Molecule

Description

Solid tumours

Description

OSI-930 is a potent inhibitor of Kit, KDR, Flt, CSF-1R, c-Raf and Lck with IC50 of 80 nM, 9 nM, 8 nM, 15 nM, 41 nM and 22 nM, respectively.

Targets

IC₅₀

In Vitro

OSI-930 inhibits the cell proliferation in the HMC-1 cell line with IC50 of 14 nM without significant effect on growth of the COLO-205 cell line that does not express a constitutively active mutant receptor tyrosine kinase. Moreover, OSI-930 also induces apoptosis in HMC-1 cell line with EC50 of 34 nM. ^[1] A recent study shows that OSI-930 inactivates purified, recombinant cytochrome P450 (P450) 3A4 with a K_i of 24 μM in a time- and concentration-dependent mode. ^[2]

In Vivo

OSI-930, administrated at the maximally efficacious dose of 200 mg/kg by oral gavage, exhibits potent antitumor activity in a broad range of preclinical xenograft models including HMC-1, NCI-SNU-5, COLO-205 and U251 xenograft models. ^[1]

Clinical Trials

OSI-930 is currently in Phase I clinical trials in patients with Advanced Solid Tumors.

Features

Description

Combination of OSI-930 and Erlotinib is currently in Phase I clinical trials in patients with Advanced Solid Tumors.

Protein kinase assays

Protein kinase assays are either done in-house by ELISA-based assay methods (Kit, KDR, PDGFRα, and PDGFRβ) or by a radiometric method. In-house ELISA assays used poly(Glu:Tyr) as the substrate bound to the surface of 96-well assay plates; phosphorylation is then detected using an antiphosphotyrosine antibody conjugated to HRP. The bound antibody is then quantitated using ABTS as the peroxidase substrate by measuring the absorbance at 405/490 nm. All assays uses purified recombinant kinase catalytic domains that are either expressed in insect cells or in bacteria. The Kit and EGFR protein used for in-house assays are prepared internally; other enzymes are obtained. Recombinant Kit protein is expressed as an NH₂-terminal glutathione S-transferase fusion protein in insect cells and is initially purified as a nonphosphorylated (nonactivated) enzyme with a relatively high K_m for ATP (400 μM). In some assays, an activated (tyrosine phosphorylated) form of the enzyme is prepared by incubation with 1 mM ATP for 1 hour at 30 °C. The phosphorylated protein is then passed through a desalting column to remove the majority of the ATP and stored at ?80 °C in buffer containing 50% glycerol. The resultant preparation has a considerably higher specific activity and a lower Km for ATP (25 μM) than the initial nonphosphorylated preparation. The inhibition of Kit autophosphorylation by OSI-930 is assayed by incubation of the nonphosphorylated enzyme at 30 °C in the presence of 200 μM ATP and various concentrations of OSI-930. The reaction is stopped by removal of aliquots into SDS-PAGE sample buffer followed by heating to 100 °C for 5 minutes. The degree of phosphorylation of Kit is then determined by immunoblotting for both total Kit and phosphorylated Kit.

Cell Lines

HMC-1 and COLO-205

Concentrations

0--1 μM

Incubation Time

48 hours

Methods

For assays of cell proliferation and apoptosis, cells are seeded into 96-well plates and incubated for 2 to 3 days in the presence of OSI-930 at various concentrations. Inhibition of cell growth is determined by luminescent quantitation of the intracellular ATP content using CellTiterGlo. Induction of caspase-dependent apoptosis by OSI-930 is quantitated by an enzymatic caspase 3/7 assay. Inhibition of angiogenesis by OSI-930 is monitored using the rat aortic ring endothelial sprout outgrowth assay. Sections of aorta are prepared from CO₂-euthanized male rats and cultured in vitro in a collagen matrix in the presence or absence of OSI-930. The collagen matrix is prepared from type 1 rat tail collagen solubilized in 0.1% acetic acid at 3 mg/mL, which is combined with 0.125 volume collagen buffer (0.05 N NaOH, 200 mM HEPES, 260 mM NaHCO₃), 0.125 volume of medium 199, 0.0125 volume of 1 M NaOH, and 1% GlutaMax. Aortic rings are embedded in 0.4 mL of this matrix in six-well plates, to which 0.5 mL endothelial basal medium and the appropriate amount of OSI-930 is added; the rings are then incubated for 10 days and the resultant angiogenic sprout outgrowth is digitally quantitated from images by measurement of the sprout-containing area within a series of concentric rings around the aortic tissue area.

Animal Models

HMC-1, NCI-SNU-5, COLO-205 and U251 cells are injected s.c. into the right flank of CD1 nu/nu mice.

Formulation

OSI-930 is dissolved in either Labrafil M 1944 CS or polyethylene glycol (PEG)-400/water (50:50).

Doses

≤200 mg/kg

Administration

Administered via p.o.