Sunitinib malate

Female nu/nu mice implanted s.c. with HT-29, A431, Colo205, H-460, SF763T, C6, A375, or MDA-MB-435, and male nu/nu mice bearing luciferase-expressing PC-3M tumors

(Z)-N-(2-(diethylamino)ethyl)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2, 4-dimethyl-1H-pyrrole-3-carboxamide , (S)-2-hydroxysuccinic acid

Dissolved in DMSO, final concentrations ca.10 uM

ca.80 mg/kg

80 nM, 80 nM, 80 nM, 80 nM, 80 nM, 80 nM

Consistent with the substantial and selective inhibition of VEGFR2 or PDGFR phosphorylation and signaling in vivo, Sunitinib (20-80 mg/kg/day) exhibits broad and potent dose-dependent anti-tumor activity against a variety of tumor xenograft models including HT-29, A431, Colo205, H-460, SF763T, C6, A375, or MDA-MB-435. Sunitinib dosing at 80 mg/kg/day for 21 days leads to complete tumor regression in six of eight mice, without tumor re-growing during a 110-day observation period after the end of treatment. Second round of treatment with Sunitinib remains efficacious against tumors that are not fully regressed during the first round of treatment. Sunitinib treatment results in significant decrease in tumor MVD, with ca.40% reduction in SF763T glioma tumors. SU11248 treatment results in a complete inhibition of additional tumor growth of luciferase-expressing PC-3M xenografts, despite no reduction in tumor size. [2] Sunitinib treatment (20 mg/kg/day) dramatically suppresses the growth subcutaneous MV4, 11 (FLT3-ITD) xenografts and prolongs survival in the FLT3-ITD bone marrow engraftment model. [3]

Biochemical Tyrosine Kinase Assays, IC50 values for Sunitinib against VEGFR2 (Flk-1) and PDGFRbeta are determined using glutathione S-transferasefusion proteins containing the complete cytoplasmic domain of the RTK. Biochemical tyrosine kinase assays to quantitate the trans-phosphorylation activity of VEGFR2 (Flk-1) and PDGFRbeta 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 the addition of 1-5% (w/v) BSA in PBS. Purified GST-fusion proteins are produced in baculovirus-infected insect cells. GST-VEGFR2 and GST-PDGFRbeta are then added to the microtiter wells in 2, 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-VEGFR2 or GST-PDGFRbeta is 50 ng/mL. Twenty-five uL of diluted Sunitinib are subsequently added to each reaction well to produce a range of inhibitor concentrations appropriate for each enzyme. 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 concentration of MnCl2 is 10 mM. The plates are incubated for 5-15 minutes 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 1:10, 000 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 antirabbit antisera conjugated with horseradish peroxidase (1:10, 000 dilution in TBST). 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.

532, 56

Data from [Biochem Bioph Res Co , 2010, 398, 205211], Sunitinib Malate (Sutent)purchased from Selleck, Sunitinib limits the colonial growth of HT-29 by downregulating HIF-1a. (A) The number and size of colonies formed in soft agar. The numbers of small colonies (50 m diameter) were not different among conditions of a serial concentration of sunitinib. On the contrary, large colonies (50 m diameter) disappeared after incubation with sunitinib. Each point represents the mean and SD from four separate experiments. (B) HIF-1a expression and hypoxia within HT-29 colony. After colonies grew for 4 weeks, HIF-1a and hypoxia were visualized by immunofluoroscence staining. Bar = 20 m.

DMSO 15 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL