Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis.

Increased vascular endothelial growth factor (VEGF) expression is associated with colon cancer metastases. We hypothesized that inhibition of VEGF receptor activity could inhibit colon cancer liver metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate metastases. Mice received daily i.p. injections of vehicle, tyrosine kinase inhibitor for Flk-1/KDR (SU5416) or tyrosine kinase inhibitor for VEGF, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). SU5416 and SU6668 respectively inhibited metastases (48.1% and 55.3%), microvessel formation (42.0% and 36.2%), and cell proliferation (24.4% and 27.3%) and increased tumor cell (by 2.6- and 4.3-fold) and endothelial cell (by 18.6- and 81.4-fold) apoptosis (P<0.001). VEGF receptor inhibitors increased endothelial cell apoptosis, suggesting that VEGF may serve as an endothelial survival factor.     

Vascular endothelial growth factor in human colon cancer: biology and therapeutic implications

Tumor growth and metastasis are dependent on angiogenesis. Vascular endothelial growth factor (VEGF) plays an important role in the angiogenesis of numerous solid malignancies including colon cancer. Evidence from preclinical and clinical studies indicates VEGF is the predominant angiogenic factor in human colon cancer and is associated with formation of metastases and poor prognosis. Based on these results, it was hypothesized that inhibition of VEGF receptor activity could inhibit colon cancer liver metastasis. To test this hypothesis, the authors evaluated the ability of a small molecule inhibitor specific for the tyrosine kinase VEGF receptor Flk-1/KDR (SU5416) or multiple tyrosine kinase receptors (SU6668) to inhibit tumor angiogenesis and metastasis in a model of colon cancer hepatic metastasis. Both SU5416 and SU6668 inhibited metastases, microvessel formation, and cell proliferation while increasing tumor cell and endothelial cell apoptosis. These results showed that targeting the VEGF receptor/ligand system is a rational approach to inhibiting tumor growth and prolonging survival.     

SU5416 and SU6668 attenuate the angiogenic effects of radiation-induced tumor cell growth factor production and amplify the direct anti-endothelial action of radiation in vitro

In recent decades, radiation research has concentrated primarily on the cancer cell compartment. Much less is known about the effect of ionizing radiation on the endothelial cell compartment and the complex interaction between tumor cells and their microenvironment. Here we report that ionizing radiation is a potent antiangiogenic agent that inhibits endothelial cell survival, proliferation, tube formation and invasion. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor were able to reduce the radiosensitivity of endothelial cells. Yet, it is also found that radiation induces angiogenic factor production by tumor cells that can be abrogated by the addition of antiangiogenic agents. Receptor tyrosine kinase inhibitors of Flk-1/KDR/VEGFR2, FGFR1 and PDGFR beta, SU5416, and SU6668 enhanced the antiangiogenic effects of direct radiation of the endothelial cells. In a coculture system of PC3 prostate cancer cells and endothelial cells, isolated irradiation of the PC3 cells enhanced endothelial cell invasiveness through a Matrigel matrix, which was inhibited by SU5416 and SU6668. Furthermore, ionizing radiation up-regulated VEGF and basic fibroblast growth factor in PC3 cells and VEGFR2 in endothelial cells. Together these findings suggest a radiation-inducible protective role for tumor cells in the support of their associated vasculature that may be down-regulated by coadministration of angiogenesis inhibitors. These results rationalize concurrent administration of angiogenesis inhibitors and radiotherapy in cancer treatment.     

The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function.

SU5416, a selective inhibitor of the tyrosine kinase activity of the vascular endothelial growth factor (VEGF) receptor Flk-1/KDR, is currently in Phase III clinical trials for the treatment of advanced malignancies. In cellular assays, SU5416 inhibits the VEGF-dependent mitogenic/proliferative response of human umbilical vein endothelial cells (HUVECs). In tumor xenograft models, SU5416 inhibits the growth of tumors from a variety of origins by inhibiting tumor angiogenesis. In three different human tumor xenograft models, infrequent (once or twice a week) administration of SU5416 is efficacious despite the fact that it has a short plasma half-life (30 min), which suggests that SU5416 has long-lasting inhibitory activity in vivo. The goal of the present study was to determine the basis for the prolonged activity of SU5416. The results indicate that a short (3 h) exposure to 5 microM SU5416 (to mimic plasma levels of the compound as measured in patients who were receiving SU5416 therapy) produced long-lasting (at least 72 h) inhibition of the VEGF-dependent proliferation of HUVECs in culture, which indicate that SU5416 has long-lasting inhibitory activity in vitro as well as in vivo. SU5416 treatment of HUVECs did not affect surface expression of Flk-1/KDR or the affinity of the receptor for VEGF. Instead, the durability of the in vitro activity of SU5416 was shown to be attributable to its long-lasting ability to specifically inhibit VEGF-dependent phosphorylation of Flk-1/KDR and subsequent downstream signaling, although SU5416 is not an irreversible inhibitor of Flk-1/KDR tyrosine kinase activity. The long-lasting inhibition of cellular responses to VEGF was attributable to the accumulation of SU5416 in cells, as shown using radiolabeled compound, such that inhibitory cellular concentrations of SU5416 are maintained long after the removal of the compound from the medium. The long-lasting inhibitory activity of SU5416 in vitro is consistent with the finding that SU5416 has demonstrated evidence of biological activity in clinical studies when administered twice a week despite a short plasma half-life.     

Inhibition of tumor growth, angiogenesis, and microcirculation by the novel Flk-1 inhibitor SU5416 as assessed by intravital multi-fluorescence videomicroscopy

Vascular endothelial growth factor (VEGF) plays a fundamental role in mediating tumor angiogenesis and tumor growth. Here we investigate the direct effect of a novel small molecule inhibitor of the Flk-1-mediated signal transduction pathway of VEGF, SU5416, on tumor angiogenesis and microhemodynamics of an experimental glioblastoma by using intravital multifluorescence videomicroscopy. SU5416 treatment significantly suppressed tumor growth. In parallel, SU5416 demonstrated a potent antiangiogenic activity, resulting in a significant reduction of both the total and functional vascular density of the tumor microvasculature, which indicates an impaired vascularization as well as significant perfusion failure in treated tumors. This malperfusion was not compensated for by changes in vessel diameter or recruitment of nonperfused vessels. Analyses of the tumor microcirculation revealed significant microhemodynamic changes after angiogenesis blockage such as a higher red blood cell velocity and blood flow in remnant tumor vessels when compared with controls. Our results demonstrate that the novel antiangiogenic concept of targeting the tyrosine kinase of Flk-1/KDR by means of a small molecule inhibitor represents an efficient strategy to control growth and progression of angiogenesis-dependent tumors. This study provides insight into microvascular consequences of Flk-1/KDR targeting in vivo and may have important implications for the future treatment of angiogenesis-dependent neoplasms.     

Inhibition of peritoneal dissemination of ovarian cancer by tyrosine kinase receptor inhibitor SU6668 (TSU-68)

SU6668 (TSU-68) is a small-molecule synthetic inhibitor of the angiogenic related receptor tyrosine kinases Flk-1/KDR, PDGFRbeta, and FGFR1. Using a mouse model of peritoneally disseminated ovarian cancer, we investigated whether SU6668 inhibits peritoneal dissemination and prolongs survival time. BALB/c nude mice were intraperitoneally (i.p.) inoculated with SHIN-3 (VEGF-hypersecretory) or KOC-2S (PDGF-hypersecretory) ovarian serous adenocarcinoma cells with marked peritoneal dissemination ability. From the day after i.p. inoculation of tumor cells, SU6668 was orally administered 6 times weekly at a daily dose of 100 mg/kg or 400 mg/kg. The SU6668-administered group and the vehicle-administered control group were compared for the number of tumor vascular endothelial cells, weight of peritoneally disseminated tumors, amount of ascitic fluid and survival time. As a result, these 3 parameters were significantly smaller in the SHIN-3-inoculated, SU6668-administered mice than in the control group (p = 0.03, p = 0.002, and p = 0.02, respectively). The mean survival time was significantly longer, at 58.1 +/- 11.2 days, in the SU6668-administered mice than that (34.5 +/- 8.8 days) in the control group (p = 0.002). Similarly, in the KOC-2S-inoculated mice, the oral administration of SU6668 significantly reduced these 3 parameters (p = 0.04, p = 0.04, and p = 0.03, respectively), and significantly prolonged survival (16.6 +/- 1.7 days vs. 11.0 +/- 0.7 days, p = 0.008). Thus, the oral administration of SU6668 inhibited angiogenesis and peritoneal dissemination and prolonged survival in mice with peritoneally disseminated ovarian cancer. These effects were observed with both the VEGF- and PDGF-hypersecretory cell lines. Our results suggest that molecular targeting with oral SU6668 will become a new therapeutic strategy targeting peritoneally disseminated ovarian cancer.     

Measuring VEGF-Flk-1 activity and consequences of VEGF-Flk-1 targeting in vivo using intravital microscopy: clinical applications

Vascular endothelial growth factor (VEGF)-Flk-1/KDR tyrosine kinase signaling pathway plays a pivotal role in tumor angiogenesis. Targeting this angiogenic signaling pathway presents a promising alternative for the treatment of neoplasms. However, recent experimental and clinical studies have suggested that VEGF-Flk-1/KDR activity is unevenly distributed throughout the tumor microvasculature. To further evaluate this phenomenon, the regional differences in VEGF-Flk-1/KDR signaling activities in vivo were studied using intravital fluorescence videomicroscopy in an experimental murine brain tumor model. Regional VEGF-Flk-1/KDR was assessed using the small molecule inhibitor SU5416, which selectively inhibits the tyrosine kinase receptor Flk-1. C(6) glioblastoma cells were implanted into the dorsal skinfold chamber preparation of nude mice. The process of tumor vascularization was repeatedly assessed over 22 days. SU5416 treatment resulted in a significant reduction in tumor vascular density (p<0.05). Regional microvascular evaluation indicated that the magnitude of this antiangiogenic effect was pronounced in the more angiogenic and better vascularized peritumoral areas than in the intratumoral areas of the tumor microvasculature. These results demonstrate regional differences in Flk-1 activity in vivo that may have significant impact on the susceptibility of tumors to compounds that target VEGF-Flk-1/KDR. This finding should be considered in upcoming clinical trials targeting individual signal transduction systems in cancer patients.     

Results of a Phase I dose-escalating study of the antiangiogenic agent, SU5416, in patients with advanced malignancies.

SU5416 is a small molecule antiangiogenic agent that inhibits vascular endothelial growth factor (VEGF) stimulation of the KDR tyrosine kinase receptor. In this Phase I dose escalation trial, a weekly dose schedule of SU5416 was tested whereby an initial 5-day loading dose was followed by weekly maintenance infusions. The start dose was 20 mg/m(2) for the loading dose followed by 65 mg/m(2) for the weekly infusions. Dose escalations occurred at 33% until a final dose of 65 mg/m(2) (loading dose) and 190 mg/m(2) (weekly infusion) was obtained. Twenty-two patients were treated at five dose levels; tumor types included gastrointestinal (8), breast (3), lung (4), sarcoma (2), and other (5). The most common serious drug-related toxicity was headache, often associated with nausea and vomiting. Grade 1 and 2 toxicities included headache, nausea, vomiting, asthenia, pain at the infusion site, phlebitis, change in voice, and fevers. Of 19 evaluable patients, 4 obtained clinical benefit as defined by tumor regression (1) or disease stabilization for at least 12 weeks (3). Pharmacokinetic data revealed that the weekly infusion schedule prevented the reported 50-60% induction in SU5416 clearance observed with either daily or twice weekly dosing. Higher baseline levels of urine VEGF were observed in the 4 patients who gained clinical benefit, suggesting this may be a useful marker for predicting response to anti-VEGF therapies. Our results suggest that a weekly schedule of SU5416 shows signs of biological activity and is well tolerated at doses up to 145 mg/m(2).     

The combination of the tyrosine kinase receptor inhibitor SU6668 with paclitaxel affects ascites formation and tumor spread in ovarian carcinoma xenografts growing orthotopically.

