Flk-1 (IC50 = 1.23 μM)

Semaxanib, with an IC50 of 1.04 μM, prevents Flk-1-overexpressing NIH 3T3 cells from phosphorylating the Flk-1 receptor in a VEGF-dependent manner. With an IC50 of 20.3 μM, semaxanib prevents PDGF-dependent autophosphorylation in NIH 3T3 cells. With an IC50 of 0.04 and 50 μM, respectively, semaxanib inhibits VEGF- and FGF-driven mitogenesis in a dose-dependent manner. The in vitro growth of C6 glioma, Calu 6 lung carcinoma, A375 melanoma, A431 epidermoid carcinoma, and SF767T glioma cells (all with IC50s > 20 μM) is unaffected by semaxanib treatment.[1]

Semaxanib dose-related suppresses the in vivo growth of the A375 tumor. Daily intraperitoneal administration of SU5416 in DMSO at Semaxanib results in a >85% inhibition of subcutaneous tumor growth with no detectable toxicity. Semaxanib exhibits a wide range of antitumor properties. With an average death rate of 2.5%, SU5416 significantly inhibits the subcutaneous growth of 8 out of 10 tumor lines tested (A431, Calu-6, C6, LNCAP, EPH4-VEGF, 3T3HER2, 488G2M2, and SF763T cells).[1] The tumor microvasculature's total and functional vascular densities are significantly reduced by semaxanib (25 mg/kg/day), which exhibits strong antiangiogenic activity.[2]

Polystyrene ELISA plates precoated with a Flk-1-specific monoclonal antibody are then filled with soluble membranes from 3T3 Flk-1 cells. Serial dilutions of SU5416 are added to the immunolocalized receptor following an overnight incubation at 4 °C with lysate. The ELISA plate wells containing serially diluted solutions of SU5416 are filled with varying concentrations of ATP in order to cause autophosphorylation of the receptor. After 60 minutes at room temperature, EDTA is used to halt the autophosphorylation process. The immunolocalized receptor is incubated with a biotinylated monoclonal antibody that is directed against phosphotyrosine to measure the amount of phosphotyrosine on the Flk-1 receptors in each individual well. Homo sapiens conjugated with avidin is added to the wells following the extraction of the unbound anti-phosphotyrosine antibody. Three, three, five, nine tetramethyl benzidine dihydrochloride in stabilized form is added to each well along with H₂O₂. H₂SO₄ is used to halt the reaction after 30 minutes of allowing the assay's color readout to progress.

HUVECs are cultivated at 37 °C for 24 hours to quiesce the cells after plating them in 96-well flat-bottomed plates (1×10⁴ cells/100 μL/well) in F-12K media containing 0.5% heat-inactivated FBS. After adding serial dilutions of the compounds made in the medium containing 1% DMSO for two hours, the media are then supplemented with mitogenic concentrations of acidic fibroblast growth factor (0.5–5 ng/mL) or VEGF (5 ng/mL or 20 ng/mL). In the assay, the final DMSO concentration is 0.25%. After a full day, the cell monolayers are incubated for an additional 24 hours with the addition of either BrdUrd or [³H]thymidine (1 μCi/well). One can use a liquid scintillation counter or a BrdUrd ELISA to quantify the uptake of [³H]thymidine or BrdUrd into cells, respectively.

Mice: At 12 weeks of age, female BALB/c nu/nu mice weighing 20–22 g are utilized. In this surgical procedure, aseptic technique is applied. The abdominal wall just above the colon has a tiny 1 cm midline incision made in it. Applying a 27-gauge needle beneath the colon's serosa allows for the implantation of C6 cells (0.5×10⁶ cells/animal). All of the exposed intestine is reinserted into the abdominal cavity following implantation. Using a 6.0 surgical suture and wound clips, the peritoneum and skin are sealed. Seven days following surgery, the wound clips are extracted. Starting one day after implantation, the animals receive a 50 μL intraperitoneal bolus injection of either DMSO or semaxinib (SU5416) once a day. The animals are put to sleep 13–16 days after implantation, and the amount of local tumor growth on the colon is measured using venier calipers or by weighing the tumors. The formula for calculating tumor volumes is length × width × height.
Rats: Five groups of sixty male Sprague Dawley rats (n = 60, 6–8 weeks) are randomly assigned to: control (Con), pneumonectomy (PNx), semaxinib (SU), semaxinib+hypoxia (SuHx), and semaxinib+PNx (SuPNx). It uses the SuHx protocol. In short, animals receive an injection of 25 mg/kg of semaxinib dissolved in carboxymethylcellulose (CMC) and are then exposed to 10% hypoxia for four weeks before being returned to normoxia. Animals from PNx had a left pneumonectomy. A 25 mg/kg injection of semaxinib is given two days after PNx surgery. Con received only the CMC that was solvent. Echocardiography is used to assess the morphometry and function of the heart at baseline (prehypoxia/presurgery), week 2, and week 6. The animals are put to sleep and their left and right ventricles' (LV and RV) pressures are measured using catheterization two and six weeks after surgery or hypoxia.

[1]. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res, 1999, 59(1), 99-106.

