical Adjuvant Breast and Bowel Project (NSABP) established tamoxifen’s efficacy in reducing the risk of developing invasive breast cancer [66-68]. The study enrolled 13,388 women randomly assigned to receive placebo or tamoxifen for 5 years. After 7 years of follow-up, the cumulative rate of invasive breast cancer was reduced from 42.5 per 1000 women in the placebo group to 24.8 per 1000 women in the tamoxifen group. Tamoxifen also reduced the risk of developing non- invasive breast cancer and osteoporotic fractures, but increased the risk of thromboembolic events and endometrial cancer.

Ophthalmic diseases: Ocular neovascularization Angiogenesis in the eye underlies the major causes of blindness in both developed and developing nations: exudative age-related macular degeneration (AMD), proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), neovascular glaucoma, corneal neovascularization (trachoma), and pterygium. In these conditions, new blood vessel growth takes place in ocular compartments that are avascular, such as the cornea, and aqueous or vitreous humour, or where the vascular pattern is highly regulated, such as the retina and subretinal (choroidal) space. Traditionally, ophthalmologists have referred to angiogenesis occurring in the eye using the interchangeable term ‘neovascularization.’ Anti-angiogenic therapy for blinding disorders is based on the targeting of angiogenic factors identified in ocular disease, such as VEGF, fibroblast growth factor (FGF), and placental growth factor (PlGF) [69]. Therapies directed against VEGF halt or regress vessels in age-related macular degeneration, slowing vision loss, or in a minority of cases, partially reversing vision loss. Because VEGF is also a permeability factor, its inhibition also decreases edema in ocular tissues.

Overview of approaches Presently approved anti-angiogenic therapies for ophthalmic conditions are biologic agents that inhibit VEGF: an anti-VEGF aptamer (pegaptanib, Macugen); and a Fab fragment of a monoclonal antibody directed against VEGF-A (ranibizumab, Lucentis). The anti-VEGF monoclonal antibody (bevacizumab, Avastin), which is approved for cancer indications, is used off-label to treat ocular neovascularization.

Age-related macular degeneration Age-related macular degeneration (AMD), a progressive eye disease that results in loss of central vision, is the leading cause of severe vision loss in adults over the age of 65 [70]. The wet form of AMD accounts for 10% of cases, and is characterized by the abnormal growth of new blood vessels from the choroidal circulation beneath the macula. These vessels leak fluid and blood, inducing scar formation and destroying vision. Ranibizumab. A Phase 3 study called MARINA (Minimally classic/occult trial of the Anti-VEGF antibody Ranibizumab in the treatment of Neovascular AMD) established the efficacy of ranibizumab in 716 patients with the wet form of age-related macular degeneration over a 2-year interval, leading to its FDA-approval [70, 71]. Patients received either 24 monthly intravitreal injections of ranibizumab (0.3 mg or 0.5 mg) or 390 William W. Li et al. sham injections. At 12 months, the percentage of patients with a loss of fewer than 15 letters was 94.5% in the group receiving 0.3 mg ranibizumab, 94.6% of those receiving 0.5 mg ranibizumab, and 62.2% of patients receiving sham injections. The percentage of patients with visual acuity improvement by 15 or more letters was 24.8% of the 0.3 mg group, 33.8% of the 0.5 mg group, and 5.0% of the sham-injection group. Visual acuity improved by a mean 6.5 letters in the 0.3 mg group and 7.2 letters in the 0.5 mg group. By contrast, visual acuity decreased by 10.4 letters in the sham injected group. At 2 years, the benefits of receiving ranibizumab remained durable. Bevacizumab. Intravitreal injections of bevacizumab are used off label to treat wet AMD. A short-term study of the efficacy of monthly intravitreal bevacizumab (1.25 mg) was performed in 102 eyes of 102 patients with wet AMD. Mean visual acuity was 20/80 before injection, and improved to 20/63 at 6 weeks post-treatment. The improvement stabilized at 10 weeks through 14 weeks at 20/50 visual acuity [72]. A head to head study comparing ranibizumab vs. bevacizumab is being sponsored by the U.S. National Institutes of Health. Pegaptanib. Pegaptanib was FDA-approved for the treatment of wet AMD in 2004. Combined analysis was performed of two concurrent, Phase 3 clinical trials enrolling 1186 patients receiving intravitreous injections of pegaptanib at three doses (0.3 mg, 1.0 mg, or 3.0 mg) or sham injections, administered every 6 weeks for 48 weeks [73]. In the 0.3 mg pegaptanib-treated group, 70% of patients lost fewer than 15 letters of vision vs. 55% for the sham-injected group. The risk of severe loss of visual acuity (loss of 30 letters or more) decreased from 22% in the sham-injected group to 10% in the 0.3 mg pegaptanib group. In the 0.3 mg pegaptanib group, 33% of patients receiving pegaptanib maintained or gained their visual acuity, compared to 23% of the sham- injected group. Clinical use in the U.S. of pegaptanib for treating AMD has declined due to its lesser efficacy compared with ranibizumab.

Dermatological disorders The healthy skin is richly vascularized, with deep dermal vessels giving rise to the superficial vascular plexus lying beneath the avascular epidermis. Except for follicular- related angiogenesis during the proliferative phase of the hair cycle, blood vessels remain quiescent in healthy skin. This state is maintained by a balance of endogenous angiogenesis stimulators (e.g. VEGF, FGFs, interleukin-8 (IL-8), tumor necrosis factor [TNF]) and angiogenesis inhibitors (e.g. thrombospondin-1 (TSP-1) and thrombospondin-2 (TSP-2)) [74-79]. The dermal-epidermal basement membrane serves as reservoir of these inhibitors, suppressing vascular proliferation in the skin under normal circumstances. In many inflammatory and neoplastic disorders of the skin, however, pathological angiogenesis is frequently observed. Angiogenesis accompanies many inflammatory skin conditions because cytokines liberated from monocytes and macrophages are potent angiogenic stimuli [80]. The hallmark skin plaques in psoriasis, for example, are characterized by an inflammatory infiltrate associated with prominent dermal microvascular expansion, where tortuous and elongated capillaries develop in the dermal papillae [81]. Neovascularization precedes epidermal proliferation and leukocyte accumulation. Additionally, the epidermis of psoriatic skin itself overexpresses many angiogenic factors [82-84]. Common treatments for psoriasis often involve drugs with anti-angiogenic mechanisms of action. Angiogenesis-based medicine 391

Benign and all malignant skin neoplasms are recognized to be angiogenesis- dependent [85]. Some lesions themselves represent vascular proliferations, such as hemangiomas of infancy, angiokeratomas, and pyogenic granulomas [86]. Angiogenesis occurs in epidermal proliferations such as skin warts. In experimental models of transgenic mice carrying bovine papillomavirus-1 (BPV-1), neovascularization occurs following release of FGF2 with malignant progression from fibromatosis to fibrosarcoma [87]. In human warts, lesion vascularity was found to increase from normal peri-lesional skin to human papilloma virus (HPV) negative warts and from these to HPV+ warts. Studies have shown lack of correlation with VEGF expression in warts, suggesting an alternate angiogenic pathway [88]. Other HPV studies have demonstrated an increased MVD in the progression of intraepithelial neoplasia to anal carcinoma, and from cervical dysplasia to carcinoma, although vessel counts of the precursor external ano-genital warts compared to adjacent tissue were not statistically significant [89-91]. Hemangiomas were the first human disease to be successfully treated with anti- angiogenic therapy using interferon alfa-2a [92, 93]. The rationale for these therapies was recognition that, during the proliferative phase, hemangiomas overexpress angiogenesis stimulators (FGF-2, VEGF), whereas during their involutional phase, endogenous angiogenesis inhibitors (Tissue inhibitor of metalloproteinase 1 (TIMP-1), interferon-beta (IFN-beta)) are upregulated [94]. Like all malignancies, cancers occurring in skin are highly angiogenic. Kaposi’s sarcoma (KS) is a cancer occurring predominantly in HIV-infected patients. An uncommon form is sporadically found in Mediterranean men. AIDS-KS lesions are composed of endothelial-like spindle cells that overexpress number of angiogenic factors (VEGF-A, VEGF-C, FGF-2, IL-6, IL-8, Angiopoietin-2 (Ang-2), Tie-1, Tie-2). The viral cause of KS, human herpes virus 8, induces endothelial cells to overexpress multiple forms of VEGF (VEGF-A, -C, and -D) [95]. Additionally, the HIV-Tat protein is an AIDS-specific angiogenic factor, and this protein has synergistic endothelial stimulatory activity with other KS-associated factors (FGF-2) [96]. Prior to the emergence of effective HIV retroviral therapies, which led to a decline in the prevalence of KS, many anti-angiogenic agents were in development for this lesion. Other skin tumors for which anti-angiogenic therapies are in clinic use are: actinic keratoses (AK), melanoma, non-melanoma skin cancers (basal cell carcinoma, BCC; and squamous cell carcinoma, SCC). Sun damage to the skin, caused by excessive exposure to ultraviolet radiation, induces dramatic alternations in the expression of angiogenic factors and cytokine in the epidermis. Within days of intense sun exposure, VEGF and FGF are upregulated in the skin, while the local expression of endogenous inhibitors (IFN-beta, TSP-1) is simultaneously reduced [97]. Even in precursor lesions to skin cancer, such as AK and atypical melanocytic nevi, the switch to the angiogenic phenotype has been induced, rendering these lesions amenable to anti-angiogenic therapy [98, 99]. The role of angiogenesis in melanoma progression is well recognized. In a seminal paper, Breslow described primary melanoma thickness as directly proportional to rate of metastases [100]. Increase in tumor thickness correlates with neovascularization, which facilitates hematogenous metastases. Melanomas > 1 mm in thickness have significantly increased MVD compared to normal dermis and severely atypical melanocytic nevi [101]. Many angiogenic mediators, including VEGF, FGF-2, IL-8, placental-derived growth factor (PlGF), Ang-2, and αvβ3 integrins are upregulated in cutaneous malignant 392 William W. Li et al. melanoma [102, 103]. Tumor dormancy of melanoma micrometastases lack of significant vascularity compared to clinical macrometastases, despite comparable rates of proliferation and apoptosis [104]. Non-melanoma skin cancers (AK, BCC, SCC) demonstrate increased angiogenesis compared to normal skin [99, 105]. A significant association exists between high MVD in these lesions with high VEGF mRNA expression. The more aggressive behavior of SCC compared to that of BCC may be due to its higher vascularity index as well as increased expression of matrix metalloproteinases in SCC. In invasive SCC, the dermal- epidermal junction basement membrane is lost beneath lesions, and there is local reduction of angiogenesis inhibitors, such as TSP-1 and TSP-2 [106].

Overview of approaches Dermatologists use a number of drugs in clinical practice that possess multiple, complex mechanisms of action, including angiogenesis inhibitory activities. Broadly, these drugs include: biologic therapies, such as etanercept (Enbrel) and infliximab (Remicade); immune response modifiers and immunosuppressants such as imiquimod (Aldara), interferon alfa-2a (Roferon), interferon alfa-2b (Intron), cyclosporine A (Neoral); anti-inflammatory agents such as diclofenac (Solaraze), and Polyphenon E; and anti-proliferative agents such as calcipotriene (Dovonex); acetretin (Soriatane), alitretinoin (Panretin), and paclitaxel (Paxene).

Psoriasis An estimated 125 million people, or 2-3% of the world’s population, suffer from psoriasis, a disfiguring diseases that compromises quality of life. Of these, 20-30% further develop psoriatic arthritis as a debilitating complication. The overall cost of treating psoriasis exceeds $3 billion annually. Calcipotriene. Calcipotriene is a vitamin D3 analog that possesses both anti- proliferative and anti-angiogenic activity [107]. The U.S. FDA approved calcipotriene 0.005% ointment in 1993 for the topical treatment of plaque-type psoriasis. The approval was based on a randomized, double-blinded, 6 week clinical trial enrolling 409 patients with stable plaque psoriasis, comparing the efficacy of two topical agents: twice daily calcipotriene (50 micrograms/gm) vs. twice daily betamethasone 17-valerate (1 mg/gm) ointments. After six weeks of treatment, an analysis of patient-reported outcomes showed clinical improvement in 61.2% of the calcipotriene patients vs. 50.5% with betamethasone [108]. Acitretin. Retinoids, derivatives of vitamin A, are commonly used for the treatment of psoriasis. In addition to its pro-differentiation effects, retinoids are anti-angiogenic via down-regulation of VEGF production by keratinocytes [109]. Acitretin (Soriatane), an oral retinoid, was approved by the U.S. FDA in 1996 for the treatment of severe psoriasis [110]. In two double-blinded, placebo-controlled studies, 275 patients with psoriasis received acitretin (25-50 mg orally per day) or a placebo. The results demonstrated significant improvements in scaling, thickness, and erythema in the acitretin-treated group, relative to baseline and to placebo. Cyclosporin A. Cyclosporin A, another commonly prescribed systemic agent with immune suppressant effects, has anti-angiogenic activity by decreasing VEGF production via a COX-2-dependent mechanism [111, 112]. Cyclosporin A microemulsion (Neoral) Angiogenesis-based medicine 393 was originally FDA-approved in 1995 for organ and bone marrow transplants, and in 1997 was approved as a treatment for psoriasis [111]. In a one year, multi-center, randomized clinical study of 400 patients with plaque psoriasis, treatment with intermittent short courses of cyclosporin A microemulsion effectively control plaque psoriasis [113]. Cyclosporin A dosage ranged from 2.5 to 5 mg/kg daily until clearance of psoriasis or a maximum of 12 weeks treatment, with additional courses of treatment with relapse. After one course of cyclosporin A treatment, the probability of achieving > 75% reduction in disease area by day 84 of treatment was 83%, decreasing the requirements for each subsequent treatment. The benefits were durable in > 30% of patients who had not relapsed 6 months after discontinuing treatment. Etanercept/Infliximab. Therapeutic approaches targeting TNF-alpha, such as etanercept, a soluble TNF-alpha receptor fusion protein, or infliximab, a chimeric monoclonal antibody which binds human TNF-alpha, has been shown to result in decreased psoriasis plaque severity. Treatments decrease angiogenesis mediators such as serum TNF-alpha, MMP-9 and E-selectin [114]. An open-label study enrolling 921 patients who had previously received etanercept (Enbrel) showed efficacy on a second course of therapy, similar to their response to initial therapy. A Phase 3 study (Evaluation of Infliximab for Psoriasis [REMICADE(R)] Efficacy and Safety Study, or EXPRESS II) of infliximab (Remicaide) was performed in 835 patients with moderate to severe psoriasis demonstrated rapid improvement and long-term clinical benefit.

Genital warts Genital warts are a common sexually transmitted disease caused by human papilloma virus (HPV type 6 or 11) infection, with a worldwide prevalence estimated to be > 30 million cases. Imiquimod. Topical imiquimod 5% cream is an immune response modifier that exerts anti-angiogenic activity through local upregulation of several endogenous angiogenesis inhibitors, interferon-(α, -β, -γ), interleukin-12, interleukin -18, TIMP, and TSP-1 in treated skin [115]. Imiquimod also downregulates local expression of FGF-2 and MMP-9, and directly promotes endothelial cell apoptosis [116]. Imiquimod was FDA-approved for treatment of external genital warts based on a study of 311 patients with external genital warts. In the study, 50% of patients receiving imiquimod 5% (n=109) cleared their lesions compared to 21% of those receiving the imiquimod 1% formulation (n=102), and only 11% of the placebo-treated group (n=100) experienced clearance [117]. Patients were treated 3 times per week for 16 weeks. A systematic review of six randomized double-blinded studies supports imiquimod’s effectiveness for genital warts [118]. Polyphenon E Ointment. Polyphenon E 15% ointment was FDA-approved in 2006 to treat external genital warts. Polyphenon E contains a defined composition of polyphenolic catechines extracted from green tea leaves (Camellia sinensis) and is the first botanical extract to receive FDA approval as a prescription drug. Epigallocatechin-3- gallate (EGCG) is the major catechin in tea and possesses potent anti-angiogenic activity [119-121]. Green tea extracts inhibit VEGF expression in squamous epithelial cells. Polyphenon E ointment was tested on 1000 patients in 15 countries in a Phase 3 trial and showed sustained clinical efficacy, with complete clearance of warts in 53% of treatment patients (p = 0.01), compared to placebo-treated controls. Wart recurrence at twelve weeks was less than 5%. 394 William W. Li et al.

Kaposi’s sarcoma Kaposi’s sarcoma (KS) is an AIDS-defining cancer that affects approximately 20% of people infected with HIV who are not taking anti-retroviral therapy. Its incidence has declined by > 80% in North America and Europe since the advent of efficacious AIDS treatments. Alitretinoin. Alitretinoin (Panretin) was approved by the U.S. FDA in 1999 as a topical treatment for AIDS-related Kaposi’s Sarcoma (KS). A Phase 3 study with 134 patients showed an overall patient response rate of 37% in patients treated with alitretinoin twice daily for 12 weeks, compared to 7% in the vehicle-treated control group [122]. Imiquimod. Imiquimod has also been reported to be effective in treating limited extent AIDS-related cutaneous KS lesions [123]. Paclitaxel. Paclitaxel infusion (Paxene) was FDA-approved in 1997 for the treatment of AIDS-related Kaposi’s sarcoma after failure of first-line chemotherapy [124]. Its efficacy was established in a study of 107 patients with advanced KS in which tumor response was seen in 57% of patients receiving paclitaxel.

Actinic keratosis Actinic keratoses (AK) are pre-malignant skin lesions that have the potential to develop into SCC if left untreated. Up to 66% of SCC cases begin as AK. Diclofenac. Diclofenac 3% gel (Solaraze) is a topical non-steroidal anti- inflammatory drug with preferential selectivity (3:1) for inhibiting the COX-2 vs. COX-1 enzyme. Due to its COX-2 inhibitory actions, diclofenac possesses anti-angiogenic activity [125, 126]. The U.S. FDA approved Diclofenac sodium gel 3% in 2000 for the treatment of AK. In a multi-center, double-blind, placebo-controlled 4-arm study, 195 patients, each with at least five AKs in up to three designated treatment blocks, were randomized to receive topical treatment with 3.0% diclofenac in 2.5% hyaluronan gel (0.5 gm) twice daily for 30 or 60 days. Patients in the placebo arm received topical treatment with 2.5% hyaluronan gel (0.5 gm) twice daily for 30 or 60 days. For the study group treated for 60 days, the patients who received diclofenac experienced significant improvement compared to those treated with placebo (64% vs. 34%, respectively), as measured using target and cumulative lesion number scores [127]. A Phase 4 open-label study of 76 patients treated with diclofenac (twice daily treatment) showed that 78% of patents had ≥ 75% AK lesion clearance on day 90, increasing to 85% by day 120. Complete clearance was seen in 41% of diclofenac-treated patients by day 90, and in 58% by day 120 [128]. Imiquimod. Imiquimod 5% cream is also FDA-approved for the treatment of AK. In a Phase 3 study of 436 patients with AKs on the face or scalp, there was complete clearance of AK lesions in 45.1% of patients applying imiquimod twice daily for 16 weeks, compared to only 3.2% of patients who received a placebo [125, 126, 129]. Imiquimod also appears to induce “immune memory” as a mechanism to minimize AK recurrence. After a median follow-up period of 16 months, 57% of patients treated twice weekly with imiquimod had no recurrence of lesions, and this number increased to 75% in patients treated 3 times per week [130]. Treatment of AK lesions using imiquimod is considered to be an anti-angiogenic cancer prevention strategy for SCC, by halting neoplastic progression at the AK level [115]. Angiogenesis-based medicine 395

Basal cell carcinoma Worldwide, basal cell carcinoma (BCC) is the most common malignancy in Caucasians, with an incidence increasing at 10% each year. Despite the low metastatic potential for BCC (estimated to range from 0.0028% to 0.55%), the lesions can be locally destructive. Metastases are associated with large lesions that have been neglected for years, and patients with metastatic BCC have a survival rate of 10%. Imiquimod. Imiquimod is approved for the treatment of superficial BCC. In two Phase 3 studies enrolling 364 patients with superficial BCC on the trunk or upper extremities, 82% of patients treated with imiquimod 5 times per week for 6 weeks achieved complete clinical and histological clearance vs. 3% of patients receiving a placebo [131]. A wide variety of imiquimod dosing regimens have been reported, but due to the heterogeneity of individual patient responses, the most effective dosing strategy involves titrating dose frequency in each patient to optimize biological effects and minimize side effects (clinical inflammation). This dosing method is termed iMTD (individualized Maximal Tolerated Dose) [132].

Cutaneous melanoma Annually, approximately 55,000 people in the US are diagnosed with cutaneous malignant melanoma, and 7900 die from the disease. The increase in the number of cases each year is rising worldwide at a faster rate than any other cancer, with a doubling of the incidence of melanoma every 10-20 years. Interferon. Interferon alfa-2b is FDA-approved for use as adjunctive therapy to surgical treatment of patients with melanoma who are at high risk of recurrence. In a study of 287 patients administered interferon alfa-2b, 20MU//m2/day intravenously for one month, and 10MU/m2 3x/week subcutaneously for 48 weeks, significant prolongation of overall survival and relapse-free survival was observed [133]. A prospective, controlled multi-center study of 252 patients with resected melanoma showed a reduction in risk of melanoma-associated death by nearly 50% following treatment with dacarbazine followed by a 6-month course of low dose interferon-alpha (3 MU 3x/week) [134].

