Influence of soluble or matrix‐bound isoforms of vascular endothelial growth factor‐A on tumor response to vascular‐targeted strategies

Antiangiogenic therapy based on blocking the actions of vascular endothelial growth factor‐A (VEGF) can lead to “normalization” of blood vessels in both animal and human tumors. Differential expression of VEGF isoforms affects tumor vascular maturity, which could influence the normalization process and response to subsequent treatment. Fibrosarcoma cells expressing only VEGF120 or VEGF188 isoforms were implanted either subcutaneously (s.c.) or in dorsal skin‐fold “window” chambers in SCID mice. VEGF120 was associated with vascular fragility and hemorrhage. Tumor‐bearing mice were treated with repeat doses of SU5416, an indolinone receptor tyrosine kinase inhibitor with activity against VEGFR‐2 and proven preclinical ability to induce tumor vascular normalization. SU5416 reduced vascularization in s.c. implants of both VEGF120 and VEGF188 tumors. However, in the window chamber, SU5416 treatment increased red cell velocity in VEGF120 (representing vascular normalization) but not VEGF188 tumors. SU5416 treatment had no effect on growth or necrosis levels in either tumor type but tended to counteract the increase in interstitial fluid pressure seen with growth of VEGF120 tumors. SU5416 pretreatment resulted in the normally fragile blood vessels in VEGF120‐expressing tumors becoming resistant to the vascular damaging effects of the tubulin‐binding vascular disrupting agent (VDA), combretastatin A4 3‐O‐phosphate (CA4P). Thus, vascular normalization induced by antiangiogenic treatment can reduce the efficacy of subsequent VDA treatment. Expression of VEGF120 made tumors particularly susceptible to vascular normalization by SU5416, which in turn made them resistant to CA4P. Therefore, VEGF isoform expression may be useful for predicting response to both antiangiogenic and vascular‐disrupting therapy.     

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.     

The multi-targeted kinase inhibitor SU5416 inhibits small cell lung cancer growth and angiogenesis, in part by blocking Kit-mediated VEGF expression

SU5416 is a multi-targeted kinase inhibitor that potentially has the ability to directly block tumor growth by inhibiting Kit signaling, as well as blocking angiogenesis by inhibiting vascular endothelial growth factor receptor (VEGFR) signaling. Previous work has demonstrated that SU5416 efficiently blocks Kit-mediated growth of small cell lung cancer (SCLC) in vitro. To determine the drug's effect on in vivo growth of SCLC, we studied its activity, alone and in combination with carboplatin, in chemotherapy-resistant H526, and chemotherapy-sensitive H209 murine xenograft models. SU5416 efficiently inhibited Kit activity in vivo when administered on a twice-weekly schedule. When administered over a 3-week period to animals bearing established tumors, it inhibited growth by at least 70%. It was at least as effective as carboplatin in suppressing growth of H526 xenografts. However, the combination with carboplatin was not superior to the most active single agent in either xenograft model at the doses and schedule utilized. SU5416 clearly inhibited growth in part by inhibiting angiogenesis, with microvessel density dropping by approximately 50% in treated xenografts. In addition to the recognized mechanism of inhibition of VEGFR, we uncovered a novel mechanism of angiogenesis suppression by demonstrating reduced VEGF expression in SU5416-treated xenografts. In vitro, stem cell factor treatment of the H526 cell line enhanced expression of VEGF, which was efficiently blocked with SU5416. Thus, we have demonstrated that SU5416 can inhibit SCLC growth by directly inhibiting tumor cell proliferation and by inhibiting angiogenesis, in part by inhibiting Kit-mediated VEGF expression. These data suggest that kinase inhibitors that target both Kit and VEGFR could play an important role in the treatment of SCLC, as well as other malignancies that express Kit.     

Flk-1特异性蛋白激酶抑制剂SU5416阻断小鼠Lewis肿瘤新生血管形成并延长生存期

Objective:To reveal the mechanism and effect of SU5416 in the treatment of mouse Lewis cancer in vivo.Methods:Lewis cell was transplanted into groin of C57/B6 mouse by subcutaneous injection,then SU5416 was administrated intraperitoneally to investigate the impact of SU5416 on tumor angiogenesis and growth in vivo.32 mice were treated with SU5416 at two different doses every day until the end-point.As a control,8 mice received no treatment and 8 mice were treated with vehicle (DMSO) only after implantation.Results:Median survival in the treated group was statistically longer compared to that in the control groups (P＜0.05) and no significant systemic adverse was observed.Histological analysis of the treated tumors showed an increase in necroses and reduced in angiogenesis compared to the control tumors.Furthermore,the percent of apoptotic cells increased in the treated tumors by FCM,the expressions of VEGF and KDR had no change after SU5416 administration by western blot,Conclusion:SU5416 may be useful therapeutics drug that specifically inhibits the enzymatic activity of KDR kinase and could down regulate the tumor angiogenesis.     

Vascular endothelial growth factor: a therapeutic target for tumors of the Ewing's sarcoma family.

PURPOSE: We have reported previously that intratumoral microvessel density (MVD) is a significant prognostic indicator of event-free survival in the Ewing's sarcoma family of tumors (ESFT). Here, the angiogenic growth factor expression profile and its relationship with MVD has been investigated in ESFT. EXPERIMENTAL DESIGN AND RESULTS: Using ESFT model systems, the potential of these factors as therapeutic targets has been evaluated. A significant correlation (P = 0.02) was observed between vascular endothelial growth factor (VEGF) expression and MVD, consistent with the hypothesis that VEGF regulates the development of microvessels in ESFT. There was no correlation between MVD and any of the other growth factors studied. All six ESFT cell lines studied produced and secreted VEGF; five of six cell lines also secreted placental growth factor, one cell line (A673) at high levels. Tumor conditioned medium induced proliferation of human umbilical vein endothelial cells. Expression of VEGF receptors Flt-1 and Flk-1/KDR was heterogeneous across the cell lines. Both receptor tyrosine kinase inhibitors SU6668 (targets Flk-1/KDR, platelet-derived growth factor receptor-beta, and fibroblast growth factor receptor 1) and SU5416 (targets Flk-1/KDR) as well as anti-VEGF agents rhuMAb-VEGF (bevacizumab) and VEGF Trap delayed s.c. growth of ESFT in mice compared with untreated groups: SU6668 (100 mg/kg/d), SU5416 (25 mg/kg/d), rhuMAb-VEGF (10 mg/kg twice weekly), and VEGF Trap (2.5 or 25 mg/kg twice weekly). CONCLUSIONS: These data suggest that VEGF is the single most important regulator of angiogenesis in ESFT and may be exploited for therapeutic advantage.     

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 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.     

Potential role of platelets in endothelial damage observed during treatment with cisplatin, gemcitabine, and the angiogenesis inhibitor SU5416.

PURPOSE: An increased incidence of thromboembolic events was observed during treatment with cisplatin-gemcitabine plus SU5416 (CG+SU5416), a tyrosine kinase inhibitor targeting the vascular endothelial growth factor (VEGF) receptor-1 and -2. Nine thromboembolic events occurred in eight of 19 patients. We performed an analysis of parameters of the coagulation cascade and vessel wall activation. MATERIALS AND METHODS: Markers for thrombin generation and endothelial cell activation were measured in three patients treated with CG+SU5416, two of whom developed a thromboembolic event. The results were compared with measurements in six patients treated with CG alone, and in 17 patients treated with SU5416 alone. RESULTS: During cycles 1 and 2 of treatment with CG+SU5416, a significant cycle-dependent activation of both the coagulation cascade and endothelial cells occurred, whereas platelet counts decreased. Change in platelet number had a significant negative predictive effect on soluble (s)-E-selectin levels. Significant activation of the coagulation cascade only was observed in the patients treated with CG alone, whereas in patients treated with SU5416 alone, significant endothelial cell activation was observed. CONCLUSION: We hypothesize that endothelial cells deprived of VEGF after exposure to SU5416 became activated and more susceptible to damage during treatment with CG+SU5416, which was aggravated by a transient decrease in platelets, which are, among other things, carriers of VEGF. These results suggests that VEGF, in addition to being a permeability, proliferation, and migration factor, also is a maintenance and protection factor for endothelial cells, and that platelets may have a role in maintaining vascular integrity.     

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.     

