o-(chloroacetylcarbamoyl)fumagillol and Neuroblastoma

Promising new antiangiogenic strategies are emerging for the treatment of cancer and the inhibition of angiogenesis could represent a powerful adjunct to traditional therapy of malignant tumors. Over the last ten years several reports have been published concerning the relationship between tumor progression and angiogenesis in neuroblastoma in experimental models in vitro and in vivo. Moreover, a high vascular index in neuroblastoma correlates with poor prognosis, suggesting dependence of aggressive tumor growth on active angiogenesis. Here, we present an overview of recent advances in antiangiogenesis in neuroblastoma and describe the most important active substances, preclinical and clinical data, as well as future perspectives.

Topics: Angiogenesis Inhibitors; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Cyclohexanes; Disease Progression; Endostatins; Humans; Liposomes; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Prognosis; Receptor, trkA; Retinoids; Sesquiterpenes; Thalidomide; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

The growth of solid tumours has been shown to be dependent on new blood vessel formation, i.e. angiogenesis. Several steps in the metastatic process have also been found to be angiogenesis dependent. The mediators of tumour angiogenesis are now being elucidated, and angiostatic agents have been developed. Some of these agents are currently undergoing clinical trials. In addition to inhibition of angiogenesis, two other clinical applications of angiogenetic research in tumour diseases are monitoring of disease activity by analyses of circulating angiogenic peptides and prediction of a poor outcome by tumour microvascular counts. Neuroblastomas grow quickly, are highly vascularised and metastasise early and hence inhibition of angiogenesis--angiostatic therapy--may be indicated in this disease. The effects of treatment with the angiostatic agent TNP-470 in an experimental model results in a significant reduction of the tumour growth rate, reduced microvascular counts and a reduced fraction of viable tumour cells compared to controls. TNP-470 as single therapy has an objective tumoristatic effect in our neuroblastoma model. Angiostatic treatment of neuroblastoma is a new and theoretically promising treatment modality that merits clinical investigations. The feasibility of assessing disease activity by repeated determinations of the levels of circulating angiogenic peptides should also be determined, as well as the use of microvascular counts to predict a poor outcome.

Topics: Animals; Antibiotics, Antineoplastic; Clinical Trials as Topic; Cyclohexanes; Humans; Immunohistochemistry; Microscopy, Electron; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Prognosis; Rats; Sesquiterpenes

Targeted expression of MYCN to the neural crest [under control of the rat tyrosine hydroxylase (TH) promoter] causes neuroblastoma in transgenic mice (TH-MYCN) and is a well-established model for this disease. Because high levels of MYCN are associated with enhanced tumor angiogenesis and poor clinical outcome in neuroblastoma, we serially characterized malignant progression, angiogenesis, and sensitivity to angiogenic blockade in tumors from these animals. Tumor cells were proliferative, secreted high levels of the angiogenic ligand vascular endothelial growth factor (VEGF), and recruited a complex vasculature expressing the angiogenic markers VEGF-R2, alpha-SMA, and matrix metalloproteinases MMP-2 and MMP-9, all of which are also expressed in human disease. Treatment of established murine tumors with the angiogenesis inhibitor TNP-470 caused near-complete ablation, with reduced proliferation, enhanced apoptosis, and vasculature disruption. Because TNP-470 has been associated with neurotoxicity, we tested the recently described water-soluble HPMA copolymer-TNP-470 conjugate (caplostatin), which showed comparable efficacy and was well tolerated without weight loss or neurotoxicity as measured by rotarod testing. This study highlights the importance of angiogenesis inhibition in a spontaneous murine tumor with native tumor-microenvironment interactions, validates the use of mice transgenic for TH-MYCN as a model for therapy in this common pediatric tumor, and supports further clinical development of caplostatin as an antiangiogenic therapy in childhood neuroblastoma.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Cyclohexanes; Disease Models, Animal; Disease Progression; Genes, myc; Mice; Mice, Transgenic; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Sesquiterpenes

Methionine aminopeptidase (MetAP)-2 has been suggested as a novel target for cancer therapy because the anticancer agent TNP-470 irreversibly inactivates the catalytic activity of this enzyme. However, the importance of MetAP2 in cell growth and tumor progression was uncertain because previous data were based on the chemically reactive TNP-470. Here we show that a rationally designed reversible MetAP2 inhibitor, A-357300, suppresses tumor growth preclinically without the toxicities observed with TNP-470. We have synthesized this bestatin-type MetAP2 inhibitor with the aid of crystal structures of the enzyme-inhibitor complexes and parallel synthesis. A-357300 induces cytostasis by cell cycle arrest at the G(1) phase selectively in endothelial cells and in a subset of tumor cells, but not in most primary cells of nonendothelial type. A-357300 inhibits angiogenesis both in vitro and in vivo and shows potent antitumor efficacy in carcinoma, sarcoma, and neuroblastoma murine models. These data affirm that MetAP2 plays a pivotal role in cell growth and establish that reversible MetAP2 inhibitors are promising novel cancer therapeutic agents.

Topics: Aminopeptidases; Animals; Antineoplastic Agents; Breast Neoplasms; Cell Cycle; Cell Division; Chlorobenzenes; Cornea; Cyclohexanes; Drug Design; Endothelium, Vascular; Female; Fibrosarcoma; Humans; Metalloendopeptidases; Mice; Mice, SCID; Models, Molecular; Neovascularization, Physiologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Protease Inhibitors; Sesquiterpenes; Xenograft Model Antitumor Assays

CHS 828 is a new chemotherapeutic drug, a pyridyl cyanoguanidine. CHS 828 has low toxicity and lacks known patterns of multidrug resistance. Here we report that oral, daily treatment with CHS 828 reduced the growth of SH-SY5Y human neuroblastoma tumors in male NMRI nu/nu mice by 82% without apparent toxicity. CHS 828 induced complete tumor regression for at least 5 weeks in four of nine animals (44%). Combination therapy with CHS 828 and the antiangiogenic drugs TNP-470 or SU5416 decreased neuroblastoma growth by a further 10 and 3%, respectively. Combination therapy induced tumor regression at d 4 with CHS plus TNP and d 6 with CHS plus SU5416, compared with d 14 with CHS 828 alone (p < 0.05), and complete tumor regression was seen in nine of 19 animals (47%). Combination treatment of CHS 828 and TNP-470 decreased the total viable tumor volume by 71% compared with treatment with CHS 828 alone. Our findings support CHS 828 as a promising new drug in treatment of childhood cancers. Furthermore, they imply efficiency of daily administration of nontoxic doses of chemotherapy, and a possible additive effect when chemotherapy is combined with angiogenesis inhibitors.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Division; Cyanides; Cyclohexanes; Guanidines; Humans; Indoles; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Pyrroles; Remission Induction; Sesquiterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Tumor vascularity is highly correlated with disease outcome in neuroblastoma. Thus, novel therapeutics that target the vascular endothelium are candidates for incorporation into clinical trials. We therefore examined the effect of TNP-470 on human neuroblastoma growth in mouse models reflecting both clinically evident and minimal disease. Mice were inoculated s.c. or by tail vein injection with 10(7) human neuroblastoma-derived CHP-134 cells and treated with TNP-470 (100 mg/kg/dose s.c. three times a week or by continuous infusion) or saline. Treatment was given as a single agent in established xenografts, 10 days after 450 mg/kg of cyclophosphamide, or 12 h after tumor inoculation. Tumor growth rate was markedly inhibited in mice receiving TNP-470 administered alone both s.c. and by continuous infusion with a treatment to control ratio (T:C) at day 16 of 0.3 (P < 0.001) and a T:C at day 30 of 0.4 (P = 0.029) for each dosing method, respectively. TNP-470 also significantly inhibited tumor growth when administered following cyclophosphamide (T:C at day 30 = 0.2, P < 0.001) and inhibited disease establishment when given shortly after xenograft inoculation (T:C at day 30 = 0.1, P < 0.001) or tail vein injection. TNP-470 was shown to directly inhibit angiogenesis by Matrigel assay (P =.010) and to increase the apoptotic index in treated tumors. These data show that TNP-470 is a potent inhibitor of human neuroblastoma growth rate and tumorigenicity. We speculate that TNP-470 may be a useful adjuvant therapy for high-risk neuroblastoma patients, particularly when used in settings of minimal disease status.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Cell Division; Cyclohexanes; Humans; Mice; Mice, Nude; Mice, SCID; Neovascularization, Pathologic; Neovascularization, Physiologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Angiogenesis plays a crucial role in the progression and metastasis of malignant solid tumors. In many types of cancer, including neuroblastoma, high tumor vascularity correlates with poor outcome. Recently, a number of angiogenic inhibitors have been identified that had antitumor activity in preclinical studies.. To investigate the effect of the antiangiogenic agent TNP-470 on neuroblastoma tumor growth, we treated nude mice with TNP-470 after they were inoculated subcutaneously with 5 x 10(6) cells from the MYCN-amplified, human neuroblastoma cell line NBL-W-N.. TNP-470 did not significantly affect tumor growth when it was administered to mice with large tumors (>600 mm3). However, when TNP-470 was administered in the setting of microscopic disease 12 hr following tumor cell inoculation, treated mice had a significantly improved tumor-free survival at 12 weeks (P < 0.001), and overall survival at 45 weeks (P < 0.001), compared to control animals.. Our studies suggest that TNP-470 treatment may be most effective if it is administered in the setting of microscopic disease. We speculate that TNP-470 may inhibit neuroblastoma growth in children if treatment is initiated following intensive multimodality therapy, when residual disease is minimal.

Topics: Angiogenesis Inhibitors; Animals; Cell Division; Cyclohexanes; Humans; Male; Mice; Mice, Nude; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Animals; Antibodies, Monoclonal; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Lewis Lung; Cyclohexanes; Cyclophosphamide; Female; Humans; Maximum Tolerated Dose; Mice; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Sesquiterpenes; Tumor Cells, Cultured; Vinblastine

TNP-470 is a strong inhibitor of angiogenesis. The present study was designed to determine whether the angiogenesis inhibitor TNP-470 inhibits metastasis of mouse neuroblastoma cells to the liver and thus increases survival.. A murine neuroblastoma cell line, C1300, and A/J mice were used in this study. First, to demonstrate the inhibitory effects of TNP-470 on angiogenesis, we quantified the area of angiogenesis on images made with SP-500 image analyzer (Olympus) 7 days after implanting a millipore chamber and compared the areas for the TNP-470-treated mice and control mice. Next, to determine the inhibitory effect of TNP-470 on metastasis of neuroblastoma cells to the liver, we made a murine hepatic metastasis model by implanting C1300 cells (1 x 10(6)) in the spleen of the mice and compared histologic findings, sizes, and weights of the livers of treated mice and control mice 14 days after the beginning of a 7-day infusion of TNP-470 (60 mg/kg). We also compared survival rates using the Kaplan-Meier method.. When the angiogenesis inhibitor TNP-470 was infused into mice that received tumor cells, the area of angiogenesis in the TNP-470-treated mice was smaller than that in the control mice (52.5 +/- 6.3 SD vs 94.1 +/- 27.6 mm(2), P < 0.001). After the same treatment in other mice, no histologic evidence of metastasis was found, whereas control mice had countless tumor cell masses. Similarly, the weight of the liver was less in TNP-470-treated mice (0.8 +/- 0.1 g vs 4.5 +/- 0.3 g, P < 0.001). Survival was longer in the TNP-470-treated mice than in controls (80% of treated mice were alive more than 60 days after treatment, whereas all control mice died by Day 20).. TNP-470 inhibits metastasis of mouse C1300 neuroblastoma cells to the liver, and thus increases survival. TNP-470 inhibits metastasis by inhibiting angiogenesis.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Body Weight; Cyclohexanes; Liver Neoplasms, Experimental; Mice; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Survival Rate; Tumor Cells, Cultured

Background and Procedure High-risk neuroblastoma disease features are correlated with tumor vascularity, suggesting that angiogenesis inhibitors may be a useful addition to current therapeutic strategies. We therefore examined the efficacy of TNP-470 (TAP Pharmaceuticals, Deerfield, IL) in human neuroblastoma xenograft models.. Tumor growth rate was markedly inhibited in mice receiving TNP-470 administered alone with a treatment to control ratio (T/C) at day 21 = 0.4 (P <.001). TNP-470 also significantly inhibited tumorigenicity when administered shortly after xenograft inoculation (T/C at day 30 = 0.1, P <.001) and when administered following cyclophosphamide (T/C at day 35 = 0.1, P <.001).. These data show that TNP-470 is a potent inhibitor of human neuroblastoma growth both alone and when given with conventional chemotherapy, suggesting that it may be a useful adjunctive therapy for high-risk neuroblastoma patients.

Topics: Angiogenesis Inhibitors; Animals; Child; Cyclohexanes; Humans; Mice; Mice, Nude; Neoplasm, Residual; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Inhibition of angiogenesis has been shown to reduce tumor growth, metastasis, and tumor microvascular density in experimental models. To these effects we would now like to add induction of differentiation, based on biological analysis of xenografted human neuroblastoma (SH-SY5Y, WAG rnu/rnu) treated with the angiogenesis inhibitor TNP-470. Treatment with TNP-470 (10 mg/kg s.c., n = 15) reduced the tumor growth by 66% and stereological vascular parameters (Lv, Vv, Sv) by 36-45%. The tumor cell apoptotic fraction increased more than threefold, resulting in a decrease in viable tumor cells by 33%. In contrast, the mean vascular diameter (29 microm) and the mean tumor cell proliferative index (49%) were unaffected. TNP-470-treated tumors exhibited striking chromaffin differentiation of neuroblastoma cells, observed as increased expression of insulin-like growth factor II gene (+88%), tyrosine hydroxylase (+96%), chromogranin A, and cellular processes. Statistical analysis revealed an inverse correlation between differentiation and angiogenesis. It is suggested that by inhibiting angiogenesis, TNP-470 induces metabolic stress, resulting in chromaffin differentiation and apoptosis in neuroblastoma. Such agonal differentiation may be the link between angiostatic therapy and tumor cell apoptosis.