Rheumatological disorders Pathological angiogenesis occurs in rheumatological diseases, such as rheumatoid arthritis (RA) and osteoarthritis (OA). In severe RA, the normally avascular joint space is invaded by the vascular pannus, composed of inflammatory cells and proliferating capillaries. In OA, joint inflammation is accompanied by angiogenesis. New blood vessels are hyperpermeable and contribute to joint pathology by leaking fluid into the space, and by delivering destructive proteases, inflammatory cells, and pro-inflammatory cytokines. Drugs with anti-angiogenic activity suppress these properties and are commonly used in the treatment of arthritis.

Overview of approaches Two broad categories of drugs with anti-angiogenic activity are used for arthritis: 1) non-steroidal anti-inflammatory drugs (NSAIDs); and 2) biologic agents directed against cytokines. The NSAIDs include many agents, but selective cyclooxygenase-2 (COX-2) inhibitors (celexocib, Celebrex) are the most potent angiogenesis inhibitors. The biologic agents are directed against tumor necrosis factor-alpha (TNFα) (etanercept, Enbrel; infliximab, Remicade). 396 William W. Li et al.

Rheumatoid arthritis RA is chronic, autoimmune, and destructive joint disease affecting 1% of the world’s population. Angiogenesis occurs in the inflamed joint, driven from local hypoxia and growth factor expression [135]. Inflammation, and angiogenesis are closely intertwined processes, and are inseparable in severe RA. Celexocib. Celecoxib has been shown to reduce joint swelling in placebo- and active-controlled clinical trials of up to 24 weeks in duration [136]. Etanercept. Etanercept is indicated for reducing signs and symptoms of moderate to severe RA, and improving physical function in patients with the condition. Etanercept is effective in 66% of adults with rheumatoid arthritis, with 55% of patients having no progression of joint damage [137]. Infliximab. Infliximab is approved for the treatment of moderate to severe RA that is symptomatic despite methotrexate therapy. A landmark clinical study (Anti-TNF Trial in Rheumatoid Arthritis with Concomitant Therapy, or ATTRACT) enrolled 428 patients with this condition to receive infliximab at 2 doses (3 mg/kg, 10 mg/kg), and methotrexate for two years. Treatments were initiated at start of study, weeks 2 and 6, and thereafter every 4-8 weeks until study completion. Patients receiving infliximab experienced improved signs and symptoms of RA, exhibited decreased joint erosion and destruction at two years, and had improved physical function by self-assessment.

Osteoarthritis Celexocib. Celecoxib has demonstrated significant reduction in joint pain in osteoarthritis compared to placebo. Celecoxib was evaluated for treatment of osteoarthritis of the knee and hip in placebo- and active-controlled clinical trials of up to 12 weeks duration [136]. Doses of 100 mg BID and 200 mg BID of celecoxib improved pain, stiffness, and functional measures associated with osteoarthritis.

Vascular tumors in children Although there are no anti-angiogenic therapies specifically FDA-approved for pediatric diseases, the drug interferon alfa-2a has been used off-label as an angiogenesis inhibitor to treat tumors in children. The anti-tumor activity of interferon includes anti-proliferative and anti-angiogenesis effects, and the ability to enhance natural-killer cell activity and upregulate tumor antigen presentation [138]. Imiquimod, an anti-angiogenic immunomodulator drug, has also been used for cutaneous vascular lesions in pediatric clinics.

Hemangiomas The vast majority of hemangiomas are harmless, appearing soon after birth as small birthmarks that proliferate (from 8 to 18 months old) and eventually regress (from 5 to 10 years old), leaving normal or mildly blemished skin [92, 139, 140]. However, if a hemangioma enlarges and affects a vital structure, it can be life-, vision-, or limb- threatening. Approximately two thirds of such dangerous hemangioma do not respond to standard corticosteroid treatment. Interferon alfa-2a. A clinical study to determine the efficacy of daily interferon alfa- 2a injections was performed with 20 infants suffering from life-threatening or vision- threatening hemangiomas that were refractory to corticosteroid therapy [92]. The lesion size of 18 of the 20 (90%) infants regressed by > 50 percent after 2 to 13 months of Angiogenesis-based medicine 397 interferon therapy. The cumulative experience of interferon treatment for hemangiomas has shown that neurological function must be monitored because of a 28% incidence of neurological abnormalities, including spastic diplegia, in the infants [141]. Imiquimod. Topical imiquimod 5% cream has been used to treat hemangiomas. In a retrospective chart review and analysis of 18 children (median age of 18 weeks old), a total of 22 hemangiomas were treated with imiquimod, applied 3 times weekly in 10 patients, and 5 times weekly in 8 patients. The mean duration of treatment was 17 weeks. Remission was achieved in 4 (18%) hemangiomas, and all superficial hemangiomas improved to some degree. In all mixed and deep hemangiomas, however, there was minimal or no change [142].

Giant cell tumors Interferon alfa2b. Interferon has been investigated off-label for the treatment of giant cell angioblastoma and giant cell tumors of the mandible [143, 144]. Giant cell angioblastoma is a rare pediatric soft tissue tumor that may ulcerate and infiltrate the surrounding tissue. A pilot study was done in using interferon alfa2b in two patients whose tumors could not be completely resected [144]. In both patients, urinary levels of basic fibroblast growth factor (FGF2) were elevated. In one patient, biopsies done 15 months after initiation of interferon treatment were negative for tumor. In the second patient, the tumor completely regressed over the course of 11 months with interferon treatment.

Therapeutic angiogenesis Therapeutic angiogenesis modalities represent a broad range of interventions that generate new blood vessel growth to promote neovascularization and tissue repair. These include: biologic agents, tissue engineered products, mechanical devices, and autologous cells. Presently, there are two major indications for which angiogenic therapies are in clinical use. These are: 1) chronic wounds (diabetic lower extremity ulcers, venous leg ulcerations, pressure ulcers, arterial ulcers); and 2) ischemic heart disease. This section briefly describes the role of angiogenesis in each disease condition, and identifies the specific therapeutic angiogenesis interventions in clinical use. Approved therapeutic angiogenesis modalities are listed in Table 2.

Wound care Wound care is the first arena in which an angiogenesis-based therapy was validated through well-designed large scale clinical trials, leading to FDA approval of a therapeutic angiogenesis product, becaplermin. Angiogenesis is an absolute requirement for successful normal wound healing, playing a central role in the development of granulation tissue. Chronic, or delayed healing wounds possess a number of defects in angiogenesis that contribute to delayed healing. Diabetic wounds are deficient in various growth factors and their receptors, as well as in their ability to recruit bone marrow- derived endothelial progenitor cells to granulation tissue [145-147]. Diabetic and venous leg ulcers secrete high and excessive levels of proteases that digest endogenous growth factors as well as destroy the extracellular matrix critical for tissue repair [148-

150]. Chronic wounds are also contaminated, colonized, or frankly infected by a variety of 398 William W. Li et al.

Table 2. Approved Therapeutic Angiogenesis Modalities.

Generic Name Indication Date Approved (Brand Name: Company) (Country) Chronic Wounds Autologous Platelet-rich Plasma Chronic Wounds 6/2003 (USA) Gel (Autologel: Cytomedix) Becaplermin 0.01% Diabetic Foot Ulcers 12/1997 (USA) (REGRANEX Gel 0.01%: Ethicon) Graftskin Venous Leg Ulcers 6/1998 (USA) (Apligraf: Organogenesis) Diabetic Foot Ulcers 6/2000 (USA) Hyperbaric Oxygen Therapy Diabetic Foot Ulcers 4/2002 (USA) (Multiple Manufacturers) Wound Healing Interactive Wound Dressing Diabetic Foot Ulcers 10/2001 (USA) (Dermagraft: Advanced BioHealing) Negative Pressure Therapy Nonhealing Wounds 3/1995 (USA) (V.A.C.: Kinetic Concepts Inc.) Chronic, Acute, Traumatic, and 1/2000 (USA) Sub-acute Wounds Flaps Grafts Porcine Small Intestinal Partial and Full-thickness Wounds 4/1998 (USA) Submucosa Pressure Ulcers (OASIS Wound Matrix: Venous Leg Ulcers Healthpoint Ltd) Chronic Vascular Ulcers Diabetic Foot Ulcers Traumatic and Surgical Wounds Cardiovascular Disease EECP; Enhanced External Congestive Heart Failure 6/2002 (USA) Counter-Pulsation Stable or Unstable Angina (Vasomedical Inc.) Pectoris Acute Myocardial Infarction Cardiogenic Shock TMR or TMLR; Severe Coronary Artery Disease 4/1998 (USA) Transmyocardial Laser Revascularization (Eclipse Surgical Technologies, PLC Medical Systems, Inc.) commensal or pathogenic bacteria. Because bacteria secrete proteases, the degree of bio- burden in chronic wounds is associated with tissue breakdown, growth factor destruction, and impaired granulation [151]. Chronic hypoxia and ischemia lead to increased collagen degradation in the wound bed and defective angiogenesis, despite increased collagen synthesis and production of growth [152]. Therefore, interventions that address these multiple defects can promote angiogenesis and accelerate wound healing. Angiogenesis-based medicine 399

Overview of approaches There are presently five major categories of wound interventions, classified as ‘advanced modalities,’ that stimulate angiogenesis: 1) growth factor therapies; 2) tissue engineered products; 3) bioactive matrices; 4) mechanical systems; and 5) hyperbaric oxygen therapy. Growth factor therapies include recombinant proteins (becaplermin, REGRANEX 0.01% Gel) and autologous growth factor preparations (AutoloGel; SmartPReP). Recombinant platelet-derived growth factor (rhPDGF-BB) is a pharmaceutical grade angiogenic factor that is topically applied to the wound bed. Autologous growth factor preparations, by contrast, are derived from isolates and chemical preparations of the patient’s own platelets. These platelet- rich preparations (PRPs) contain multiple endogenous growth factors, although there may be inter-individual variation of quantities and bioactivities of the growth factors between patients due to age, sex, and underlying medical conditions. Because platelets also contain some angiogenesis inhibitory factors, such as endostatin, PRPs may concentrate these molecules as well, although this has not been well-studied. Tissue engineered products include bi-layered skin substitutes (Graftskin, Apligraf) and fibroblast-seeded scaffolds (Dermagraft). These products contain living cells capable of secreting multiple angiogenic growth factors into the wound bed. The matrix provides a scaffold upon which proliferating cells migrate within the wound bed. Bioactive matrices involve products based on collagen, such as oxidized regenerated cellulose (ORC)-collagen dressings (Promogran; PRISMA Matrix) and porcine small intestines submucosa (OASIS Wound Matrix). The collagen in these matrices supports cell migration and stimulates fibroblast proliferation. ORC-collagen forms a gel that binds and inactivates proteases from chronic wound fluid, and protects and releases growth factors, such as PDGF [148, 153]. Medical devices delivering physical forces to cells in the wound bed can stimulate angiogenesis. These include negative pressure therapy systems (V.A.C. Therapy System; V1STA), low frequency ultrasound systems (MIST Therapy), and electrical stimulation systems (E-stim). Negative pressure therapy applies suction to cells in the wound bed, creating cellular microdeformations that activate endothelial proliferation, stimulate capillary formation, and reduce protease expression [154-156]. Removal of wound exudates by suction decreases local protease levels and growth factor destruction in the wound. Electrical stimulation is also used for healing pressure ulcers and diabetic wounds [157]. Angiogenesis is stimulated when electrical current is applied to endothelial cells in vitro as well as in vivo [158, 159]. Hyperbaric oxygen (HBO) therapy is another advanced modality used to treat chronic wounds in the clinical setting. Periodic exposure of wounds to hyperoxia increases growth factor expression, angiogenesis, microvascular perfusion, and healing [160-162]. Experimental evidence suggests that HBO also mobilizes endothelial progenitor cells from the bone marrow as another mechanism for stimulating wound angiogenesis [163].

Diabetic lower extremity ulcers Diabetic foot ulcers (DFU) result in more than 85,000 lower extremity amputations each year in the US, and between 25 - 50% inpatient costs related to diabetes care are directly attributable to diabetic foot wounds [164]. Worldwide, an amputation due to diabetes takes place every 30 seconds. 400 William W. Li et al.

Becaplermin (rhPDGF-BB, REGRANEX Gel). Becaplermin is the only FDA- approved prescription drug for treating diabetic lower extremity ulcers. The approval was based on a multi-center, double-blinded, placebo-controlled Phase 3 trial comparing the incidence of complete healing in 382 diabetic patients with chronic lower extremity ulcers (> 8 weeks in duration without closure). Patients were treated with either topical becaplermin or a placebo gel [165]. Daily treatment, following sharp debridement of the ulcer in association with good wound care, continued until complete wound closure, or for a period up to 20 weeks. In the context of good standard care, becaplermin treatment resulted in complete closure in 50% of patients, compared to 35% closure in patients receiving placebo gel. The time to complete wound closure in the patients treated with becaplermin gel was 86 days vs. 127 days for those receiving best standard care plus placebo [165, 166]. Margolis and colleagues performed a landmark retrospective cohort study of 24,898 individuals with diabetic foot ulcers, of which 9.6% received becaplermin. Patients were stratified into 5 groups determined by those least likely to receive becaplermin, to those most likely to receive rhPDGF. The relative risk for complete healing with becaplermin treatment was 1.33. The relative risk for amputation after receiving becaplermin was 0.65, compared to those who did not. The drug’s effectiveness in a large uncontrolled population is therefore similar to its efficacy in a carefully controlled clinical trial [166]. Wound debridement is an absolute requirement for effective treatment of chronic wounds with becaplermin [167]. This was first established in a study by Steed and colleagues of 118 diabetic patients whose wounds were treated with becaplermin or placebo gel. All patients had aggressive sharp debridement before randomization to treatment and debridement was repeated as needed. Of patients treated with rhPDGF, 48% healed vs. only 25% of patients who received placebo. Lower rates of healing were observed when less frequent debridement occurred, and response rate improved with more frequent debridement in both groups. Clinical use of becaplermin has been limited due to concerns over drug cost [164]. The actual cost of incorporating becaplermin into wound therapy to attain complete closure, compared to the direct costs of a chronically nonhealing wound treated with conservative care has not yet been systematically studied. Effectiveness data for diabetic foot ulcers of 26,599 patients from published clinical trials, meta-analyses, and a clinical database were evaluated, however, to determine the cost effectiveness of standard wound care vs. becaplermin or a platelet releasate. Baseline effectiveness for standard care, becaplermin, platelet releasate were 30.9%, 43.0%, and 36.8%, respectively. To increase the odds of ulcer healing by 1% compared to standard care, the incremental cost for using platelet releasate and becaplermin was determined to be $414.40 vs. $36.59, respectively. Autologous Platelet-rich Plasma Gel (AutoloGel). A randomized clinical study enrolling 72 patients with nonhealing diabetic foot ulcers was performed to determine the safety and efficacy of autologous platelet-rich plasma gel vs. standard wound care. Healing was confirmed one week following closure and monitored for another 11 weeks. Of the 40 patients evaluable for analysis in the study, 68.4% of patients treated with the platelet-rich plasma gel healed, compared to 42.9% patients receiving standard care [168]. Graftskin (Apligraf). The use of graftskin in treating diabetic foot ulcers has been extensively reviewed by Dinh and Veves [169]. A randomized clinical trial evaluated the efficacy of graftskin plus standard care compared to standard care alone in 208 patients Angiogenesis-based medicine 401 with noninfected nonischemic chronic plantar diabetic foot ulcers [170]. Graftskin was applied weekly for a maximum of 5 applications or less if complete healing occurred. At 12 weeks, 56% of the graftskin-treated patients achieved complete wound healing vs. 38% in the standard care alone group. The median time to complete closure was 65 days for the graftskin group vs. 90 days for the standard care group (P = 0.0026). Osteomyelitis and lower limb amputations were reported to be less frequent in the graftskin-treated group. The cost-effectiveness of graftskin for treating diabetic foot ulcers was evaluated in a study comparing its use with standard wound care, using a Markov-based simulation model. Transition probabilities were based on clinical trial results and cost estimates were based on estimates obtained in the Netherlands. The study concluded that treatment with graftskin plus good wound care reduced wound care costs over the first year of treatment by 12% over good wound care alone, primarily due to increased ulcer-free time and a reduced risk of amputation [171]. Interactive Wound Dressing (Dermagraft). A randomized study of 314 patients with chronic diabetic foot ulcers was performed to evaluate the ability of Dermagraft plus standard wound care vs. standard care alone to facilitate wound closure at 12 weeks [172]. At 12 weeks, complete wound closure was observed in 30.0% of patients who received Dermagraft, compared with 18.3% closure in patients who received standard care. The Dermagraft-treated group also experienced fewer wound-related adverse events. V.A.C. Therapy System. A randomized clinical trial of 162 patients with diabetic foot ulcers was performed to study the efficacy of negative pressure wound therapy (NPWT) delivered through the Vacuum Assisted Closure (VAC) Therapy System compared to standard wound care [173]. Wounds were treated for 112 days or until complete closure. In the NPWT group 56% of patients healed, compared to 39% in the standard care only group. The rates of wound healing and of granulation tissue formation were also faster in the NPWT group than in the control group. ORC-Collagen (PROMOGRAN Matrix). ORC-collagen is a wound dressing consisting of a bio-adsorbable matrix of collagen and oxidized regenerated cellulose. This dressing was compared to moistened gauze in a randomized trial enrolling 276 patients with diabetic foot ulcers [174]. After 12 weeks of treatment, in patients with ulcers < 6 months duration, 45% of patients treated with ORC-collagen healed compared to 33% of the controls [174]. The cost effectiveness of ORC-collagen has been evaluated in four European countries (France, Germany, Switzerland and UK) in the setting of non- superficial diabetic foot ulcers [175]. Small Intestine Submucosa (SIS, OASIS Wound Matrix). A randomized trial of SIS was performed in 73 patients with diabetic foot ulcers, compared with becaplermin gel. After 12 weeks of treatment, 49% of patients treated with SIS experienced complete wound closure, a result not statistically different from that of patients treated with becaplermin [176].

Venous leg ulcers The efficacy of angiogenesis-stimulating advanced wound care products have also been validated in the treatment of venous leg ulcers (VLU). These wounds result from chronic venous insufficiency, a condition estimated to affect 10-15% of adults in Western countries. Conservative therapy for VLU is only 50-80% successful at six months of care. 402 William W. Li et al.

Graftskin (Apligraf). A clinical trial evaluating the effectiveness of graftskin in VLU determined the tissue engineered product to be well tolerated and more efficacious than standard therapy alone. In a subgroup of patients with hard-to-heal ulcers, graftskin’s wound healing efficacy advantage was even more significant (47% vs. 19%) and the median time to healing was significantly improved (181 days vs. healing not attained) [177]. A review of 13 patients with nonhealing VLUs was conducted to evaluate the cost advantage of graftskin treatment [178]. Graftskin treatment decreased mean ulcer size by 2.37 cm2 per week, compared to an increase in mean ulcer size by 0.72 cm2 per week. Twenty-one ulcers treated with graftskin exhibited > 75% reduction in size. The individual patient ulcer-related medical costs per unit change in ulcer size were lower following graftskin treatment relative to similar costs associated with conventional therapy. Graftskin is a bilayered product, with a bovine collagen matrix containing neonatal fibroblasts, overlaid by epithelium containing living keratinocytes. Questions have been raised concerning the viability of the product in situ after clinical application [179, 180]. A study was conducted in 10 patients to evaluate the persistence of graftskin fibroblasts and keratinocytes in acute partial-thickness wounds treated with the tissue engineered product [179]. Another 10 patients with VLUs treated with graftskin were also studied. Allogeneic DNA was detected in 2 of 8 (25%) patients one month after initial grafting. At 2 months post-grafting, neither patient showed persistence of allogeneic DNA. Both studies demonstrate that allogeneic cells from graftskin do not survive permanently after placement on a wound. ORC-Collagen (PROMOGRAN Matrix). A clinical trial of 73 patients with stagnating venous leg ulcers were randomized to receive either ORC-collagen or Adaptic dressing to compare their efficacy [181]. Target wounds were between 2 and 10 cm in any one dimension. 62% of ulcers in the ORC-collagen-treated group healed or improved, compared with 42% in the Adaptic-treated group. Median decrease in surface area was 82.4% with ORC-collagen vs. 44.6% in the Adaptic group. Small Intestine Submucosa (SIS, OASIS Wound Matrix). A trial of 120 patients with one or more VLUs was performed comparing the efficacy of SIS with standard care (compression) vs. standard care alone [182]. Weekly healing assessments were conducted for 12 weeks, and ulcer recurrence was assessed after 6 months. At 12 weeks, 55% of wounds in the SIS-treated group healed, compared to 34% in the standard-care group. No ulcers recurred at 6 months in patients treated with SIS.

Cardiovascular disease Ischemic cardiovascular diseases are angiogenesis-dependent, including coronary artery disease, peripheral arterial disease (intermittent claudication, critical limb ischemia, hand ischemia, Buerger’s disease), and stroke. In these conditions, multiple signals, including regional hypoxia, inflammation, cytokines, and growth factors released into ischemic tissues stimulate angiogenesis to generate a compensatory increase in perfusion. Mobilization and recruitment of endothelial progenitor cells (EPCs) from the bone marrow plays a key role in ischemia-induced angiogenesis [183]. A study of 514 patients with coronary artery disease demonstrated that increased levels of circulating EPCs correlated significantly, as a cellular biomarker, with decreased risk of death from cardiovascular causes [184, 185]. Arteriogenesis, the process of forming arterioles and arteries from existing Angiogenesis-based medicine 403 small vessels, is closely related to the angiogenesis and ultimately generates collateral channels [186-188]. The goals of therapeutic angiogenesis encompass vasculogenesis, angiogenesis, and arteriogenesis in ischemic tissue. Early attempts to develop therapeutic angiogenesis for chronic myocardial ischemia and critical limb ischemia focused on the delivery of growth factors peptides or their genes [189-192]. Despite promising trends and individual outcomes, Phase II clinical trials of FGF2, VEGF, and VEGF gene transfer failed to meet their pre-specified endpoints, and the programs were discontinued [193, 194]. A major challenge discovered through these studies is the profound impact of a placebo effect that ultimately blunted improvements seen in the treatment group. Growth factor delivery by gene therapy to ischemic myocardium and ischemic limbs continue to advance in clinical development. Cellular therapies based on the delivery of mobilized autologous adult stem cells from bone marrow have generated promising early clinical results [13, 195-202].

Overview of approaches Two available clinical interventions for ischemic heart disease provide clinical benefit by stimulating myocardial angiogenesis: 1) laser revascularization (transmyocardial laser revascularization, TMR, The Heart Laser CO2 TMR System; Eclipse TMR Holmium YAG Laser System); and 2) enhanced external counterpulsation (EECP). Neither modality is specific or optimal for inducing therapeutic angiogenesis. TMR is a surgical procedure, requiring patients to be placed under general anesthesia. A high-energy laser is employed to drill 20 - 40 one-millimeter wide channels directly into the ischemic region of myocardium through to the ventricle. Hemostasis is manually achieved at the entry sites of the channels. Angiogenesis occurs within the myocardium, driven by processes similar to wound healing [203-206]. Symptomatic relief in patients with refractory angina is thought to occur secondary to sympathetic denervation in the heart caused by laser injury, as well as due to improved myocardial perfusion. EECP is a non-invasive procedure developed to treat ischemic heart failure. Pressure cuffs are inflated around the patient’s legs during diastole to compress the vascular bed of the extremities in order to increase diastolic blood flow to the coronary arteries and increase venous return. Experimental dog studies show that EECP increases microvessel density in ischemic myocardial territories, associated with VEGF expression and improved myocardial perfusion [207]. EECP also increases plasma nitric oxide levels and improves systemic endothelial function in patients with symptomatic coronary artery disease [208, 209].

Ischemic heart disease Transmyocardial Revascularization. In a prospective, controlled, multicenter trial, 202 patients with refractory angina and left ventricular free-wall ischemia that precluded coronary-artery bypass surgery or percutaneous transluminal coronary angioplasty were randomly assigned to undergo TMR with a carbon dioxide laser, or to receive continued medical treatment [210]. At 12 months, 72% of the TMR patients experienced an improvement of two or more angina classes, compared to 13% of the patients assigned to only medical therapy. Patients treated with TMR experienced improved quality of life, increased myocardial perfusion, longer survival, and fewer hospitalizations due to unstable angina (2% vs. 69%), compared to control patients. 404 William W. Li et al.

The long-term efficacy of TMR was evaluated in a clinical study enrolling 212 patients with angina and diffuse coronary artery disease who were not candidates for conventional therapy. The patients were randomized to receive holmium:yttrium- aluminum-garnet transmyocardial revascularization (TMR) or continued medical management [211]. After an average follow-up of 5.7 years, 88% of the TMR patients experienced two or more class improvement in angina, while only 44% of patients on medical management improved. The average annual mortality beyond one year was 8% for TMR patients vs. 13% for control patients. There was a 5-year efficacy and survival advantage for patients treated with TMR vs. those receiving only medical management. Additional studies showed improved long-term outcomes for patients receiving TMR. In a single arm study, 78 patients with severe angina not amenable to conventional revascularization were treated with a TMR carbon dioxide laser [212]. After an average follow-up period of 5 years, 68% of the patients had a decrease of 2 or more angina classes. TMR performed adjunctively to coronary artery bypass grafting (CABG) in patients who would be incompletely revascularized by CABG alone also resulted in significantly improved clinical outcomes [213]. Enhanced external counterpulsation. Multiple clinical studies and large treatment registries of patients with refractory angina established that EECP can reduce angina, enhance exercise tolerance and quality of life, and improve objective measures of myocardial ischemia [214]. In 1999, the results of the first randomized, placebo- controlled, multi-center trial evaluating the efficacy of EECP in patients with refractory angina were published [215]. 139 patients with end-stage coronary artery disease and persistent ischemic symptoms were randomized to receive 35 one-hour sessions over 4 to 7 weeks of either EECP or an inactive sham. Those patients receiving EECP demonstrated an increase in the time to 1 mm ST-segment depression, a decrease in anginal episodes, and a trend toward decreased nitroglycerin use. In a recent study of the International Enhanced External Counterpulsation Patient Registry (IEPR), 1458 patients with Canadian Cardiovascular Society (CCS) class II-IV angina with no option for further invasive coronary revascularization procedures were evaluated [216]. At a follow-up 2 years post-treatment, 74% of class II and 70% of class III-IV patients demonstrated a durable improvement in CCS class and remained free of major adverse cardiovascular events. The PEECH (Prospective Evaluation of Enhanced External Counterpulsation in Congestive Heart Failure) study evaluated EECP for the treatment of patients with mild- to-moderate heart failure [217]. 187 patients with mild-to-moderate symptoms of heart failure were randomized to receive either EECP and protocol-defined pharmacologic therapy or pharmacologic therapy alone. Three months post-treatment, both the New York Heart Association (NYHA) functional class and the Minnesota Living with Heart Failure score improved significantly in the EECP group, compared to the control group. Six months post-treatment, 35% of the EECP-treated group increased their exercise time by at least 60 seconds, compared to 25% of the control group.

Future directions Rapid research progress in the angiogenesis field is actively generating new strategies and indications for angiogenesis-based medicine. The interventions described in this chapter are accurately characterized as first-generation therapies. Of these, only Angiogenesis-based medicine 405 six oncology drugs (bevacizumab, endostatin, lenalidomide, sorafenib, sunitinib, thalidomide), and two ophthalmic therapies (pegaptanib, ranibizumab) were specifically designed to treat angiogenesis. The other drugs and technologies have become integrated into the field through post-approval recognition of angiogenesis mechanisms of action that contribute to their observed clinical benefits.

Next generation therapies The next generation of angiogenesis-based therapies are under intensive development in oncology, ophthalmology, wound care, and cardiovascular medicine. Advancements to existing clinical strategies are being made along multiple lines of development:

1) Creation of agonists/antagonists of new targets in the angiogenesis cascade (i.e., PDGF, FGF1, FGF2, PlGF, mTOR, Tie-2, Nrp1, SDF-1, Akt pathway, sonic hedgehog, integrins, Notch-1/Dll4), to be tested alone, or in combination with an already approved therapy [69, 218-223]. 2) Improvements on the delivery of validated targets for stimulating (i.e., gene delivery of PDGF) or inhibiting angiogenesis (i.e., novel strategies for blockading VEGF signaling pathways) [224, 225]. This is combined with efforts to validate cellular or molecular biomarkers, or imaging strategies that can predict or effectively monitor the disease course and treatment response. 3) Clinical testing of combinatorial strategies involving two or more anti-angiogenic agents (i.e., bevacizumab + erlotinib, bevacizumab + sorafenib + temsirolimus, low dose metronomic chemotherapy regimens) or combining multiple angiogenesis stimulatory modalities (i.e., negative pressure therapy + PDGF). Such combinations may involve simultaneous use of treatments, or sequential applications, which may be required for enhanced efficacy, or for addressing compensatory disease mechanisms, i.e., ‘escape’ from anti-VEGF therapy. 4) Design of cellular therapies to achieve therapeutic angiogenesis in a variety of tissue repair and regenerative settings. These efforts are presently focused on three cell categories: unselected bone marrow-derived mononuclear cells; selected CD34+ EPCs; and human adipose-derived stromal cells (hASC) [226-228]. Circulating endothelial cells (CECs) are also being evaluated for their utility in assessing the efficacy of anti-angiogenic therapy [229]. 5) Identification and study of dietary-derived angiogenesis inhibitory compounds (i.e., ellagic acid, ECGC, genistein, resveratrol, brassinin, delphinidin, quercetin, isoliquitrin) that may be used for three primary purposes: to guide new synthetic drug discovery and design; for non-pharmacological suppression of pathological angiogenesis through dietary strategies, as an adjunct to drug therapy; and for developing novel preventative approaches to disease via dietary selection, or through the rationale use of bioactive supplements [230-235].

Additionally, an ambitious project is underway to map the entire human vasculature by phage display [236]. The goal is to identify unique epitopes, ligands, and markers that can differentiate diseased from normal endothelium in different tissues and organs, and that may serve as distinct endothelial targets present only in specific types of diseased endothelium. This information would powerfully guide future efforts in developing targeted therapies, as well as targeted imaging modalities, for angiogenesis-based diseases. 406 William W. Li et al.

A number of new potential clinical indications for angiogenesis-based therapies are beginning to emerge from translational research examining the role of endothelial cell growth in various disease states. Notable examples of these future indications include neurodegenerative conditions, reproductive disorders, and obesity:

Neurodegenerative disorders Angiogenesis, neurogenesis, and neuronal maintenance, are closely related processes, with each sharing anatomical and molecular similarities [237]. There is increasing evidence that VEGF and other angiogenic factors play a role in neurodegenerative diseases, such as amyotrophic lateral sclerosis (Lou Gehrig’s disease), peripheral neuropathies, and erectile dysfunction [238-244]. VEGF serves as a neuroprotective factor in these conditions, and its expression is important for neuronal survival as well as perineural angiogenesis in the vasa nervorum [245]. Experimental delivery of VEGF to animal models of these conditions leads to quantitative improvement of pathological changes, including reversal or delay of neuropathy and preservation of erectile function. A number of neurotrophins, including nerve growth factor (NGF) and secretoneurin, have been identified as angiogenic factors [246, 247]. In Alzheimer’s disease (AD), on the other hand, proliferating endothelial cells secrete a peptide neurotoxin that is selective for cortical neurons [248, 249]. The beta amyloid peptides found in Alzheimer’s plaques are highly angiogenic and contribute to pathological neovascularization [250, 251]. For AD, inhibition of pathological angiogenesis, or restoration of the normal phenotype of brain endothelium, may ameliorate disease progression.

Preeclampsia Diseases of the reproductive system have been examined for dysregulation of angiogenesis. Preeclampsia, a condition complicating up to 5% of all pregnancies, is associated with both maternal and neonatal morbidity and mortality. Recent investigations have shown a casual link between preeclampsia and placental overexpression of soluble Flt-1 (sFlt1), also known as VEGFR-1. This soluble receptor is considered a circulating angiogenesis inhibitor, because of its ability to bind to and reduce serum levels of VEGF and placental growth factor (PlGF). Reduction of these factors is now thought to cause fetal compromise [252]. When supernormal levels of sFlt1 pass into the maternal circulation, maternal VEGF declines, leading to the onset of hypertension and proteinuria. Remarkably, these hallmark phenomena of preeclampsia are also well-known side effects of anti-VEGF therapies used to treat cancer patients. Pregnant women at risk for preeclampsia also exhibit low serum and urinary levels of the angiogenic factor PlGF. Elevated levels of soluble endoglin, another endogenous anti-angiogenic factor, also appear to rise several months prior to the onset of preeclampsia [253, 254]. Increased mobilization of endothelial progenitor cells may occur in response to signals released during acute preeclampsia [255]. Collectively, these observations suggest that certain angiogenesis-associated markers may be useful to predict the risk for and onset of this disease.

Obesity Adipose tissue is angiogenesis-dependent, and capillary growth in fat controls tissue plasticity [256, 257]. In genetic models of experimental obesity, angiogenesis inhibition Angiogenesis-based medicine 407 induces weight loss although the effect appears to be limited to a physiological set point for body weight [258-260]. The same effect can be achieved in experimental obesity resulting from excessive dietary intake. Interestingly, early observations of side effect with some experimental anti-angiogenic agents, such as TNP-470, included significant weight loss in animals [261]. A growing number of fat-associated factors have been studied for their ability to stimulate angiogenesis. Leptin, Ang-1, Ang-2, and PlGF regulate angiogenesis in adipose tissue, suggesting they may be targets for anti-obesity therapy [256, 262, 263]. Adipose- derived stromal cells (hASC) harvested from human fat by liposuction possess angiogenic activity, and this novel stem cell population is being examined for its potential for therapeutic angiogenesis in ischemic cardiovascular disease [197, 200]. Alternatively, hASCs may also serve as potential targets for anti-obesity therapy. In conclusion, angiogenesis-based medicine is now a unifying approach to the treatment of a growing number of diseases. Although VEGF and PDGF are the first angiogenic factors to be validated clinically, many new targets are becoming elucidated and are being studied in clinical trials. As the next generation of anti-angiogenic and therapeutic angiogenesis modalities emerge, a number of key questions must be addressed: Which patients represent best responding populations, and are there predictive markers for this group? Are combinations of angiogenesis-based therapies superior to single agent strategies, or will sequential application of individual agents prove to be more efficacious? What are the mechanisms for disease progression through therapy? Does this reflect induction of compensatory escape mechanisms, or does this reflect true drug resistance? What surrogate markers can be validated and used to monitor treatment efficacy in the context of long-term therapy? What toxicities may emerge from long-term suppression of angiogenesis as a chronic disease management strategy? How can angiogenesis-based strategies best become validated for early disease intervention or chemoprevention? These and other questions are laying the groundwork for future avenues of angiogenesis research and its clinical translation for conquering disease.

Acknowledgements The authors (WWL, VWL) dedicate this chapter to the mourning of Dr. Judah Folkman who provided them with inspiration, mentorship, encouragement, and wisdom for over 20 years.

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