Orantinib (SU6668)

Mouse (Female, BALB/c, nu/nu) xenograft models of A375, Colo205, H460, Calu-6, C6, SF763T, and SKOV3TP5 tumor cells

(Z)-3-(2, 4-dimethyl-5-((2-oxoindolin-3-ylidene)methyl)-1H-pyrrol-3-yl)propanoic acid

0.03-10 uM , dissolved in DMSO as 10 mM stock solution

75–200 mg/kg

2.1 uM (Ki), 2.1 uM (Ki), 2.1 uM (Ki), 2.1 uM (Ki), 2.1 uM (Ki), 2.1 uM (Ki)

TSU-68 (75–200 mg/kg) induces tumor growth inhibition against a broad range of tumor types in xenograft models in athymic mice, including A375, Colo205, H460, Calu-6, C6, SF763T, and SKOV3TP5 cells. TSU-68 (75 mg/kg) also suppresses tumor angiogenesis of C6 glioma xenografts. [1] In a tumor model of HT29 human colon carcinoma, TSU-68 (200 mg/kg) decreases the average vessel permeability and average fractional plasma volume in the tumor rim and core. TSU-68 promotes abnormal stromal development at the periphery of carcinomas. [3] In a rabbit VX2 liver tumor model, TSU-68 (200 mg/kg) augments the effect of chemotherapeutic infusion. [4]

trans-Phosphorylation Reactions, Tyrosine kinase assays to quantitate the trans-phosphorylation activity of Flk-1 and FGFR1 are performed in 96-well microtiter plates precoated (20 ug/well in PBS, incubated overnight at 4 C) with the peptide substrate poly-Glu, Tyr (4:1). Excess protein binding sites are blocked with 1–5% (w/v) BSA in PBS. Purified GST-FGFR1 (kinase domain) or GST-Flk-1 (cytoplasmic domain) fusion proteins are then added to the microtiter wells in 2 x concentration kinase dilution buffer consisting of 100 mM HEPES, 50 mM NaCl, 40 uM NaVO4, and 0.02% (w/v) BSA. The final enzyme concentration for GST-Flk-1 and GST-FGFR1 is 50 ng/mL. SU6668 is dissolved in DMSO at 100x the final required concentration and diluted 1:25 in H2O. Twenty-five uL of diluted SU6668 are subsequently added to each reaction well. The kinase reaction is initiated by the addition of different concentrations of ATP in a solution of MnCl2 so that the final ATP concentrations spanned the Km for the enzyme, and the final conntration of MnCl2 is 10 mM. The plates are incubated for 5-15 min at room temperature before stopping the reaction with the addition of EDTA. The plates are then washed three times with TBST. Rabbit polyclonal antiphosphotyrosine antisera are added to the wells at a 1: 10000 dilution in TBST containing 0.5% (w/v) BSA, 0.025% (w/v) nonfat dry milk, and 100 uM NaVO4 and incubated for 1 hour at 37 C. The plates are then washed three times with TBST, followed by the addition of goat anti-rabbit antisera conjugated with HRP. The plates are incubated for 1 hour at 37 C and then washed three times with TBST. The amount of phosphotyrosine in each well is quantitated after the addition of 2, 2'-azino-di-[3-ethylbenzthiazoline sulfonate] as substrate.

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Data from [Angiogenesis, 2012, ahead of print], TSU-68 (SU6668)purchased from Selleck, (A)Tumor growth curve of s.c. HuH-7 tumor-bearing mice treated with daily i.p.injections of TSU-68 (75 mg/kg/d in 50l of DMSO) or the vehicle alone(50 l of DMSO). Data are presented as the mean SD of tumor volumes. Day 0: the day before treatment, days 1-14: treatment days, day 15: 1 day after treatment. (B) Changes in the body weight of TSU-68- and vehicle-treated mice. (C) Image of TSU-68- and vehicle-treated tumors excised on day 15.(D) Representative immuno uorescence staining of tumor sections from TSU-68-and vehicle-treated tumors using anti-mouse CD31 antibody (green). Scalebar = 1, 000 m. (E) The tumor MVD presented as the percentage of the CD31-positive area was compared between tumors from TSU-68- and vehicle-treated mice. All data presented in (A-E) are from the same set of experimental groups (n = 6 for each group).

DMSO 62 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL