Combination of Antiangiogenic Therapy With Other Anticancer Therapies: Results, Challenges, and Open Questions

http://www.100md.com 《临床肿瘤学》

     the Division of Medical Oncology, S. Filippo Neri Hospital, Rome, Italy

    Genzyme Corporation, Framingham, MA

    ABSTRACT

    INTRODUCTION

    Angiogenesis can occur by sprouting, nonsprouting, or intussusception.1,2 The abnormality of tumor vasculature and the value of working with endothelial cells (ECs) isolated from solid tumors have been recognized.3 There are at least four potential mechanisms leading to tumor angiogenesis: secretion by tumor and/or stromal cells of proangiogenic factors, co-option of pre-existing vasculature, vasculogenesis from circulating AC133+/CD34+ endothelial precursor cells (EPCs), and vascular mimicry, that is the formation of vascular channels by tumor cells.1–4 Angiogenesis, the process of formation of new vessels arising from sprouts of existing vessels is distinct from vasculogenesis, vessels arising from EPCs (angioblasts).5 Asahara et al6 isolated angioblasts from human peripheral blood undergoing postnatal vasculogenesis and pathologic neovascularization. Studies in allogeneic bone marrow transplant recipients confirmed that circulating EPCs in peripheral blood originate from bone marrow.7 Recent studies have formally tied circulating EPCs to tumor angiogenesis.8 In mice bearing human breast carcinoma xenografts, both circulating and intratumor proliferating EPCs have been detected.9 NOD/SCID mice transplanted with human bone marrow and bearing human Namalwa or Granta 519 Burkitt's lymphoma xenografts had a seven-fold increase in circulating EPCs compared with non–tumor-bearing mice.10

    Physiologic angiogenesis is tightly regulated by pro- and antiendothelial growth factors and occurs by a series of complex and interrelated steps.1,2 Proangiogenic growth factors, such as VEGF, fibroblast growth factors, and platelet-derived growth factor, are released into the microenvironment by malignant, inflammatory, and other stromal cells in response to various stimuli. The released growth factors activate local ECs and EPCs from bone marrow that enter circulation to generate new blood vessels.1,2 Activated ECs, as well as local stromal cells and EPCs, secrete several enzymes, including metalloproteinases (MMPs) that break down extracellular matrix and allow ECs to invade surrounding tissue, proliferate in response to growth factors, and migrate toward the malignant stimulus.1,11 Plasmin generated by the urokinase plasminogen activator, regulated by the urokinase plasminogen activator receptor and plasminogen activator inhibitor-1, is a key mediator of these processes.12 The migration of both ECs and EPCs is regulated by adhesion molecules. The receptors of integrins {alpha}v3 and {alpha}v5 overexpressed on the surface of activated ECs are important for differentiation and survival of blood vessels.13 Several studies have reported that anti-integrin {alpha}v3 agents inhibit angiogenesis in preclinical models.1,13 Interestingly, genetic ablation of the genes encoding these integrins fails to block angiogenesis in the embryo and in some cases even enhance it.14 Various mechanisms may contribute to this negative regulatory function: stimulation of TSP-1, increased synthesis of TIMPs and inhibition of MMPs, direct activation of MMP-2 and release of antiangiogenic matrix fragments, downregulation of VEGF receptor 2 (VEGFR2), and transdominant inhibition of the proangiogenic integrins {alpha}51 and {alpha}11/{alpha}21.1,2

    ANTIANGIOGENIC THERAPY FOR MALIGNANT DISEASE: PHARMACOLOGIC AND BIOLOGIC RATIONALE

    Antiangiogenic therapy may represent a new promising anticancer therapeutic strategy. First, like most normal tissues, normal ECs are quiescent under physiologic conditions, whereas tumor ECs and EPCs are actively proliferating and with an angiogenic phenotype.2,18 Consequently, AIs tend to have moderate toxicity, because angiogenesis is infrequent in adults, except during inflammation, ovulation, pregnancy, wound healing, and ischemia.2

    ECs are quiescent in hypoxic and necrotic regions, whereas in the areas of progressive malignant disease, they are active, proliferating, and more sensitive to therapy.1 Second, ECs are genetically stable, although under the influence of the malignant environment, tumor ECs express an abnormal phenotype.1,2,20 Third, tumor endothelium expresses high levels of specific molecular targets and antigens that may be targeted by selective inhibitors.2 Fourth, ECs are readily accessible to selective AIs given by systemic administration.2 Finally, like other anticancer therapies, some AIs may have synergistic therapeutic effects in combination with conventional cytotoxic therapies.21

    POTENTIAL MECHANISMS OF GENETIC AND EPIGENETIC RESISTANCE TO AIS

    CLASSIFICATION OF AIS

    Folkman32 proposed a classification system based on the efficacy of AIs in preclinical tumor models: first-generation AIs, such as interferons, TNP-470, thalidomide, and matrix metalloproteinases inhibitors (MMPIs), only slow tumor growth; second-generation AIs, such as anti-VEGF and anti-integrin {alpha}v3 antibodies, frequently produce tumor regression; third-generation AIs, such as angiostatin, endostatin, and TSP-1, can be curative in experimental tumors.2

    However, the results of experimental studies on endostatin are controversial in part because of the instability of the molecule and the production from different laboratories of lots with diverse activity.33 A recent Italian study suggests that endostatin sequence exhibits peptides with both angiosuppressive and angiostimulatory effects. Two fragments with high angioinhibitory activity have been sequenced.34

    Human endostatin (h-Endostatin) is the NC1 domain of the alpha-1 chain of type XVIII collagen, and it is cleaved by proteases such as elastase and cathepsins. It circulates in the blood at concentrations of 20 to 35 ng/mol. h-Endostatin inhibits EC migration, whereas mouse endostatin blocks migration and causes Gap1 EC cycle arrest. The reason for this difference is presently unknown. h-Endostatin binds to several cell surface proteins, including heparan sulfate proteoglycans, glypicans, VEGFR2, and integrins. Although many different intracellular pathways have been identified as possible mediators of h-Endostatin action, a functional receptor has not been identified.33 Recently, Sudhakar et al33 demonstrated that h-Endostatin binds {alpha}51 integrins with inhibition of focal adhesion kinase/c-Raf/MEK1–2/p38/ERK-1 mitogen-activated protein kinase pathway. Another recent study suggests that h-Endostatin, TSP-1, fumagillin, and TNP-470 modify the phosphorylation state and subcellular localization of cofilin and hsp 27, two proteins involved in actin cytoskeleton and focal adhesion of ECs.35

    AIs can also be classified by the mechanism of action: inhibitors of angiogenic factors secretion, inhibitors of EC intracellular signaling transduction, inhibitors of EC proliferation, inhibitors of MMPs, agents cytotoxic toward ECs, and inhibitors of mobilization of EPCs from bone marrow.36

    PRECLINICAL FINDINGS

    As initial step, the activity of potential AIs was tested using assays focused on normal neoangiogenesis in a variety of models, including inhibition of the chicken chorioallantoic membrane neovascularization,38 inhibition of vascularization of a matrigel plug containing angiogenic factors implanted in a mouse that can be quantified histologically,39 and inhibition of vascular growth toward an angiogenic stimulus implanted in the rat or mouse cornea.40 It has not been established whether these assays involving developing vasculature in the embryo or the neovascularization induced by an angiogenic stimulus are really comparable with in vivo tumor angiogenesis. A second generation of matrigel plug assay uses human EPCs suspended in the matrigel before the implant onto the animal.2

    It is possible to observe the effects of blocking angiogenesis on tumor transplanted onto animals.41 The activity of AIs on the primary tumor as well as on metastasis is usually evaluated by comparing the diameter and number of the lesions of the treated animals versus those receiving placebo.42 A major drawback of these models is that many experiments have not been performed in orthotopic models and that the tested vasculature is murine. Therefore, the EC molecular targets are the mouse homologs of the desired human protein targets. Although the mouse protein and the human protein targets often have high homology, an incomplete cross-reactivity of antibodies may occur in certain circumstances. Several approaches of incorporating human EC target molecules in mouse include development of transgenic animals, transplantation of immunodeficient mice with human bone marrow, transfusion of immunodeficient mice with human EPCs, and transplantation of human foreskin onto immunodeficient mice.

    PRECLINICAL STUDIES OF COMBINATION REGIMENS

    SU5416 was under development as a selective inhibitor of VEGFR2 (Flk-1, KDR) kinase activity; SU6668 and SU11248 are under development as broad-spectrum receptor kinase inhibitors being able to block VEGFR2, bFGF factor receptors, and platelet-derived growth factor receptor kinase activities.66,67 Early in vivo work with SU5416 suffered from the use of dimethyl sulfoxide as a vehicle for the compound administered by intraperitoneal injection in mice.68 Gang et al69 found that SU5416 increases the sensitivity of murine B16 melanoma and murine GL261 glioma to radiation therapy. SU5416 and SU6668 have been tested as single agents or in combination with fractionated radiation therapy in C3H mice bearing SCC VII squamous carcinomas.70 Like STI571, SU5416, SU6668, and SU11248 inhibit c-kit (KIT), the stem-cell factor receptor tyrosine kinase, and FLT3.66,67,71 C-kit is a key factor for development of normal hematopoietic cells and has a functional role in acute myeloid leukemia. The potential therapeutic utility of SU11248, alone or in combination with docetaxel, fluorouracil, or doxorubicin, was evaluated in different models of breast carcinoma: MMTV-v-Ha-ras transgenic mouse, DMBA carcinogen-induced rat mammary cancer, MX-1 human breast carcinoma subcutaneously implanted xenograft, and MDA-MB-435 human breast carcinoma subline, and in the 435/HAL-Luc line, selected from bone metastases. The combined regimens resulted in longer survival times than either single chemotherapeutic agent or SU11248 alone.66,67 Other small molecule tyrosine kinase inhibitors showing promising activity in early clinical trial include PTK787/ZK222584 and ZD6474.72–74 Daily oral treatment with PTK787/ZK222584 resulted in a significant decrease in primary murine renal cell carcinoma grown in the surrenal capsule of Balb/c mice. The occurrence of lung metastases was reduced 98% and 78% on days 14 and 21, respectively, and development of lymph node metastases was delayed.73

    Protein kinase C isoforms are involved in the signaling transduction pathways that regulate cell cycle, apoptosis, angiogenesis, differentiation, invasiveness, senescence, and drug efflux.75,76 Nude mice bearing human SW2 small-cell lung carcinoma subcutaneous xenografts treated with LY317615, a potent and selective inhibitor of protein kinase C, show a dose-dependent decrease in tumor microvessel density.77 Plasma VEGF levels in LY317615-treated SW2-bearing animals were significantly lower as compared with the control group.78 VEGF levels in the control Caki-1 renal cell carcinoma–bearing nude mice treated with LY317615 remained suppressed throughout all the treatment period (d21–39) and until day 53, when the experiment was terminated.78 Treatment of SW2 small-cell lung carcinoma bearing nude mice with paclitaxel followed by LY317615 resulted in more than 60 days of tumor growth delay and a 2.5-fold increased duration of tumor response. The antitumor activity of LY317615 alone and in combination with cytotoxic agents has been explored in several human tumor xenografts in nude mice.77,79 In most tumor models, LY317615, as a single agent, induced tumor growth delay. The combined schedules suggested higher activity and LY317615 has currently completed phase I clinical studies.80

    A major strategy to inhibit VEGF signaling pathway consists of VEGF neutralizing monoclonal antibodies.81–83 Bevacizumab, a recombinant humanized anti-VEGF monoclonal antibody, is showing promise in clinical trial.84 Indeed, preclinical studies have shown that the antitumor activity of some cytotoxic agents is potentiated by cyclo-oxygenase 2 (COX-2) inhibitors.85

    CLINICAL STUDIES WITH COMBINED REGIMENS

    INDIRECT AIS AND MIXED ANTIANGIOGENIC/ANTITUMOR AGENTS

    Several small molecules that selectively block phosphorylation of VEGF receptors entered clinical evaluation. The first tested in humans was SU5416 (semoxinal).90–93 In untreated patients with metastatic colorectal cancer, SU5416 was administered in combination with fluorouracil/leucovorin (Roswell Park or Mayo Clinic regimens), at two different dose levels, 85 and 145 mg/m2 twice weekly. The toxicity observed was that expected for the fluorouracil/leucovorin regimen (mucositis), with only few patients reporting mild headache. Six patients achieved a major objective response, and another nine patients had durable stable disease.90 In a randomized, international, multicenter prospective phase III trial in untreated metastatic colorectal cancer patients, SU5416 was administered with fluorouracil/leucovorin (Roswell Park regimen) compared with fluorouracil/leucovorin alone. The final analysis after the enrollment of 737 patients indicated no improvement of clinical outcome in the SU5416 arm.2 The combination of SU5416 at the dose of 145 mg/m2 biweekly with cisplatin and gemcitabine in patients with advanced solid tumors was associated with a surprisingly high toxicity, resulting in a severe rate of thromboembolic events.91 SU5416 may also be useful for the treatment of patients with von Hippel-Lindau syndrome,94 but, taking into account the negative results of phase II/III clinical trials, the compound is no longer in clinical development.

    SU11248, a multitargeted receptor tyrosine kinase inhibitor, blocks the kinase activity of VEGFR2, platelet-derived growth factor receptor, KIT, and Flt3.95–100 In four phase I clinical studies, decreased levels of circulated VEGF were observed in treated patients.95,96,98,99 Responses were observed in two phase I studies, especially in renal cell carcinoma, neuroendocrine tumor, and thyroid cancer, and nearly 50% of patients had stable disease.95,96 In a phase I study of gastrointestinal stromal tumors in patients resistant to imatinib (Gleevec; Novartis Pharmaceuticals Corp, East Hanover, NJ), five of the 32 treated cases had measurable responses, and approximately 60% of the patients had stable disease for more than 4 months.97,98

    Clinical phase I studies of PTK787/ZK222584 alone or in combination with cytotoxic agents have been conducted in patients with metastatic colorectal cancer, renal cell carcinoma, brain tumors, and acute myelogenous leukemia or myelodysplastic syndrome.101–106 The once per day oral dose of PTK787/ZK222584 tolerated in most combination regimens was 1,200 to 1,250 mg/d. International multicenter phase III trials are comparing the bolus fluorouracil plus leucovorin, oxaliplatin, and infused fluorouracil (FOLFOX-4) regimens versus the same regimen plus PTK787/ZK222584 in previously untreated or treated patients with advanced colorectal cancer. The phase I study of ZD6474 concluded that 100 to 300 mg/d would be the appropriate dose range for phase II testing.107

    Marimastat, a synthetic inhibitor of MMP-1, -2, -3, -7, and -9, was the first orally bioavailable MMPI tested in the clinic.108–116 Marimastat was evaluated in phase I and II studies at doses from 2 to 100 mg bid, either alone or in combination with chemotherapy.108–112,116 The dose-limiting toxicity of marimastat is musculoskeletal disorders at doses of 10 mg or greater twice daily. In a phase III trial, 414 patients with advanced pancreatic cancer were randomly assigned to receive marimastat (5 mg v 10 mg v 25 mg twice a day) or gemcitabine. Forty-four percent of the patients treated with marimastat experienced musculoskeletal toxicity versus 12% of those who received gemcitabine. Survival was not significantly different in the two arms, whereas a better progression-free survival was reported in the gemcitabine group.109 A large randomized study of marimastat as maintenance therapy of small-cell lung cancer was conducted.111 Among the 532 eligible patients (266 patients receiving marimastat and 266 patients receiving placebo), the median TTP for patients receiving marimastat was 4.3 months compared with 4.4 months for placebo (P = .81). Median survival for marimastat and placebo was 9.3 months and 9.7 months, respectively (P = .90). Toxicity was generally limited to musculoskeletal symptoms (18% grade 3/4 for marimastat). Patients receiving marimastat had significantly poorer quality of life at 3 and 6 months. Despite promising preclinical data, results of clinical phase III trials have been disappointing because of the narrow therapeutic index of MMPIs. There are currently five MMPIs in clinical development: marimastat in radically resected pancreatic cancer, BMS-275291 in advanced NSCLC, prinomastat in diverse tumor types and earlier stages of disease, Metastat in Kaposi's sarcoma, and Neovastat in unresectable renal cell carcinoma.114

    DIRECT ANTIANGIOGENIC AGENTS

    OTHER ANTIANGIOGENIC AGENTS

    Several phase II studies have found a moderate activity of thalidomide in renal cell carcinoma.129,130 Dose levels of 800 to 1,200 mg/d were achieved; however, toxicities including somnolence, constipation, fatigue, neuropathy, and thromboembolism occurred. The addition of thalidomide to gemcitabine and fluorouracil did not improve the objective response rate observed with gemcitabine and fluorouracil, but added significant toxicity.129,130 In 17 assessable patients with recurrent glioblastoma, thalidomide, at 400 mg/d, was well tolerated, with constipation, somnolence, and peripheral neuropathy being the most common side effects.131 One minimal response and eight cases of stable disease were observed, with an overall clinical benefit of 52.9%. Median TTP and overall survival for responders were 25 and 36 weeks, respectively.131 Phase II clinical trials of thalidomide alone or in combination regimens have been reported in prostate cancer, head and neck cancer, malignant melanoma, and brain tumors.124,132–136 Most studies observed little activity with thalidomide alone, but suggest that further investigation in combination regimens may be justified with close follow-up of toxicities, especially peripheral neuropathy.

    Numerous clinical trials are ongoing to test the efficacy of nonsteroidal anti-inflammatory COX-2 inhibitors in combination regimens for therapy of advanced solid tumors.85 These compounds exhibit anti-inflammatory, analgesic, and antipyretic activities, as well as block of angiogenesis in animal models. Phase II clinical studies have combined celecoxib with a taxane, either docetaxel or paclitaxel, for treatment of NSCLC.85 Each study found the combination to be well tolerated, with response rates trending toward improved activity with celecoxib, without additional toxicity. In breast cancer, celecoxib in combination with exemestane has been reported.85 The combination was well tolerated, with a trend toward more efficacy for the combination. Celecoxib combinations have been studied for therapy of esophageal cancer with irinotecan/cisplatin/concurrent radiation therapy, pancreatic cancer with gemcitabine, renal cell carcinoma with low-dose cyclophosphamide, and malignant glioma with temozolomide.85 Celecoxib was well tolerated in all of the combination regimens.

    In a phase II study, Altorki et al137 evaluated the combination of celecoxib with paclitaxel/carboplatin regimen as preoperative chemotherapy in early-stage NSCLC. In comparison with historically reported data, the addition of celecoxib enhanced response rate and normalized the prostaglandin E2 tissue levels. This is the first published study suggesting a possible additive therapeutic effect by combining chemotherapy and anti–COX-2 agents in human solid tumors.

    Promising are the preliminary results of a phase I/II study ongoing at the San Filippo Neri Hospital in Rome, Italy, testing the combination of rofecoxib (50 mg/d) with weekly irinotecan and infusional fluorouracil. The dose-finding study on 15 cases demonstrated a good tolerability up to the irinotecan dose of 125 mg/m2/wk. The phase II study enrolled up to now 37 cases, and among the 30 assessable patients, the objective response rate was 36.7%, with a clinical benefit of 76.7%. Median TTP and overall survival were 4+ and 9+ months, respectively. The combination seems to be feasible and safe, with a reduced rate of mucositis and diarrhea.85,138 In an ongoing phase II study, the activity and tolerability of weekly paclitaxel and celecoxib was tested in 58 pretreated patients with NSCLC. The preliminary analysis on the 48 assessable patients showed a response rate of 27% and a stable disease rate of 48%, with a TTP and an overall survival of 4+ and 6+ months, respectively. This schedule was well tolerated, with a low incidence of grade 3/4 neutropenia and peripheral neuropathy.85

    The rationale supporting the antitumor activity of selective inhibitors of COX-2 and an overview of preliminary data of the phase I/II clinical studies of combined therapy of anti–COX-2 agents with chemotherapy in advanced solid tumors have been recently reviewed.85 The main clinical trials with AIs combination are listed in Table 2.

    METRONOMIC CHEMOTHERAPY

    There are currently no published clinical studies that compare a true metronomic schedule of chemotherapy with conventional schedules. Several phase I and II studies were carried out involving oral, low, continuous doses of cytotoxic agents, with interesting results.140 There are some theoretical advantages to be explored with regard to this new schedule of chemotherapy (Table 3), but there are also potential problems and challenges in terms of appropriate experimental study design and clinical testing.62 cDNA microarrays and proteomic studies will better clarify the genetic basis of responsiveness of tumors to metronomic, antiangiogenic schedules.141 Finally, the identification of more specific surrogate markers is warranted, allowing the selection of patients for such treatments and the monitoring of the biologic effects as early or intermediate end points of treatment efficacy.142

    IN VIVO NONINVASIVE ASSESSMENT OF TUMOR VASCULARIZATION

    PET is another approach used to assess blood flow in human tumors.148 Two radiolabeled molecules are of particular interest: a radioactive form of water labeled with 15O, used to calculate blood flow within tumors, and radiolabeled carbon monoxide (11CO) that irreversibly binds RBCs and distributes in accordance with vascular volume.

    Also, membrane proteins were selectively expressed by tumor ECs, such as integrins ({alpha}v3, {alpha}v5), endoglins (CD105), and VEGF receptors. Dynamic MRI with paramagnetic contrast agents targeted to integrin {alpha}v3 has been used by Sipkins et al149 and novel PET tracers using 18F- labeled glycopeptides containing RGD sequences are also available to target {alpha}v3 and {alpha}v5 integrins.150

    The utility of MRI and PET are under clinical evaluation in phase I/II studies. Preliminary results with AIs monitored with dynamic MRI or PET imaging demonstrated changes in vascular permeability, volume fraction, or metabolism after therapy.151 However, these changes do not always predict clinical efficacy of AIs. In patients with advanced colorectal cancer, Morgan et al have demonstrated a significant reduction in dynamic contrast-enhanced MRI parameters within a few hours after administration of PTK787/ZK222584152; moreover, there was a significant relationship between reduction of contrast enhancement and tumor regression.

    A phase I trial was performed with combretastatin A4 phosphate in 34 patients with different solid tumors measuring tumor blood flow parameters by either PET or dynamic MRI.153,154 Significant dose-dependent reduction in tumor blood flow perfusion was seen with PET or MRI a few hours after therapy.

    These two studies suggest that functional imaging obtained by PET or dynamic MRI could help to assess whether the drug achieves the target, as demonstrated by the reduction of tumor perfusion; select an adequate dosage for phase II studies, in relation to the identification of the doses able to reduce tumor perfusion; identify the better schedule of administration for phase II studies; and, finally, distinguish responsive versus unresponsive patients to AIs.

    STUDY DESIGN AND SELECTION OF END POINTS

    The study design could be improved in several ways: first, by including a surrogate marker whose expression is confirmed in each tumor (eg, the expression and concentration of the therapeutic target [VEGF receptors]); second, by including pharmacodynamic indicators able to confirm that the schedule of the experimental agent is optimal to maintain a therapeutic concentration at the molecular target; and third, by conducting phase I/II clinical studies to establish the optimal dose/schedule of the experimental agent combined with cytotoxic drugs. Betensky et al155 suggested that mistaken assumptions or lack of information regarding the molecular characteristics of tumors can lead to negative results, even in large randomized phase III trials, as observed in randomized phase III clinical trials with experimental MMPIs without selection of the patients based on the expression of related surrogate biomarkers.156

    As far as clinical end points are concerned, the assumption is that it may be difficult to demonstrate a conventional antitumor response (ie, objective response) with antiangiogenic therapies in cohorts of patients with advanced disease resistant to conventional therapy. For many AIs, the more appropriate clinical settings may be chemopreventive, adjuvant, or maintenance therapy, once satisfactory tolerability and activity has been proven by phase I studies in patients with advanced disease. The ideal clinical end points for AIs would be as follows.

    In phase I clinical study, the ideal clinical end points for AIs would be identification of the pharmacodynamic and pharmacokinetic parameters associated with the experimental agent, the development of surrogate biomarkers to confirm the therapeutic target in tumor tissue or biologic fluids or in vivo dynamic studies for determination of the modifications induced in tumor blood flow, and definition of the range of biologically active doses. The MTD of an AI may be higher than that required to achieve the maximum desired biologic activity. Determination of an optimal biologic dose (OBD) would provide more useful information for further drug development. Dose-finding study design for combined therapy is needed to identify the dose-limiting toxicity and OBD of the schedule. Ideally, a phase I clinical trial should be designed to determine the plasma concentration of the experimental agent required to achieve the maximum inhibition of the antiangiogenic target in vivo.157,158

    Phase II trials designed to demonstrate the clinical activity (including durable stable disease) and toxicity of antiangiogenic therapy with the biologic modulation of the target along with assessment of tumor response by imaging and clinical examination would be ideal. The initial interest was focused on serum and/or urinary levels of angiogenic factors such as VEGF or bFGF as surrogates for the clinical activity of AIs. However, several studies failed to demonstrate significant correlation of circulating angiogenic factors with clinical outcome.159 Other possible clinical activity correlates under investigation include dynamic imaging parameters using PET, MRI, and Doppler ultrasound techniques or decreased circulating EPCs.119,121,160 In addition to standard clinical study end points, TTP may be particularly informative.157,159

    Phase III clinical trials are designed to prove increased clinical benefit of a new agent or combined schedule as compared with standard therapy. The principal end points are survival, TTP, and quality of life. Ideally, patients with tumors having confirmed expression of the molecular target will be selected for the phase III trials, and surrogate biomarkers of angiogenesis determinable with standardized, reproducible laboratory tests will need to be studied. The biologic response criteria should be included along with conventional criteria for assessment of efficacy. The expected advantage of antiangiogenic therapies is to achieve long-term biologic benefits by inducing a dormant state in residual tumor foci without unacceptable toxic effects.161

    OPEN QUESTIONS AND FUTURE PROSPECTS

    First, can therapy with AIs alone be active in human advanced tumors Current data suggest this is likely not the case.

    Second, which are the most promising antiangiogenic therapies Direct EC inhibitors present the theoretical advantage to selectively target tumor-activated endothelium but have the disadvantage that no surrogate markers are available for clinical correlates. The indirect AIs have the advantage that the molecular targets can be identified by surrogate marker assays, but the predictive value of these correlates remains to be proven.36

    Third, are the therapeutic strategies based on vascular-targeting or hypoxic cell selective cytotoxicity more effective than antiangiogenic therapy Would it be more effective to block tumor angiogenesis with a combination of AIs against multiple molecular targets (multitargeted therapy), thus inhibiting multiple steps in the angiogenic cascade (Table 4) Studies of angiomics and the development of selective microarrays to define the angiogenesis-related genes in individual tumors, and at different stages of therapy and tumor progression, may allow improved therapeutic efficacy.2,85 Recently, Comoglio et al162 identified a novel molecular pathway by which hypoxia promotes tumor growth by transcriptional activation of the met proto-oncogene. The study provides evidence of a second type of cellular response to oxygen deprivation, complementary and independent to the angiogenic response mediated by upregulation of VEGF. These results raise the possibility that antiangiogenic therapy per se, by reducing tumor vascularization in the primary tumor, would promote the spread of cancer cells toward a more oxygenated environment in distant tissues (ie, metastasis). On the other hand, a novel therapeutic paradigm of tumor suffocation was proposed, based on the combination of selective AIs with met or hepatocyte growth factor (also known as scatter factor-1) inhibitors (Fig 2). Experimental and clinical studies are warranted to prove the efficacy of the above proposed therapeutic strategy.

    Fourth, should angiogenesis inhibition be considered a tumor-eradicating therapy Preclinical studies suggest that AIs can produce growth inhibition of early steps of tumor growth and delay the growth of established tumors. The efficacy of AIs is generally inversely correlated to tumor burden.2

    Fifth, what is the optimal scheduling of chemotherapy in combination with AIs Ideally, clinical development of antiangiogenic therapies should be molecularly targeted and coupled with laboratory tools to confirm target expression and target inhibition to avoid negative studies and/or toxicity in large numbers of patients. Although some preclinical studies suggest that frequent low doses of chemotherapy can be more efficacious than intermittent high-dose bolus regimens, the total dose of chemotherapy administered by frequent schedule is often greater than that administered by the intermittent schedule; in addition, many of these preclinical studies lack pharmacokinetic data.163

    Regarding cytotoxic agents, most of the combinations tested in preclinical and clinical studies evaluated chemotherapy given with conventional doses and schedules. Following such a strategy, certain phase I or III clinical studies have been associated with unacceptable toxicity2,91 or negative results.114 The results of a few experimental studies, testing metronomic schedules of chemotherapy combined with anti-VEGF compounds, suggest high antitumor activity64; however, no clinical data are available yet.

    Recent phase I/II clinical studies aimed to evaluate tolerability and activity of selective anti–COX-2 compounds associated with cytotoxic agents are testing also dose-dense chemotherapeutic regimens, in particular with taxanes and camptothecins.62 However, the optimal strategy of combining chemotherapy with AIs is presently an unresolved issue, and prospective randomized trials are needed to properly compare conventional versus metronomic versus dose-dense schedules.

    There are several challenges facing clinical trials with AIs. These challenges are outlined below.

    Determining the OBD

    The optimal biological dose (OBD) may be different from the MTD. Ideally, clinical OBD could be determined using a validated, standardized assay with quality-controlled, biologic surrogate biomarkers developed during the preclinical tests of the agent. Preclinical studies suggest that the kinetics of antiangiogenic antitumor effects are quite slow and may take weeks or months to manifest. For this reason, antiangiogenic therapy needs to be administered for long periods of time, as semichronic/chronic therapy.

    Scheduling of Antiangiogenic Agents

    The optimal dosage and scheduling of AIs should be based on the knowledge of the pharmacokinetic characteristics of the tested compound as well as of the required concentration at the target level. Phase I studies should evaluate different schedules, taking into account that most of the experimental studies suggest that chronic administration is needed to obtain the maximum antitumor effect of AIs. Indeed, the possible pharmacologic interactions with cytotoxic agents are to be properly evaluated in phase I studies with pharmacokinetic analysis to prevent further development of potentially toxic combinations.91

    Surrogate Biomarker Correlate Assays

    The development of surrogate end points to assess biologic activity of AIs is a key point. The determination of intratumoral microvessel density before and after antiangiogenic treatment has been tested as a surrogate end point in several clinical studies.132 Intratumoral microvessel density may be uninformative as a surrogate end point because it is the result of the balance between the apoptosis rates of ECs and tumor cells, related to the tumor cell/capillary distance.2 The disappearance of EPCs from circulation and EC shedding from tumor vasculature are being evaluated as possible predictive surrogate biomarkers.2,8,121 Methods are available to detect ECs and EPCs in the circulation of patients.119,164

    Optimal Clinical Setting

    Like many antitumor therapies, preclinical studies suggest the highest antitumor activity of AIs when the tumor burden is small. Therefore, patients who have a high likelihood of tumor recurrence after radical surgery would be ideal candidates for antiangiogenic therapies. Patients with metastatic disease may take more benefit of AIs as maintenance therapy after achieving response to standard therapy. In fact, the vasculature of advanced solid tumors may be heterogeneous and more difficult to impact with antiangiogenic therapy than the vasculature of microscopic or early-stage disease.

    Tailored Therapy

    Tumor angiogenesis, similarly to other malignant processes, is dynamic during tumor progression and it is altered by anticancer therapies. VEGF was the only angiogenic factor produced by early-stage human breast cancer; however, during progression, tumors secrete concurrently many angiogenic factors: bFGF, transforming growth factor beta-1, placental growth factor (PIGF), PDGF, and pleiotrophin.165 Therefore, antiangiogenic therapy should be tailored depending on the angiogenic phenotype and expression of endothelial growth factors in each single tumor. A combination of antiangiogenic therapies, affecting different targets, might produce a synergistic antitumor effect.

    Toxicity

    AIs may interfere with normal angiogenic processes such as wound healing, ovulation, and pregnancy. Ischemic diseases could potentially be exacerbated by these agents. Hemorrhagic and/or thrombotic events have been reported in early trials of anti-VEGF antibodies in patients with colorectal and lung cancers.85,86,88 In preclinical studies in mice, angiostatin and endostatin had little effect on wound healing.2 Studies evaluating gene expression patterns in ECs during wound healing with tumor ECs may elucidate differences between these two angiogenesis-dependent processes.

    In conclusion, rational clinical evaluation of AIs would be facilitated by the availability of surrogate biomarkers enabling the identification of the patients most likely to benefit of therapy. The development of feasible assays allowing the determination of circulating and tissue levels of AIs is also important to ensure that these agents are given at optimum concentrations at the molecular target.

    Once the phase I clinical trial identifies a clinically active AI with a good therapeutic index, translational research should include combined therapy testing: (1) combinations of antiangiogenic therapies with different mechanisms of action or acting against diverse molecular targets; (2) combinations of antiangiogenic therapies with conventional chemotherapy or radiation therapy to block the reciprocal growth stimulation between tumor parenchyma and stroma; (3) combinations with other molecular-targeted therapies; (4) combinations of AIs with met or hepatocyte growth factor antagonists to prove the paradigm of tumor suffocation, as recently proposed by Comoglio et al.162

    The improvement of clinical study design is of paramount importance. New therapeutic approaches may fail if inappropriate clinical studies are performed.155 Metronomic chemotherapy warrants appropriate clinical evaluation to validate its activity and feasibility for long-term therapy in combination with AIs.140,163 The antiangiogenic activity of inhibitors of COX-2 is currently being assessed in many phase II/III clinical trials, with encouraging preliminary results.85 Strategies that target hypoxic cells may synergize with AIs.166 The next advances in antiangiogenic therapies will come from data obtained from molecular analysis of human clinical disease that will further improve our understanding of the mechanisms supporting angiogenesis in malignant disease, the development of standardized methods to determine surrogate predictive markers of response, the appropriate selection of the patients to be treated, and the capability of performing rigorous, informative clinical studies.

    Authors' Disclosures of Potential Conflicts of Interest

    NOTES

    Authors' disclosures of potential conflicts of interest are found at the end of this article.

    REFERENCES

    1. Bergers G, Benjamin LE: Tumorigenesis and the angiogenic switch. Nat Rev Cancer 3:401–410, 2003

    2. Longo R, Sarmiento R, Fanelli M, et al: Anti-angiogenic therapy: Rationale, challenges and clinical studies. Angiogenesis 5:237–256, 2002

    3. St Croix B, Rago C, Velculescu V, et al: Genes expressed in human tumor and endothelium. Science 289:1197–1202, 2000

    4. Bagley R, Walter-Yohrling J, Cao X, et al: Endothelial precursor cells as a model of tumor endothelium: Characterization and comparison to mature endothelial cells. Cancer Res 63:5866–5873, 2003

    5. Luttun A, Carmeliet G, Carmeliet P: Vascular progenitors: From biology to treatment. Trends Cardiovasc Med 12:88–96, 2002

    6. Asahara T, Masuda H, Takahashi T, et al: Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization. Circ Res 85:221–228, 1999

    7. Lin Y, Weisdorf DJ, Solovey A, et al: Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 105:71–77, 2000

    8. Rafii S: Circulating endothelial precursors: Mystery, reality, and promise. J Clin Invest 105:17–19, 2000

    9. Shirakawa K, Furuhata S, Watanabe I, et al: Induction of vasculogenesis in breast cancer models. Br J Cancer 87:1454–1461, 2002

    10. Capillo M, Mancuso P, Gobbi A, et al: Continuous infusion of endostatin inhibits differentiation, mobilization, and clonogenic potential of endothelial cell progenitors. Clin Cancer Res 9:377–382, 2003

    11. Jackson C: Matrix metalloproteinases and angiogenesis. Curr Opin Nephrol Hypertens 11:295–299, 2002

    12. Pepper MS: Role of the matrix metalloproteinases and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol 21:1104–1117, 2001

    13. Eliceiri BP, Cheresh DA: The role of alpha-v integrin during angiogenesis: Insights into potential mechanisms of action and clinical development. J Clin Invest 103:1227–1230, 1999

    14. Hynes RO: A reevaluation of integrins as regulators of angiogenesis. Nat Med 8:918–921, 2002

    15. Brooks PC, Clark R, Cheresh DA: Requirement of vascular integrin alpha v beta 3 for angiogenesis. Science 193: 264:569–571, 1994

    16. Burrows FJ, Derbyshire J, Tazzari PL, et al: Up-regulation of endoglin on vascular endothelial cells in human solid tumors: Implications for diagnosis and therapy. Clin Cancer Res 1:1623–1634, 1995

    17. Shalaby F, Ho J, Stanford WL, et al: A requirement for Flk1 in primitive and definitive hematopoiesis and vasculogenesis. Cell 89:981–990, 1997

    18. Gasparini G: The rationale and future potential of angiogenesis inhibitors in neoplasia. Drugs 58:17–38, 1999

    19. Kerbel RS: Inhibition of tumor angiogenesis as a strategy to circumvent acquired resistance to anti-cancer therapeutic agents. Bioessays 13:31–36, 1991

    20. Rak J, Kerbel RS: Treating cancer by inhibiting angiogenesis: New hopes and potential pitfalls. Cancer Metastasis Rev 15:231–236, 1996

    21. Kakeji Y, Teicher BA: Preclinical studies of the combination of angiogenic inhibitors with cytotoxic agents. Invest New Drugs 15:39–48, 1997

    22. Yu JL, Rak JW, Coomber BL, et al: Effect of p53 status on tumor response to anti-angiogenic therapy. Science 295:1526–1528, 2002

    23. Liu W, Ahmad SA, Reinmuth N, et al: Endothelial cell survival and apoptosis in tumor vasculature. Apoptosis 5:323–328, 2000

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

    25. Nor JE, Polverini PJ: Role of endothelial cell survival and death signals in angiogenesis. Angiogenesis 3:101–116, 1999

    26. Tran J, Rak J, Sheehan C, et al: Marked induction of the IAP family anti-apoptotic proteins survivin and XIAP by VEGF in vascular endothelial cells. Biochem Biophys Res Commun 264:781–788, 1999

    27. Tran J, Master Z, Yu JL, et al: A role for survivin in chemoresistance of endothelial cells mediated by VEGF. Proc Natl Acad Sci U S A 99:4349–4354, 2002

    28. Rybak SM, Sanovich E, Hollingshead MG, et al: ‘Vasocrine’ formation of tumor cell-lined vascular spaces: Implications for rational design of antiangiogenic therapies. Cancer Res 63:2812–2819, 2003

    29. Yu JL, Rak JW, Carmeliet P, et al: Heterogeneous vascular dependence of tumor cell populations. Am J Pathol 158:1325–1334, 2001

    30. Carmeliet P, Dor Y, Herbert JM, et al: Role of HIF-1 alpha in hypoxic-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394:485–490, 1998

    31. Blagosklonny MV: Hypoxia-inducible factor: Achilles' heel of angiogenic cancer therapy. Int J Oncol 19:257–262, 2001

    32. Folkman J: Looking for a good endothelial address. Cancer Cell 1:113–115, 2002

    33. Sudhakar A, Sugimoto H, Yang C, et al: Human tumstatin and human endostatin exhibit distinct antiangiogenic activities mediated by alpha v beta-3 and alpha-5 beta-1 integrins. Proc Natl Acad Sci U S A 100:4766–4771, 2003

    34. Morbidelli L, Donnini S, Chillemi F, et al: Angiosuppressive and angiostimulatory effects exerted by synthetic partial sequences of endostatin. Clin Cancer Res 9:5358–5369, 2003

    35. Keezer SM, Ivie SE, Krutzsch HC, et al: Angiogenesis inhibitors target the endothelial cell cytoskeleton through altered regulation of heat shock protein 27 and cofilin. Cancer Res 63:6405–6412, 2003

    36. Kerbel RS: Clinical trials of antiangiogenic drugs: Opportunities, problems, and assessment of initial results. J Clin Oncol 19: 45s–51s, 2001 (suppl)

    37. Maier J, Delia D, Thorpe PE, et al: In vitro inhibition of endothelial cell growth by the antiangiogenic drug AGM-1470 (TNP-470) and the anti-endoglin antibody TEC-11. Anticancer Drugs 8:238–244, 1997

    38. Auerbach R, Kubai L, Knioghton D, et al: A simple procedure for the long-term cultivation of chicken embryos. Dev Biol 41:391–394, 1974

    39. Grant DS, Tashiro K, Segui-Real B, et al: Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 58:933–943, 1989

    40. Ziche M, Donnini S, Morbidelli L, et al: Linomide blocks angiogenesis by breast carcinoma VEGF transfectants. Br J Cancer 77:1123–1129, 1998

    41. O'Reilly MS, Holmgren L, Shing Y, et al: Angiostatin: A novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 79:315–328, 1994

    42. Fidler IJ: Angiogenesis and cancer metastasis. Cancer J 6:S134–S141, 2000 (suppl 2)

    43. Sweeney CJ, Miller KD, Sissons SE, et al: The antiangiogenic property of docetaxel is synergistic with a recombinant humanized monoclonal antibody against vascular endothelial growth factor or 2-methoxyestradiol but antagonized by endothelial growth factors. Cancer Res 61:3369–3372, 2001

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

    45. Yokoyama Y, Dhanabal M, Griffioen AW, et al: Synergy between angiostatin and endostatin: Inhibition of ovarian cancer growth. Cancer Res 60:2190–2196, 2000

    46. Teicher BA: A systems approach to cancer therapy: Antiangiogenics + standard cytotoxics mechanism(s) of interaction. Cancer Metastasis Rev 15:247–272, 1996

    47. Herbst RS, Takeuchi H, Teicher BA: Paclitaxel/carboplatin administration along with antiangiogenic therapy in non-small cell lung and breast carcinoma models. Cancer Chemother Pharmacol 41:497–504, 1998

    48. Laird AD, Cherrington JM: Small molecule tyrosine kinase inhibitors: Clinical development of anticancer agents. Expert Opin Investig Drugs 12:51–64, 2003

    49. Gorski DH, Beckett MA, Jaskowiak NT, et al: Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res 59:3374–3378, 1999

    50. Teicher BA, Dupuis NP, Robinson M, et al: Antiangiogenic treatment (TNP-470/minocycline) increases tissue levels of anticancer drugs in mice bearing Lewis lung carcinoma. Oncol Res 7:237–243, 1995

    51. Teicher BA, Holden SA, Ara G, et al: Potentiation of cytotoxic cancer therapies by TNP-470 alone and with other antiangiogenic agents. Int J Cancer 57:920–925, 1994

    52. Teicher BA, Alvarez E, Huang ZD: Antiangiogenic agents potentiate cytotoxic cancer therapies against primary and metastatic disease. Cancer Res 52:6702–6704, 1992

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

    54. Inoue K, Chikazawa M, Fukata S, et al: Docetaxel enhances the therapeutic effect of the angiogenesis inhibitor TNP-470 (AGM-1470) in metastatic human transitional cell carcinoma. Clin Cancer Res 9:886–899, 2003

    55. Satoh H, Ishikawa H, Fujimoto M, et al: Combined effects of TNP-470 and taxol in human non-small cell lung cancer cell lines. Anticancer Res 18:1027–1030, 1998

    56. Shishido T, Yasoshima T, Denno R, et al: Inhibition of liver metastasis of human pancreatic carcinoma by angiogenesis inhibitor TNP-470 in combination with cisplatin. Jpn J Cancer Res 89:963–969, 1998

    57. Ogawa H, Sato Y, Kondo M, et al: Combined treatment with TNP-470 and 5-fluorouracil effectively inhibits growth of murine colon cancer cells in vitro and liver metastasis in vivo. Oncol Rep 7:467–472, 2000

    58. Inoue K, Slaton JW, Perrotte P, et al: Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma. Clin Cancer Res 6:4874–4884, 2000

    59. Inoue K, Slaton JW, Davis DW, et al: Treatment of human metastatic transitional cell carcinoma of the bladder in a murine model with the anti-vascular endothelial growth factor receptor monoclonal antibody DC 101 and paclitaxel. Clin Cancer Res 6:2635–2643, 2000

    60. Kim SJ, Uehara H, Karashima T, et al: Blockage of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice. Clin Cancer Res 9:1200–1210, 2003

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

    62. Gasparini G: Metronomic scheduling: The future of chemotherapy Lancet Oncol 2:733–740, 2001

    63. Browder T, Butterfield CE, Kraling BM, et al: Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 60:1878–1886, 2000

    64. Klement G, Baruchel S, Rak J, et al: Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 105:15–24, 2000

    65. Hida T, Kozaki K, Muramatsu H, et al: Cyclo-oxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines. Clin Cancer Res 6:2006–2011, 2000

    66. Mendel DB, Laird AD, Xin X, et al: In vivo antitumor activity of SU11248, a novel tyrosine kinase inhibitor targeting vascular endothelial growth factor and platelet-derived growth factor receptors: Determination of a pharmacokinetic/pharmacodynamic relationship. Clin Cancer Res 9:327–337, 2003

    67. O'Farrell AM, Abrams TJ, Yuen HA, et al: SU11248 is a novel FLT3 tyrosine kinase inhibitor with potent activity in vitro and in vivo. Blood 101:3597–3605, 2003

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

    69. Geng L, Donnelly E, McMahon G, et al: Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 61:2413–2419, 2001

    70. Ning S, Laird D, Cherrington JM, et al: The antiangiogenic agents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation. Radiat Res 157:45–51, 2002

    71. Krystal GW, Honsawek S, Kiewlich D, et al: Indoline tyrosine kinase inhibitors block kit activation and growth of small cell lung cancer cells. Cancer Res 61:3660–3668, 2001

    72. Wood JM, Bold G, Buchdunger E, et al: PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 60:2178–2189, 2000

    73. Drevs J, Hofmann I, Hugenschmidt H, et al: Effects of PTK787/ZK 222584, a specific inhibitor of vascular endothelial growth factor receptor tyrosine kinases, on primary tumor, metastasis, vessel density and blood flow in a murine renal cell carcinoma model. Cancer Res 60:4819–4824, 2000

    74. Hurwitz H, Holden SN, Eckhardt SG, et al: Clinical evaluation of ZD6474, an orally active inhibitor of VEGF signaling, in patients with solid tumors. Proc Am Soc Clin Oncol 21:82a, 2002 (abstr 325)

    75. Goekjian PG, Jirousek MR: Protein kinase C inhibitors as novel anticancer drugs. Expert Opin Investig Drugs 10:2117–2140, 2001

    76. Swannie HC, Kaye SB: Protein kinase C inhibitors. Curr Oncol Rep 4:37–46, 2002

    77. Teicher BA, Alvarez E, Menon K, et al: Antiangiogenic effects of a protein kinase C beta-selective small molecule. Cancer Chemother Pharmacol 49:69–77, 2002

    78. Keyes K, Mann L, Cox K, et al: Circulating angiogenic growth factor levels in mice bearing human tumors using Luminex Multiplex technology. Cancer Chemother Pharmacol 51:321–327, 2003

    79. Teicher BA, Menon K, Alvarez E, et al: Antiangiogenic and antitumor effects of a protein kinase C beta inhibitor in human T98G glioblastoma multiforme xenografts. Clin Cancer Res 7:634–640, 2001

    80. Herbst RS, Thornton DE, Kies MS, et al: Phase 1 study of LY317615, a protein kinase C beta inhibitor. Proc Am Soc Clin Oncol 21:82a, 2002 (abstr 326)

    81. Ferrara N: Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: Therapeutic implications. Semin Oncol 29:10–14, 2002

    82. Margolin K, Gordon MS, Holmgren E, et al: Phase Ib trial of intravenous recombinant humanized monoclonal antibody to vascular endothelial growth factor in combination with chemotherapy in patients with advanced cancer: Pharmacologic and long-term safety data. J Clin Oncol 19:851–856, 2001

    83. Gordon MS, Margolin K, Talpaz M, et al: Phase I safety and pharmacokinetic study of recombinant human anti-vascular endothelial growth factor in patients with advanced cancer. J Clin Oncol 19:843–850, 2001

    84. Kabbinavar F, Hurwitz H, Fehrenbacher L, et al: Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 21:60–65, 2003

    85. Gasparini G, Longo R, Sarmiento R, et al: COX-2 inhibitors (Coxibs): A new class of anticancer agents Lancet Oncol 4:605–615, 2003

    86. Hurwitz H, Fehrenbacher L, Cartwright T, et al: Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 350:2335–2342, 2004

    87. De Vore RF, Fehrenbacher Rs, Herbst RS: A randomized phase II trial comparing rhumab VEGF (recombinant humanized monoclonal antibody to vascular endothelial cell growth factor) plus carboplatin/paclitaxel (CP) to CP alone in patients with stage IIIB/IV NSCLC. Proc Am Soc Clin Oncol 19:485a, 2000 (abstr 1896)

    88. Sledge GW Jr: Breast cancer in the clinic: Treatments past, treatments future. J Mammary Gland Biol Neoplasia 6:487–495, 2001

    89. Yang JC, Haworth L, Sherry RM, et al: A randomized trial of bevacizumab, an anti-vascular endothelial growth factor antibody, for metastatic renal cancer. N Engl J Med 349:427–434, 2003

    90. Rosen LS: Clinical experience with angiogenesis signaling inhibitors: Focus on vascular endothelial growth factor (VEGF) blockers. Cancer Control 9:36–44, 2002 (suppl 2)

    91. Kuenen BC, Rosen L, Smit EF, et al: Dose-finding and pharmacokinetics study of cisplatin, gemcitabine and SU5416 in patients with solid tumors. J Clin Oncol 20:1657–1667, 2002

    92. Hoekman K, Kuenen B, Levi M, et al: Activation of the coagulation cascade and endothelial cell perturbation during treatment with cisplatin, gemcitabine, and the angiogenesis inhibitor SU5416. Proc Am Soc Clin Oncol 21:6a, 2002 (abstr 21)

    93. Aklilu M, Kindler HL, Gajewski TF, et al: Toxicities of the antiangiogenic agent SU5416 in phase II studies. Proc Am Soc Clin Oncol 21:28b, 2002 (abstr 1921)

    94. Harris AL: Von Hippel-Landau syndrome: Target for anti-vascular endothelial growth factor (VEGF) receptor therapy. Oncologist 5:32–36, 2000

    95. Raymond E, Faivre S, Vera K, et al: Final results of a phase I and pharmacokinetic study of SU11248, a novel multi-targeted tyrosine kinase inhibitor, in patients with advanced cancers. Proc Am Soc Clin Oncol 22:192, 2003 (abstr 769)

    96. Rosen L, Mulay M, Long J, et al: Phase I trial of SU11248, a novel tyrosine kinase inhibitor in advanced solid tumors. Proc Am Soc Clin Oncol 22:191, 2003 (abstr 765)

    97. Demetri GD, George S, Heinrich MC, et al: Clinical activity and tolerability of the multi-targeted tyrosine kinase inhibitor SU11248 in patients (pts) with metastatic gastrointestinal stromal tumor (gist) refractory to imatinib. Proc Am Soc Clin Oncol 22:814, 2003 (abstr 3273)

    98. Manning WC, Bello CL, Deprimo SE, et al: Pharmacokinetic and pharmacodynamic evaluation of SU11248 in a phase I clinical trial of patients (pts) with imatinib-resistant gastrointestinal stromal tumor (GIST). Proc Am Soc Clin Oncol 22:192, 2003 (abstr 768)

    99. O'Farrell A-M, Deprimo SE, Manning WC, et al: Analysis of biomarkers of SU11248 action in an exploratory study in patients with advanced malignancies. Proc Am Soc Clin Oncol 22:234, 2003 (abstr 939)

    100. Toner GC, Mitchell PL, De Boer R, et al: PET imaging study of SU11248 in patients with advanced malignancies. Proc Am Soc Clin Oncol 22:191, 2003 (abstr 767)

    101. Steward WP, Thomas AL, Morgan TB, et al: Extended phase I study of the oral vascular endothelial growth factor (VEGF) receptor inhibitor PTK787/ZK222584 in combination with oxaliplatin/5-fluorouracil (5-FU)/leucovorin as first line treatment for metastatic colorectal cancer. Proc Am Soc Clin Oncol 22:274, 2003 (abstr 1098)

    102. Trarbach T, Schleucher N, Riedel U, et al: Phase I study of the oral vascular endothelial growth factor (VEGF) receptor inhibitor PTK787/ZK222584 (PTK/ZK) in combination with irinotecan/5-fluorouracil/leucovorin in patients with metastatic colorectal cancer. Proc Am Soc Clin Oncol 22:285, 2003 (abstr 1144)

    103. Drevs J, Mross K, Medinger M, et al: Phase I dose-escalation and pharmacokinetic (PK) study of the VEGF inhibitor PTK787/ZK22584 (PTK/ZK) in patients with liver metastases. Proc Am Soc Clin Oncol 22:284, 2003 (abstr 1142)

    104. George D, Michaelson D, Oh WK, et al: Phase I study of PTK787/ZK222584 (PTK/ZK) in metastatic renal cell carcinoma. Proc Am Soc Clin Oncol 22:385, 2003 (abstr 1548)

    105. Reardon D, Friedman HS, Yung WKA, et al: A phase I trial of PTK787/ZK222584 (PTK/ZK), an oral VEGF tyrosine kinase inhibitor, in combination with either temozolomide or lomustine for patients with recurrent glioblastoma multiforme (GBM). Proc Am Soc Clin Oncol 22:103, 2003 (abstr 412)

    106. Roboz GJ, Giles FJ, List AF, et al: Phase I trial PTK787/ZK222584 (PTK/ZK), an inhibitor of vascular endothelial growth factor receptor tyrosine kinases, in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Proc Am Soc Clin Oncol 22:568, 2003 (abstr 2284)

    107. Minami H, Ebi H, Tahara M, et al: A phase I study of an oral VEGF receptor tyrosine kinase inhibitor ZD6474, in Japanese patients with solid tumors. Proc Am Soc Clin Oncol 22:194, 2003 (abstr 778)

    108. King J, Zhao J, Clingan P, et al: Randomized double placebo control study of adjuvant treatment with the metalloproteinase inhibitor, marimastat in patients with operable colorectal hepatic metastases: Significant survival advantage in patients with musculoskeletal side-effects. Anticancer Res 23:639–645, 2003

    109. Bramhall SR, Schulz J, Nemunaitis J, et al: A double-blind placebo-controlled, randomized study comparing gemcitabine and marimastat with gemcitabine and placebo as first line therapy in patients with advanced pancreatic cancer. Br J Cancer 87:161–167, 2002

    110. Anderson I, Supko J, Eder J: Pilot pharmacokinetic study of marimastat (MAR) in combination with carboplatin (C)/Paclitaxel (T) in patients with metastatic or locally advanced inoperable non-small lung cancer (NSCLC). Proc Am Soc Clin Oncol 18:187a, 1999 (abstr 719)

    111. Shephard FA, Giaccone G, Seymour L, et al: Prospective, randomized, double-blind, placebo-controlled trial of marimastat after response to first-line chemotherapy in patients with small-cell lung cancer: A trial of the National Cancer Institute of Canada-Clinical trials group and the European Organization for Research and Treatment of Cancer. J Clin Oncol 20:4434–4439, 2002

    112. Eisenberger M, Sinibaldi V, Laufer M: Phase I/II pharmacokinetic evaluation of marimastat in patients (pts) with advanced prostate cancer (PC): Identification of the biological active dose. Proc Am Soc Clin Oncol 19:336a, 2000 (abstr 1320)

    113. Bonomi P: Matrix metalloproteinases and matrix inhibitors in lung cancer. Semin Oncol 29:78–86, 2002

    114. Coussens LM, Fingleton B, Matrisian LM: Matrix metalloproteinse inhibitors and cancer trials and tribulations. Science 295:2387–2392, 2002

    115. Hidalgo M, Eckhardt SG: Development of matrix metalloproteinase inhibitors in cancer therapy. J Natl Cancer Inst 93:178–193, 2001

    116. Rosenbaum E, Sinibaldi V, Carducci MA, et al: Evidence of a dose/response relationship with marimastat in patients with biochemically relapsed prostate cancer (pca). Proc Am Soc Clin Oncol 22:436, 2003 (abstr 1762)

    117. DeMoraes ED, Fogler WE, Grant D: Recombinant human angiostatin (rhA): A phase I clinical trial assessing safety, pharmacokinetics (PK) and pharmacodynamics (PD). Proc Am Soc Clin Oncol 20:3a, 2001 (abstr 10)

    118. Eder JP, Supko JG, Clark JW, et al: Phase I clinical trial of recombinant human Endostatin administered as a short intravenous infusion repeated daily. J Clin Oncol 20:3772–3784, 2002

    119. Herbst RS, Hess KR, Tran HT, et al: Phase I study of recombinant human Endostatin in patients with advanced solid tumors. J Clin Oncol 20:3792–3803, 2002

    120. Thomas JP, Arzoomanian RZ, Alberti D, et al: A phase I pharmacokinetic and pharmacodynamic study of recombinant human endostatin in patients with advanced solid tumors. J Clin Oncol 21:223–231, 2003

    121. Heymach J, Kulke MH, Fuchs CS, et al: Circulating endothelial cells as a surrogate marker of antiangiogenic activity in patients treated with endostatin. Proc Am Soc Clin Oncol 22:244, 2003 (abstr 979)

    122. Kulke M, Bergsland E, Ryan DP, et al: A phase II, open-label, safety, pharmacokinetic and efficacy study of recombinant human endostatin in patients with advanced neuroendocrine tumors. Proc Am Soc Clin Oncol 22:239, 2003 (abstr 958)

    123. Eisterer W, Jiang X, Bachelot T, et al: Unfulfilled promise of endostatin in a gene therapy-xenotransplant model of human acute lymphocytic leukemia. Mol Ther 5:352–359, 2002

    124. Gasparini G, Morabito A, Magnani E, et al: Thalidomide: An old sedative-hypnotic with anticancer activity Current Opin Investig Drugs 2:1302–1308, 2001

    125. Neben K, Moehler T, Benner A, et al: Dose-dependent effect of thalidomide on overall survival in relapsed multiple myeloma. Clin Cancer Res 8:3377–3382, 2002

    126. Barlogie B, Desikan R, Eddlemon P, et al: Extended survival in advanced and refractory multiple myeloma after single-agent thalidomide: Identification of prognostic factors in a phase 2 study of 169 patients. Blood 98:492–494, 2001

    127. Weber D, Rankin K, Gavino M, et al: Thalidomide alone or with dexamethasone for previously untreated multiple myeloma. J Clin Oncol 21:16–19, 2003

    128. Cortes J, Kantarjian H, Albitar M, et al: A randomized trial of liposomal daunorubicin and cytarabine versus liposomal daunorubicin and topotecan with or without thalidomide as initial therapy for patients with poor prognosis acute myelogenous leukemia or myelodysplastic syndrome. Cancer 97:1234–1241, 2003

    129. Eisen T, Boshoff C, Mak I: Continuous low dose thalidomide: A phase II study in advanced melanoma, renal cell, ovarian and breast cancer. Br J Cancer 82:812–817, 2000

    130. Motzer RJ, Berg W, Ginsberg M, et al: Phase II trial of thalidomide for patients with advanced renal cell carcinoma. J Clin Oncol 20:302–306, 2002

    131. Morabito A, Fanelli M, Carillio G, et al: Thalidomide prolongs disease stabilization after conventional therapy in patients with recurrent glioblastoma. Oncol Rep 11:93–95, 2004

    132. Drake MJ, Robson W, Mehta P, et al: An open-label phase II study of low-dose thalidomide in androgen-independent prostate cancer. Br J Cancer 88:822–827, 2003

    133. Tseng JE, Glisson BS, Khuri FR, et al: Phase II study of the antiangiogenesis agent thalidomide in recurrent or metastatic squamous cell carcinoma of the head and neck. Cancer 92:2364–2373, 2001

    134. Fine HA, Figg WD, Jaeckle K, et al: Phase II trial of the angiogenic agent thalidomide in patients with recurrent high-grade gliomas. J Clin Oncol 18:708–715, 2000

    135. Fine HA, Wen PY, Maher EA, et al: Phase II trial of thalidomide and carmustine for patients with recurrent high-grade gliomas. J Clin Oncol 21:2299–2304, 2003

    136. Hwu WJ, Krown SE, Panageas KS, et al: Temozolomide plus thalidomide in patients with advanced melanoma: Results of a dose-finding trial. J Clin Oncol 20:2610–2615, 2002

    137. Altorki NK, Keresztes RS, Port JL, et al: Celecoxib, a selective cyclo-oxygenase-2 inhibitor, enhances the response to preoperative paclitaxel and carboplatin in early-stage non-small-cell lung cancer. J Clin Oncol 21:2645–2650, 2003

    138. Morabito A, Gattuso D, Sarmiento R, et al: Rofecoxib associated with an antiangiogenic schedule of weekly irinotecan and infusional 5-fluorouracil as second line treatment of patients with metastatic colorectal cancer: Results of a dose-finding study. Proc Am Soc Clin Oncol 22:326, 2003 (abstr 1311)

    139. Hanahan D, Bergers G, Bergsland E: Less is more, regularly: Metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest, 105:1045–1047, 2000

    140. Gately S, Kerbel RS: Antiangiogenic scheduling of lower dose cancer chemotherapy. Cancer J 7:427–436, 2001

    141. Khan J, Wei JS, Ringner M, et al: Classification and diagnostic prediction of cancers using gene expression profiling and artificial neural network. Nat Med 7:673–679, 2001

    142. Miklos GLG, Maleszka R: Integrating molecular medicine with functional proteomics: Realities and expectations. Proteomics 1:30–41, 2001

    143. Weissleder R: Scaling down imaging: Molecular mapping of cancer in mice. Nat Rev Cancer 2:11–18, 2002

    144. Pearlman JD, Laham RJ, Post M, et al: Medical imaging techniques in the evaluation of strategies for therapeutic angiogenesis. Curr Pharm Des 8:1467–1496, 2002

    145. Costouros NG, Diehn FE, Libutti SK: Molecular imaging of tumor angiogenesis. J Cell Biochem 39:72–78, 2002 (suppl)

    146. Weissleder R, Ntziachristos V: Shedding light onto live molecular targets. Nat Med 9:123–128, 2003

    147. Degani N, Gusis v, Weinstein D, et al: Mapping pathophysiological features of breast tumors by MRI at high spatial resolution. Nat Med 3:780–782, 1997

    148. Bacharac SL, Sundaram SK: 18F-FDG in cardiology and oncology: The bitter with the sweet. J Nucl Med 43:1542–1544, 2002

    149. Sipkins DA, Cheresm DA, Kazemi MR, et al: Detection of tumor angiogenesis in vivo by alpha v beta-3 target magnetic resonance imaging. Nat Med 4:623–626, 1998

    150. Haubner R, Wester HJ, Weber WA, et al: Non invasive imaging of alpha v beta-3 integrin expression using 18F- labeled RGD-containing glycopeptide and positron emission tomography. Cancer Res 61:1781–1785, 2001

    151. Herbst RS, Mullani NA, Davis DW, et al: Development of biologic markers of response and assessment of antiangiogenic activity in a clinical trial of human recombinant endostatin. J Clin Oncol 20:3804–3814, 2002

    152. Morgan B, Thomas AL, Drevs j, et al: Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: Results from two phase I studies. J Clin Oncol 21:3955–3964, 2003

    153. Anderson LH, Yap JT, Miller MP, et al: Assessment of pharmacodynamic vascular response in a phase I trial of Combretastatin A4 phosphate. J Clin Oncol 21:2823–2830, 2003

    154. Galbraith SM, Maxwell RJ, Lodge MA, et al: Combretastatin A4 phosphate has tumor antivascular activity in rat and man as demonstrated by dynamic magnetic resonance imaging. J Clin Oncol 21:2831–2842, 2003

    155. Betensky RA, Louis DN, Cairncross JG: Influence of unrecognized molecular heterogeneity on randomized clinical trials. J Clin Oncol 20:2495–2499, 2002

    156. Fox E, Curt GA, Balis FM: Clinical trial design for target-based therapy. Oncologist 7:401–409, 2002

    157. Deplanque G, Harris AL: Anti-angiogenic agents: Clinical trial design and therapies in development. Eur J Cancer 36:1713–1724, 2000

    158. Collins JM: Functional imaging in phase I studies: Decorations or decision making J Clin Oncol 21:2807–2809, 2003

    159. Gasparini G, Gion M: Molecular-targeted anticancer therapy: Challenges related to study-design and choice of proper end-points. Cancer J 6:117–131, 2000

    160. Ruoslathi E: Antiangiogenics meet nanotechnology. Cancer Cell 2:97–98, 2002

    161. Isner JM, Asahara T: Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization. J Clin Invest 103:1231–1236, 1999

    162. Pennacchietti S, Michieli P, Galluzzo M, et al: Hypoxia promotes invasive growth by transcriptional activation of the met protooncogene. Cancer Cell 3:347–361, 2003

    163. Bertolini F, Paul S, Mancuso P, et al: Maximum tolerable dose and low-dose metronomic chemotherapy have opposite effects on the mobilization and viability of circulating endothelial progenitor cells. Cancer Res 63:4342–4346, 2003

    164. Mullani N, Herbst R, Abbruzzese J, et al: First pass FDG measured blood flow in tumors: A comparative with O-15 labeled water measured blood flow. Clin Positron Imaging 3:153, 2000

    165. Relf M, LeJeune S, Scott PA, et al: Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis. Cancer Res 57:963–969, 1997

    166. Brown JM: The hypoxic cell: A target for selective cancer therapy—Eighteenth Bruce F Cain Memorial Award lecture. Cancer Res 59:5863–5870, 1999(Giampietro Gasparini, Raf)

    http://www.100md.com/html/DirDu/2007/01/01/32/93/70.htm