Molecule

Description

Cancer

Description

Tie2 kinase inhibitor is a potent and selective Tie2 inhibitor with IC50 of 0.25 μM.

Targets

IC₅₀

In Vitro

Tie2 kinase inhibitor exhibits a moderate inhibitory activity against Tie2 tyrosine kinase. Tie2 kinase inhibitor also shows moderate cellular activities with IC50 of 232 nM in HEL cells. Furthermore, Tie2 kinase inhibitor has a remarkable selectivity for Tie2 over p38 (IC50=50 μM) and a >10-fold selectivity over VEGFR2, VEGFR3, and PDGFR1β. ^[1]

In Vivo

In a Matrigel mouse model of angiogenesis, Tie2 kinase inhibitor, at doses of 25 and 50 mg/kg (i.p., b.i.d), leads to a reduction of 41% and 70% of angiogenesis, respectively. In a MOPC-315 plasmacytoma xenograft model, Tie2 kinase inhibitor treatment results in a modest dose dependent delay in tumor growth. ^[1]

Clinical Trials

Features

Tie2 kinase activity assay

Turn on an incubator shaker and adjust temperature to 30 °C. Add 20 μL of 3× kinase buffer (final 20 mM Tris-HCl, pH 7, 100 mM NaCl, 12 mM MgCl₂, 1 mM DTT) per well to the Flashplate. Add 20 μL of protein per well except for background. Add Tie2 kinase inhibitor, typically 1-2 μL in DMSO stocks. Add 20 μL of a mixture of gamma ³³p-ATP and cold ATP(1:1 v/v ) per well. Cover with transparent polyester film. Incubate at 30 °C for 2 hours in shaker, wash five times. Read plate on TopCount or other counting instrument, results are calculated as IC50 using normal methods.

Tie2 receptor signal transduction assay

HEL cells are cultured at between 1 and 5 × l0⁵ mL in RPMI-1640 medium supplemented with 2 mM glutamine and 10% FBS as a suspension culture. Sixteen to thirty-six hours prior to an experiment, the necessary number of cells are passaged into 0.5% FBS/ RPMI medium. On the day of an experiment, cells are harvested and resuspended at a density of 0.5-1.0 × l0⁷ cells mL in 0.5% FBS RPMI and seeded at 2-3 mL/well in six well plates. Alternatively, Human Umbilical Vein Endothelial Cells (HUVECs) may be used for the assay. HUVECs between passages 2 and 12, are plated at 2 × l0⁵ and 1 × l0⁶ cells per well in a six well plate in supplemented EGM. After 24 hours the media is changed to EBM containing 3% BSA, and the cells are cultured overnight and used for assay the following day. Cells are treated with inhibitory compounds at appropriate concentrations for 30-45 minutes. The contents of the wells are mixed briefly on a rocker (approx 30 seconds) and then incubated at 37 °C. The cells are then treated with a source of native ligand, such as serum or fibroblast conditioned medium for 10 minutes. At the end of 10 minute incubation period the plate is placed on ice. The cells are harvested and the media is removed. The cells are lysed in denaturing sample buffer. The suspension is sonicated for 5 pulses at a medium setting and returned to ice. The phosphorylation state of the Tie2 receptor is determined by Western blotting and detection by an anti-phospho-Tie2 antibody. Thirty μL of the lysate are run on a 7% SDS/polyacrylamide gel. The gel is then transferred to a nitrocellulose or PVDF membrane for Western blotting. The blots are washed with PBS/0.05% Tween-20 and then blocked with 3% BSA/PBS/Tween for 1 hour at room temperature. The blots are then incubated with 1 μg/mL anti-phospho-Tie2 antibody in PBS/0.05% Tween for 1 hour. The blot is then washed 4 times with PBS/Tween for 5 minutes each. The blot is incubated with an anti-mouse-HRP conjugate secondary antibody at the dilution recommended by the manufacturer, in PBS/Tween for 1 hour. The blot is washed in PBS/Tween, 4 times for 5 minutes each. After the last wash, the blot is developed by the ECL method or some equivalent. Using a densitometer or graphics program, each blot is scanned. The Tie-2 band is isolated and "boxed out" for each lane. The pixel volume or comparable measure for each sample is analyzed. Also, an appropriate background region of the same dimensions is determined for each sample. After adjusting for background, phosphorylation is expressed as the ratio of phosphotyrosine staining, relative to the untreated control. Reduced phosphorylation indicates inhibition of tie-2 kinase.

Animal Models

MOPC-315 plasmacytoma xenograft model.

Formulation

Tie2 kinase inhibitor is dissolved in 5% EtOH+5%cremophor+90% water.

Doses

≤50 mg

Administration

Administered via i.p.