Topics: Animals; Antibiotics, Antineoplastic; Apoptosis; Brain Neoplasms; Cell Differentiation; Chromaffin Cells; Chromogranin A; Chromogranins; Cyclohexanes; Female; Humans; Insulin-Like Growth Factor II; Male; Neoplasm Transplantation; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Rats, Nude; Sesquiterpenes; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Angiogenesis plays an important role in the growth and metastasis of malignant tumors. We have previously reported that in children with neuroblastoma (NB), tumor vascularity directly correlates with metastatic disease, MYCN amplification, and poor outcome. The angiogenesis inhibitor TNP-470 has been shown to reduce the rate of NB growth in rodents with macroscopic tumors without ultimately impacting survival. To investigate whether TNP-470 could more effectively inhibit NB growth in animals with a low tumor burden, we treated 30 nude mice with minimal disease with this angiogenesis inhibitor (supplied by TAP Pharmaceuticals, Inc.). Therapy was initiated before tumors were clinically evident after s.c. inoculation of 5 x 10(6) cells from the MYCN-amplified NB cell line NBL-W-N. TNP-470 was administered 3 days/week, and after 12 weeks of treatment, 53% of the treated mice remained tumor free, whereas 100% of the control mice developed tumors (P < 0.0001). To further assess the relationship between the efficacy of TNP-470 treatment and tumor burden, TNP-470 was also administered s.c., 3 days/week, to mice with clinically evident small (<400 mm3; n = 15) and large (>400 mm3; n = 11) tumors. For animals with small tumors, the mean rate of growth was significantly decreased in the treated mice compared to the controls (P = 0.02). In contrast, there was no difference in the mean rate of tumor growth between animals with large tumors treated with TNP-470 and controls (P = 0.64). Our studies demonstrate that the effectiveness of TNP-470 inversely correlates with tumor burden. We speculate that TNP-470 may most effectively inhibit NB tumor growth in children with a low tumor burden.

Topics: Angiogenesis Inhibitors; Animals; Antibiotics, Antineoplastic; Cell Division; Cyclohexanes; Growth Inhibitors; Humans; Male; Mice; Mice, Nude; Neoplasm Staging; Neoplasm Transplantation; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Transplantation, Heterologous; Tumor Cells, Cultured

The finding that angiogenesis plays an important role in the progression and metastasis of malignant tumors has led to the development of several antiangiogenic drugs. The authors report here an examination of the effect of the antiangiogenic agent TNP-470 on the growth, metastases, and survival of two differing murine neuroblastoma cell lines, TBJ and C1300. We found that TNP-470 significantly reduced primary tumor volumes in mice injected with either cell line. In addition, antiangiogenic therapy significantly reduced the size of axillary lymph node metastases in both groups as well as decreased the size of liver metastases in mice receiving TBJ neuroblastoma. TNP-470 treatment also improved animal survival. These data suggest that antiangiogenic therapy retards the growth of primary and metastatic murine neuroblastoma. We speculate that antiangiogenic therapy may be a useful therapeutic modality in the treatment of advanced neuroblastoma once side effects and appropriate dosage requirements are determined.

Topics: Animals; Antibiotics, Antineoplastic; Cyclohexanes; Male; Mice; Mice, Inbred Strains; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Sesquiterpenes; Tumor Cells, Cultured

A new animal experimental model of human neuroblastoma is described. The model involves xenotransplantation of a poorly differentiated human neuroblastoma cell line (SH-SY5Y) to the subcutaneous tissue in the hind leg of nude rats (WAG mu/rnu). Injection of 20 million cells suspended in 0.2 mL of medium in each hind leg yielded an 89% tumor take (41/46) in 23 nude rats. Tumor take was evident after 2 wk. The tumors grew exponentially and reached a volume of 5.2 +/- 1.6 mL 4 wk after transplantation. The tumor cells retained their morphologic phenotype at the ultrastructural level after transplantation and were immunohistochemically positive for neuron-specific enolase and for chromogranins A and B. Subcutaneous injections of the angiogenesis inhibitor TNP-470 (10 mg/kg of body weight) every other day gave a treated/control quotient for mean tumor volume of 0.34 after 12 d of treatment. This implies that angiogenesis inhibition may be of value as a complement to chemotherapy in the treatment of human neuroblastoma. The presented animal experimental model is designed for investigations of the effects of chemotherapy, angiogenesis inhibitors, radiotherapy, and/or surgery on the growth rate of human neuroblastoma.

Topics: Animals; Antibiotics, Antineoplastic; Autonomic Nervous System Diseases; Cell Survival; Cyclohexanes; Female; Humans; Immunohistochemistry; Male; Microscopy, Electron; Neovascularization, Pathologic; Neuroblastoma; O-(Chloroacetylcarbamoyl)fumagillol; Rats; Rats, Nude; Sesquiterpenes; Transplantation, Heterologous