[2]. Inhibition of tumor growth, angiogenesis, and microcirculation by the novel Flk-1 inhibitor SU5416 as assessed by intravital multi-fluorescence videomicroscopy. Neoplasia, 1999, 1(1), 31-41.

[3]. Pneumonectomy combined with SU5416 induces severe pulmonary hypertension in rats.Am J Physiol Lung Cell Mol Physiol. 2016 Jun 1;310(11):L1088-97.

[4]. Metabolic Reprogramming in the Heart and Lung in a Murine Model of Pulmonary Arterial Hypertension. Front Cardiovasc Med. 2018 Aug 15;5:110.

Semaxanib is an oxindole that is 3-methyleneoxindole in which one of the hydrogens of the methylene group is replaced by a 3,5-dimethylpyrrol-2-yl group. It has a role as an antineoplastic agent, a vascular endothelial growth factor receptor antagonist, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an angiogenesis modulating agent. It is a member of pyrroles, a member of oxindoles and an olefinic compound. It is functionally related to a 3-methyleneoxindole.
Semaxanib is a quinolone derivative with potential antineoplastic activity. Semaxanib reversibly inhibits ATP binding to the tyrosine kinase domain of vascular endothelial growth factor receptor 2 (VEGFR2), which may inhibit VEGF-stimulated endothelial cell migration and proliferation and reduce the tumor microvasculature. This agent also inhibits the phosphorylation of the stem cell factor receptor tyrosine kinase c-kit, often expressed in acute myelogenous leukemia cells.

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Drug Indication
Investigated for use/treatment in colorectal cancer and lung cancer.

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SU5416, a novel synthetic compound, is a potent and selective inhibitor of the Flk-1/KDR receptor tyrosine kinase that is presently under evaluation in Phase I clinical studies for the treatment of human cancers. SU5416 was shown to inhibit vascular endothelial growth factor-dependent mitogenesis of human endothelial cells without inhibiting the growth of a variety of tumor cells in vitro. In contrast, systemic administration of SU5416 at nontoxic doses in mice resulted in inhibition of subcutaneous tumor growth of cells derived from various tissue origins. The antitumor effect of SU5416 was accompanied by the appearance of pale white tumors that were resected from drug-treated animals, supporting the antiangiogenic property of this agent. These findings support that pharmacological inhibition of the enzymatic activity of the vascular endothelial growth factor receptor represents a novel strategy for limiting the growth of a wide variety of tumor types.[1]

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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.[2]

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The SU5416 + hypoxia (SuHx) rat model is a commonly used model of severe pulmonary arterial hypertension. While it is known that exposure to hypoxia can be replaced by another type of hit (e.g., ovalbumin sensitization) it is unknown whether abnormal pulmonary blood flow (PBF), which has long been known to invoke pathological changes in the pulmonary vasculature, can replace the hypoxic exposure. Here we studied if a combination of SU5416 administration combined with pneumonectomy (PNx), to induce abnormal PBF in the contralateral lung, is sufficient to induce severe pulmonary arterial hypertension (PAH) in rats. Sprague Dawley rats were subjected to SuPNx protocol (SU5416 + combined with left pneumonectomy) or standard SuHx protocol, and comparisons between models were made at week 2 and 6 postinitiation. Both SuHx and SuPNx models displayed extensive obliterative vascular remodeling leading to an increased right ventricular systolic pressure at week 6 Similar inflammatory response in the lung vasculature of both models was observed alongside increased endothelial cell proliferation and apoptosis. This study describes the SuPNx model, which features severe PAH at 6 wk and could serve as an alternative to the SuHx model. Our study, together with previous studies on experimental models of pulmonary hypertension, shows that the typical histopathological findings of PAH, including obliterative lesions, inflammation, increased cell turnover, and ongoing apoptosis, represent a final common pathway of a disease that can evolve as a consequence of a variety of insults to the lung vasculature. [3]

C15H14N2O

238.28

238.11

C, 75.61; H, 5.92; N, 11.76; O, 6.71

204005-46-9

(Z)-Semaxanib;194413-58-6; 204005-46-9; 1055412-47-9 (Semaxanib analog/chlorinated)

5329098

Yellow to orange solid powder

1.3±0.1 g/cm3

481.4±45.0 °C at 760 mmHg

244.9±28.7 °C

0.0±1.2 mmHg at 25°C

1.684

2.87

2

1

1

18

377

0

O=C1/C(=C(/[H])\C2=C(C([H])([H])[H])C([H])=C(C([H])([H])[H])N2[H])/C2=C([H])C([H])=C([H])C([H])=C2N1[H]

WUWDLXZGHZSWQZ-WQLSENKSSA-N

InChI=1S/C15H14N2O/c1-9-7-10(2)16-14(9)8-12-11-5-3-4-6-13(11)17-15(12)18/h3-8,16H,1-2H3,(H,17,18)/b12-8-

(3Z)-3-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-1H-indol-2-one

Sugen 5416; Sugen5416; Sugen-5416; semoxind; SU5416; SU-5416; SU 5416; Semaxanib; Semaxinib; 204005-46-9; 194413-58-6

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.49 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: 2.25 mg/mL (9.44 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 22.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)