PURPOSE: The purpose of this study was to investigate the antitumor activity of SU6668, tyrosine kinase inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2), fibroblast growth factor receptor 1 (FGFR1), and platelet-derived growth factor receptor beta (PDGFRbeta), as single-agent therapy and in combination with paclitaxel on ovarian carcinoma xenograft models transplanted in the peritoneal cavity of nude mice. EXPERIMENTAL DESIGN: HOC22 and HOC79 ascites-producing human ovarian carcinoma xenografts were transplanted i.p. into nude mice. SU6668 was given p.o. (200 mg/kg, daily) as a single agent or in combination with paclitaxel i.v. (6 mg/kg/dose every other day or 20 mg/kg/dose weekly). Tumor burden was evaluated at the end of the treatment period as ascites volume and tumor cells, VEGF, FGF-2, and PDGF levels in ascites, and involvement of the organ of the peritoneal cavity. Response was evaluated as percentage increment of life span (%ILS). RESULTS: SU6668 affected ascites formation and tumor burden in the peritoneal cavity of nude mice bearing HOC22 and HOC79 xenografts. Decreased levels of VEGF and PDGF in ascites paralleled this effect. The overall survival of the mice bearing HOC xenograft (HOC79 less response than HOC22) was significantly increased by the treatment with SU6668. The magnitude of the effects depended on the length of treatment and tumor burden at the beginning of treatment. The combination of SU6668 with paclitaxel significantly prolonged the survival of mice bearing HOC79, compared with single therapies. SU6668-based combination therapy was more effective with paclitaxel given at the optimal dose and schedule (20 mg/kg every 7 days for 3 doses) than at the same total dose but split (6 mg/kg every 2 days for 10 doses). However, a similar outcome was observed when giving high-dose paclitaxel (20 mg/kg every 7 days for 3 doses) in monotherapy or split low-dose paclitaxel (6 mg/kg every 2 days for 10 doses) but in combination with SU6668. The addition of paclitaxel, by either schedule, to SU6668 treatment inhibited tumor spread in the peritoneal organs (omentum, pancreas, and diaphragm) even at low doses of paclitaxel. A greater effect was observed with prolonged treatments. CONCLUSIONS: This study shows that SU6668 in combination with paclitaxel inhibits ovarian carcinoma progression in the peritoneal cavity, by blocking ascites formation and tumor spread. Because an adequate schedule and dose of the combination might be as effective as conventional chemotherapy, this should be considered as a therapeutic alternative. These findings provide a rationale for the clinical evaluation of combination therapies affecting multiple biological targets in this tumor type.     

Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy

Certain refractory neoplasms, such as glioblastoma multiforme (GBM) and melanoma, demonstrate a resistant tumor phenotype in vivo. We observed that these refractory tumor models (GBM and melanoma) contain blood vessels that are relatively resistant to radiotherapy. To determine whether the vascular endothelial growth factor receptor-2 (Flk-1/KDR) may be a therapeutic target to improve the effects of radiotherapy, we used the soluble extracellular component of Flk-1 (ExFlk), which blocks vascular endothelial growth factor binding to Flk-1 receptor expressed on the tumor endothelium. Both sFlk-1 and the Flk-1-specifc inhibitor SU5416 eliminated the resistance phenotype in GBM and melanoma microvasculature as determined by both the vascular window and Doppler blood flow methods. Human microendothelial cells and human umbilical vein endothelial cells showed minimal radiation-induced apoptosis. The Flk-1 antagonists sFlk-1 and SU5416 reverted these cell models to apoptosis-prone phenotype. Flk-1 antagonists also reverted GBM and melanoma tumor models to radiation-sensitive phenotype after treatment with 3 Gy. These findings demonstrate that the tumor microenvironment including the survival of tumor-associated endothelial cells contributes to tumor blood vessel resistance to therapy.     

SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors

Vascular endothelial growth factor, fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) and their cognate receptor tyrosine kinases are strongly implicated in angiogenesis associated with solid tumors. Using rational drug design coupled with traditional screening technologies, we have discovered SU6668, a novel inhibitor of these receptors. Biochemical kinetic studies using isolated Flk-1, FGF receptor 1, and PDGF receptor beta kinases revealed that SU6668 has competitive inhibitory properties with respect to ATP. Cocrystallographic studies of SU6668 in the catalytic domain of FGF receptor 1 substantiated the adenine mimetic properties of its oxindole core. Molecular modeling of SU6668 in the ATP binding pockets of the FIk-1/KDR and PDGF receptor kinases provided insight to explain the relative potency and selectivity of SU6668 for these receptors. In cellular systems, SU6668 inhibited receptor tyrosine phosphorylation and mitogenesis after stimulation of cells by appropriate ligands. Oral or i.p. administration of SU6668 in athymic mice resulted in significant growth inhibition of a diverse panel of human tumor xenografts of glioma, melanoma, lung, colon, ovarian, and epidermoid origin. Furthermore, intravital multifluorescence videomicroscopy of C6 glioma xenografts in the dorsal skinfold chamber model revealed that SU6668 treatment suppressed tumor angiogenesis. Finally, SU6668 treatment induced striking regression of large established human tumor xenografts. Investigations of SU6668 activity in cancer patients are ongoing in Phase I clinical trials.     

Expression of vascular endothelial growth factor and its receptor, KDR/Flk-1, in soft tissue sarcomas

The aim of the present study was to evaluate the association of vascular endothelial growth factor (VEGF) and its receptor, KDR/Flk-1, with tumor grade, microvessel density (MVD) and clinical outcome in patients with soft tissue sarcomas (STSs). Tissue specimens of 28 patients with STSs were analyzed immunohistochemically using specific monoclonal antibodies. Tissue samples were obtained prior to any treatment. Half of the STSs exhibited strong expression of VEGF that was associated statistically significantly with high tumor grade. Strong expression of KDR/Flk-1 was detected in only 2 sarcomas. No association was demonstrated between VEGF and KDR/Flk-1 expression. MVD was significantly associated with tumor grade and was higher in sarcomas with strong VEGF expression. Limited data on clinical outcome precluded solid analyses for an association with disease progression. This study provides further evidence on the role of VEGF and MVD in tumor aggressiveness in STSs.     

Improved effect of an antiangiogenic tyrosine kinase inhibitor (SU5416) by combinations with fractionated radiotherapy or low molecular weight heparin

The effect of combining SU5416 with fractionated radiotherapy or with low molecular weight (LMW) heparin (dalteparin) was studied in U87 human glioblastoma xenografts in nude mice. SU5416 is antiangiogenic by a specific inhibition of the vascular endothelial growth factor receptor 2 (VEGFR-2), and heparins are assumed to bind VEGF. Both SU5416 (100 mg/kg every second day in 5 days) and 3 Gyx5 produced moderate, yet significant, growth inhibition. Tumors treated with concomitant irradiation and short-term SU5416 maintained a lower growth rate during regrowth than the other treatment groups (P=.007). Dalteparin (1000 IE/kg subcutaneously once a day) had no growth-inhibitory effect on its own, but when this LMW heparin was added to the SU5416 schedule, a significantly enhanced growth inhibition was obtained. VEGF protein content in tumors was not significantly altered by SU5416, but a significant decrease in VEGF levels was found in tumors treated with concomitant dalteparin and SU5416 compared with controls (P=.03). We conclude that: 1) an additive growth-inhibitory effect is obtained by combining SU5416 and fractionated radiotherapy; and 2) LMW heparin (dalteparin), in combination with SU5416, decreases the level of VEGF in tumors and increases the growth-inhibitory effect of SU5416.     

Enhancing the therapeutic responsiveness of photodynamic therapy with the antiangiogenic agents SU5416 and SU6668 in murine nasopharyngeal carcinoma models

Background: Photodynamic therapy (PDT) is a promising therapeutic modality using a tumor localizing photosensitizer and light to destroy tumor cells. A major limitation of PDT is tumor recurrence, which is partly due to neovascularization. Purpose: The objective of the present study was to determine whether combination therapy with PDT and antiangiogenic agents (i.e. SU5416 and SU6668) would be more effective in controlling tumor recurrence in a mouse model of human CNE2 poorly differentiated nasopharyngeal carcinoma compared with PDT or antiangiogenic agents administered alone. Methods: Athymic mice bearing CNE2 tumor xenografts received daily i.p. injections of 20 mg/kg SU5416 or 100 mg/kg SU6668 for 28 consecutive days either alone or following a single hypericin-PDT treatment. Results: Significant inhibition of CNE2 tumor growth was observed in all treatment groups. Differences in 4× tumor growth time, the number of mice with 4× tumor growth, tumor growth inhibition as well as the percent of mice surviving were not statistically significant among individual treatment groups. However, the number of mice with 4× tumor growth observed in SU6668 monotherapy and combined PDT and SU6668 treatment groups was significantly less than that in the control group (P<0.05 and 0.01, respectively). Moreover, compared with the control group, only the combined PDT and SU6668 treatment significantly extended survival of tumor-bearing host mice (P<0.05). The semiquantitative RT-PCR results showed that the expression of HIF-1, VEGF, COX-2 and bFGF were increased in PDT-treated tumor samples collected 24 h post-PDT, suggesting that PDT-induced damage to tumor microvasculature and the resultant hypoxia upregulate the expression of certain proangiogenic factors. Conclusions: The effectiveness of PDT can be enhanced by antiangiogenic treatment with the synthetic RTK inhibitors. Of the two synthetic RTK inhibitors tested, SU6668 was more effective than SU5416 in enhancing tumor responsiveness to PDT.     

A randomized Phase II trial of the antiangiogenic agent SU5416 in hormone-refractory prostate cancer.

PURPOSE: To assess the activity of the antiangiogenic agent and VEGFR2 inhibitor SU5416 in hormone-refractory prostate cancer. Patients and Methods: Thirty-six chemotherapy na?ve patients were randomized to treatment with SU5416 (145 mg/m(2)) and dexamethasone premedication or dexamethasone alone. Patients in the control arm could cross over to experimental therapy after progression. Prostate-specific antigen (PSA) was measured every 2 weeks, and radiological evaluation was performed every 8 weeks. In vitro assessment of SU5416 on PSA secretion was assessed in the LNCaP cell line. Baseline serum basic fibroblast growth factor and plasma vascular endothelial growth factor (VEGF) were explored as prognostic factors. RESULTS: VEGF receptor-2 expression is detectable in prostate cancer cell lines, and SU5416 inhibited in vitro PSA secretion. No effect of SU5416 on PSA secretion or time to progression is detectable in patients. VEGF and basic fibroblast growth factor were not prognostic. Headache and fatigue were the most common SU5416 toxicities, but hyperglycemia, hyponatremia, lymphopenia, infection, and adrenal suppression, all attributable to steroids and the required central line, were common. CONCLUSION: No disease modifying effects of SU5416 were detectable in this small study. Modest toxicity, an inconvenient administration schedule, and availability of other VEGFR-targeted agents support the decision to halt further evaluation of SU5416 in prostate cancer.     

Vascular endothelial growth factor (VEGF) inhibition by small molecules

Angiogenesis is essential for primary tumours to grow and metastasise, and is driven by the production of positive angiogenic factors. The Vascular Endothelial Growth Factor (VEGF) family is central to the process of angiogenesis and comprises 5 molecules designated A, B, C, D and E. VEGF is overexpressed in several solid malignancies. The actions of VEGF are mediated through receptors possessing tyrosine kinase activity: VEGFR-1 (Flt-1), VEGFR-2 (Kdr/Flk-1) and VEGFR-3 (Flt-4). Anti-VEGF strategies include the use of antibodies to VEGF or its receptors, the use of ribozymes to decrease receptor expression, and the use of inhibitors of tyrosine kinase to reduce receptor activation and downstream signalling. The focus of this review is small molecule inhibitors of VEGF receptors which target their intrinsic tyrosine kinase activity. The clinical development of the following agents is discussed: SU5416, SU11248, SU6668, PTK/ZK, ZD6474.     

Combined therapy of local and metastatic 4T1 breast tumor in mice using SU6668, an inhibitor of angiogenic receptor tyrosine kinases,and the immunostimulator B7.2-IgG fusion protein

The therapeutic efficacy of combined antiangiogenic and immune therapy was tested against weakly immunogenic and highly metastatic 4T1 breast tumor using SU6668, an angiogenesis inhibitor and recombinant murine (rm) B7.2-IgG fusion protein, an immunostimulator. SU6668 inhibits the tyrosine kinase activity of three angiogenic receptors VEGFR2 (Flk-1/KDR), PDGFRbeta, and FGFR1, which play a crucial role in tumor-induced vascularization. rmB7.2-IgG is a fusion protein of the extracellular domain of B7.2, and the hinge and constant domains of murine IgG2a. Intermolecule disulfide linkages are maintained so that it forms a dimer. Our studies showed that three weekly immunizations of BALB/c mice bearing 0.5-0.8 cm 4T1 breast tumors with rmB7.2-IgG and irradiated 4T1 tumor cells (B7.2-IgG/TC) resulted in a significant inhibition of tumor growth and formation of pulmonary metastases. T-cell depletion experiments revealed that both CD4(+) and CD8(+) T lymphocytes are required for stimulation of the antitumor and antimetastatic immune response by B7.2-IgG/TC. Treatment of mice with SU6668 substantially inhibited tumor vascularization but did not inhibit tumor infiltration by T lymphocytes or the T-cell response to rmB7.2-IgG therapy. On the contrary, tumors in mice immunized with B7.2-IgG/TC and treated with SU6668 had higher numbers of tumor infiltrating T cells than tumors of other groups. SU6668 treatments initiated either on day 3 or day 10 after inoculation of 4T1 tumor cells resulted in a significant inhibition of tumor growth. Similarly, three weekly immunizations with B7.2-IgG/TC starting either on day 7 or 12 inhibited growth of 4T1 tumors. However, the most potent antitumor effects were observed in mice treated with a combination of SU6668 and B7.2-IgG/TC. Analysis of pulmonary metastases revealed that combined therapy also had a more potent antimetastatic effect than each modality alone. These results indicate that antiangiogenic and immune therapies using SU6668 and B7.2-IgG/TC are compatible, and manifest complementary antitumor and antimetastatic effects. Combined antiangiogenic and immune therapy might represent a new strategy for cancer treatment.     

Plasma and cerebrospinal fluid pharmacokinetics of SU5416 after intravenous administration in nonhuman primates

Purpose: SU5416 is a small, lipophilic synthetic molecule that selectively inhibits the tyrosine kinase activity of the VEGF receptor Flk-1/KDR. The role of this agent in brain tumors is currently being investigated. Pharmacokinetic studies of SU5416 have been performed in humans; however, there have been no studies of its penetration in the cerebrospinal fluid (CSF). We studied the pharmacokinetics of SU5416 in plasma and CSF after intravenous (i.v.) administration using a nonhuman primate model that is highly predictive of the CSF penetration in humans.Experimental design:SU5416 (85 mg/m², about 3.8 mg/kg) was administered i.v. over 20 min to four nonhuman primates. Serial plasma and CSF samples were obtained prior to, during, and after completion of the infusion, for determination of SU5416 concentrations. SU5416 was measured in plasma and CSF using high-performance liquid chromatography (HPLC). Concentration-versus-time data were modeled using model-independent and model-dependent methods.Results: Peak plasma concentrations ranged from 6.3 to 14.5 μM and the mean plasma AUC was 620±180 μM min. Disappearance of SU5416 from the plasma was best described by a one-compartment model with a half-life of 39±2.9 min. The volume of distribution was 36±11 l/m² and the clerance was 0.62±0.2 l/min per m². SU4516 was not quantifiable in the CSF.Conclusions: There is minimal penetration of SU5416 into the CSF after i.v. administration. The very low CNS exposure to SU5416 after i.v. dosing suggests that this agent is not optimal for the treatment of leptomeningeal tumors. Published online: 9 October 2003     

Antiangiogenic agent SU6668 suppresses the tumor growth of xenografted A-431 cells

SU6668 is an antiangiogenic agent that acts as a tyrosine kinase inhibitor for the vascular endothelial growth factor (VEGF) receptor. We treated xenografted A-431, a human squamous cell carcinoma cell line, with SU6668 to investigate the efficacy of tumor dormant therapy using angiogenesis inhibitors for patients with squamous cell carcinoma. A-431 cells were transplanted into severe combined immunodeficient (SCID) mice. The treatment group was given SU6668 at a dose of 200 mg/kg orally twice a day for 3 weeks; the control group was given only the vehicle in the same manner and intervals. SU6668 suppressed tumor growth of A-431 cells in the xenograft model. Furthermore, tumor volume and the number of vessels were significantly reduced in the treatment group compared with those of the control. No significant differences in body weight were found between the treatment and control groups, and no toxic reactions occurred during the experiment. We concluded that this agent was safe, efficient and potentially useful for the treatment of patients with squamous cell carcinoma.