Flk-1特异性蛋白激酶抑制剂SU5416阻断小鼠Lewis肿瘤新生血管形成并延长生存期

Objective  To reveal the mechanism and effect of SU5416 in the treatment of mouse Lewis cancer in vivo. Methods  Lewis cell was transplanted into groin of C57/B6 mouse by subcutaneous injection, then SU5416 was administrated intraperitoneally to investigate the impact of SU5416 on tumor angiogenesis and growth in vivo. 32 mice were treated with SU5416 at two different doses every day until the end-point. As a control, 8 mice received no treatment and 8 mice were treated with vehicle (DMSO) only after implantation. Results  Median survival in the treated group was statistically longer compared to that in the control groups (P < 0.05) and no significant systemic adverse was observed. Histological analysis of the treated tumors showed an increase in necroses and reduced in angiogenesis compared to the control tumors. Furthermore, the percent of apoptotic cells increased in the treated tumors by FCM, the expressions of VEGF and KDR had no change after SU5416 administration by western blot. Conclusion  SU5416 may be useful therapeutics drug that specifically inhibits the enzymatic activity of KDR kinase and could down regulate the tumor angiogenesis. Supported by a grant from the National Key Project of Scientific and Technical Supporting Programs funded by Ministry of Science & Technology of China (No. 2006BAI02A05).     

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.     

Phase II study of the antiangiogenic agent SU5416 in patients with advanced soft tissue sarcomas.

PURPOSE: SU5416 (semaxanib) is a small molecule inhibitor of the vascular endothelial growth factor (VEGF) receptor-2 and KIT receptor tyrosine kinases. This Phase II study was conducted to investigate the safety and efficacy of SU5416 for patients with soft tissue sarcomas. EXPERIMENTAL DESIGN: Thirteen patients with locally advanced or metastatic soft tissue sarcomas were treated with SU5416 via intravenous infusion at a dose of 145 mg/m(2) twice weekly. In selected cases tumor biopsies were taken before and after 2 months of treatment. RESULTS: The median progression-free survival was 1.8 months. Median overall survival was 22.8 months. No objective tumor responses were observed. There was evidence of shorter survival among patients with high baseline urine VEGF levels (P = 0.04). No grade 4 toxicities were observed. The most common grade 3 toxicities were headache and thrombosis. Other less serious toxicities included fatigue, nausea, and abdominal pain. The median systolic blood pressure increased from 118 mmHg at baseline to 133 after 1 month of treatment (P = 0.01). Post-treatment tumor biopsies showed no significant decreases in VEGF receptor phosphorylation compared with baseline in 3 evaluable patients. One patient with gastrointestinal stromal tumor who had rapid progression during SU5416 treatment was subsequently treated with another KIT inhibitor, imatinib mesylate, and had a partial response lasting >36 months. CONCLUSIONS: SU5416 was relatively well tolerated but did not demonstrate significant antitumor activity against advanced soft tissue sarcoma. Correlative studies suggest that VEGF receptor or KIT inhibition was incomplete in at least some cases, providing a possible explanation for the observed lack of activity.     

Targeting angiogenesis inhibits tumor infiltration and expression of the pro-invasive protein SPARC

The solid growth of high-grade glioma appears to be critically dependent on tumor angiogenesis. It remains unknown, however, whether the diffuse infiltration of glioma cells into healthy adjacent tissue is also dependent on the formation of new tumor vessels. Here, we analyze the relationship between tumor angiogenesis and tumor cell infiltration in an experimental glioma model. C6 cells were implanted into the dorsal skinfold chamber of nude mice, and tumor angiogenesis was monitored by intravital fluorescence videomicroscopy. Glioma infiltration was assessed by the extent of tumor cell invasion into the adjacent chamber tissue and by expression of SPARC, a cellular marker of glioma invasiveness. To test the hypothesis that glioma angiogenesis and glioma infiltration are codependent, we assessed tumor infiltration in both the presence and the absence of the angiogenesis inhibitor SU5416. SU5416 is a selective inhibitor of the VEGF/Flk-1 signal-transduction pathway, a critical pathway implicated in angiogenesis. Control tumors demonstrated both high angiogenic activity and tumor cell invasion accompanied by strong expression of SPARC in invading tumor cells at the tumor-host tissue border. SU5416-treated tumors demonstrated reduced vascular density and vascular surface in the tumor periphery accompanied by marked inhibition of glioma invasion and decreased SPARC expression. A direct effect of SU5416 on glioma cell motility and invasiveness was excluded by in vitro migration and invasion assays. These results suggest a crucial role for glioma-induced angiogenesis as a prerequisite for diffuse tumor invasion and a possible therapeutic role for anti-angiogenic compounds as inhibitors of both solid and diffuse infiltrative tumor growth.     

Mechanism of reoxygenation after antiangiogenic therapy using SU5416 and its importance for guiding combined antitumor therapy

Emerging preclinical studies support the concept of a transient "normalization" of tumor vasculature during the early stage of antiangiogenic treatment, with possible beneficial effects on associated radiotherapy or chemotherapy. One key issue in this area of research is to determine whether this feature is common to all antiangiogenic drugs and whether the phenomenon occurs in all types of tumors. In the present study, we characterized the evolution of the tumor oxygenation (in transplantable liver tumor and FSAII tumor models) after administration of SU5416, an antagonist of the vascular endothelial growth factor receptor. SU5416 induced an early increase in tumor oxygenation [measured by electronic paramagnetic resonance (EPR)], which did not correlate with remodeling of the tumor vasculature (assessed by CD31 labeling using immunohistochemistry) or with tumor perfusion (measured by dynamic contrast enhanced-magnetic resonance imaging). Inhibition of mitochondrial respiration (measured by EPR) was responsible for this early reoxygenation. Consistent with these unique findings in the tumor microenvironment, we found that SU5416 potentiated tumor response to radiotherapy but not to chemotherapy. In addition to the fact that the characterization of the tumor oxygenation is essential to enable correct application of combined therapies, our results show that the long-term inhibition of oxygen consumption is a potential novel target in this class of compounds.     

Inhibition of angiogenesis by blocking activation of the vascular endothelial growth factor receptor 2 leads to decreased growth of neurogenic sarcomas.

Neurogenic sarcomas are incurable, common malignant human peripheral nerve tumors subject to local recurrence and systemic metastasis. In this study, the vascularity, vascular endothelial growth factor (VEGF) expression, and effects of inhibiting VEGF receptor on growth of neurogenic sarcomas were examined. Vascularization and VEGF expression were 6.4- and 15-fold higher in tumors than in normal nerves. The small molecule inhibitor (SU5416) of VEGF receptor 2 had no effect on neurogenic sarcoma cell lines in vitro, but the growth of a human tumor explant xenograft model was reduced by 54.8% compared to vehicle. Reduction in tumor growth was due to decreased tumor angiogenesis, leading to reduction of tumor cell proliferation and increased apoptosis. Inhibiting VEGF function may therefore be a useful adjuvant therapy for neurogenic sarcomas.     

Irradiation combined with SU5416: microvascular changes and growth delay in a human xenograft glioblastoma tumor line

PURPOSE: The combination of irradiation and the antiangiogenic compound SU5416 was tested and compared with irradiation alone in a human glioblastoma tumor line xenografted in nude mice. The aim of this study was to monitor microenvironmental changes and growth delay. METHODS AND MATERIALS: A human glioblastoma xenograft tumor line was implanted in nude mice. Irradiations consisted of 10 Gy or 20 Gy with and without SU5416. Several microenvironmental parameters (tumor cell hypoxia, tumor blood perfusion, vascular volume, and microvascular density) were analyzed after imunohistochemical staining. Tumor growth delay was monitored for up to 200 days after treatment. RESULTS: SU5416, when combined with irradiation, has an additive effect over treatment with irradiation alone. Analysis of the tumor microenvironment showed a decreased vascular density during treatment with SU5416. In tumors regrowing after reaching only a partial remission, vascular characteristics normalized shortly after cessation of SU5416. However, in tumors regrowing after reaching a complete remission, permanent microenvironmental changes and an increase of tumor necrosis with a subsequent slower tumor regrowth was found. CONCLUSIONS: Permanent vascular changes were seen after combined treatment resulting in complete remission. Antiangiogenic treatment with SU5416 when combined with irradiation has an additive effect over treatment with irradiation or antiangiogenic treatment alone.     

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     

Effects of SU5416, a small molecule tyrosine kinase receptor inhibitor, on FLT3 expression and phosphorylation in patients with refractory acute myeloid leukemia

Acute myeloid leukemia (AML) is associated with dysregulated hematopoietic cell proliferation and increased bone marrow angiogenesis, each regulated by signaling through receptor tyrosine kinases (RTKs). SU5416 is a small molecule inhibitor of VEGF receptors, c-kit and FLT3 and therefore provides a novel opportunity to target both angiogenesis and proliferation in AML. SU5416 was assessed in a phase II hematological malignancy trial in the US, where partial responses were observed in two of 33 patients. Since AML provides a unique platform to evaluate mechanism of action of small molecule inhibitors, investigation of the effect of SU5416 on FLT3 expression and phosphorylation in blood and bone marrow was an additional focus of this trial. Phosphorylated FLT3 was detected by immunoprecipitation/Western analysis in peripheral blood samples from 17 of 22 patients, and seven exhibited strong inhibition of phosphorylation immediately following a 1h SU5416 infusion, demonstrating that SU5416 can modulate RTK phosphorylation in humans. Although no clear correlation with clinical response was observed, analysis of patient plasma drug levels suggested that a threshold SU5416 concentration of 15 microM was associated with FLT3 inhibition. This observation was supported by data from an ex vivo model where AML cells were spiked into human blood, established to mimic the clinical setting and enable more rigorous analysis of effect of SU5416. In addition, FLT3 protein levels were downregulated in patient bone marrow samples, analyzed by an RIA assay. To identify putative predictors of response, patient plasma was analyzed for levels of secreted ligands of SU5416 targets; SCF and FLT3 ligand. Baseline levels of SCF in patients with stable or progressive disease were significantly higher than those in normal donors, whereas FLT3 ligand levels in patients who exhibited progressive disease were significantly lower than those in normal donors. The translational and clinical analyses described in this report provide some insights into the mechanism and duration of action of SU5416.     

The role of vascular endothelial growth factor (VEGF) in AIDS-related Kaposi's sarcoma

Kaposi's sarcoma (KS) is the most common neoplasm associated with human immunodeficiency virus-1 (HIV-1) infection. KS involves the skin and mucous membranes as well as other organs and can lead to tumor-associated edema and ulcerations. Despite therapy with highly active antiviral agents, most patients with HIV-1-related KS eventually develop disseminated disease. In the treatment of KS, a strong rationale exists for the use of agents that inhibit vascular endothelial growth factor (VEGF). Angiogenesis appears to be an important feature of this disease, and recent experimental studies have demonstrated the role of VEGF and its receptors in the pathogenesis of KS. Thus, therapeutic agents that target the VEGF pathway may be an effective strategy in reducing the tumor growth and edema associated with KS. Phase I study results with SU5416, a synthetic low molecular-weight inhibitor of the VEGF-Flk-1/KDR receptor tyrosine kinase, demonstrate that this agent is well tolerated. Preliminary results show that in a majority of patients with autoimmune deficiency syndrome (AIDS)-related disease, SU5416 clearly has biological activity (it flattens, shrinks, or dissolves lesions and reduces or resolves edema) or stabilizes the disease. Angiogenesis inhibition with SU5416 is a promising therapeutic approach in treating patients with KS, and further clinical evaluation is currently under way.     

SU5416 delays wound healing through inhibition of TGF-beta 1 activation

Angiogenesis, development of new blood vessels, is essential for wound healing and tumor growth. A potentially important side effect of anti-angiogenic therapy can be delayed wound healing. In this study we address this issue by using a novel in vivo method utilizing fibrin containing dual porous plexiglass chambers (Fibrin Z-Chambers; F-ZC) to investigate wound healing in rats administered with SU5416 (inhibitor of Flk-1 and Flt-1, at 20 mg/kg i.p.). SU5416 treated F-ZCs developed 45% less granulation tissue (p = 0.0076) and showed a 10% reduction in microvessel density (p = 0.0009) than controls treated with drug carrier alone. The granulation tissue showed distinctly decreased collagen deposition (p = 0.0006) in SU5416 treated animals that was associated with 90% reduction in active TGF-beta 1 level. We found that tissue transglutaminase (TG), a cross-linking enzyme involved in TGF-beta 1 activation and matrix stabilization, was inhibited by SU5416. These results suggest that SU5416 delays wound healing by reducing matrix synthesis and stabilization through inhibition of TGF-beta 1 activation. This study was made feasible via the development of a unique method to study anti-angiogenic compounds that provides highly reproducible and quantitative results. Further studies are needed to evaluate in vivo whether anti-angiogenic agents alter wound healing.