OSI-930

 

Distributor Tel: +886-2-2827-1197jimmy@new.abscience.com.tw

Biological Activity

Description	OSI-930 is a potent inhibitor of Kit, KDR and CSF-1R with IC50 of 80 nM, 9 nM and 15 nM, respectively; also potent to Flt-1, c-Raf and Lck and low activity against PDGFRα/β, Flt-3 and Abl. Phase 1.
Targets	Kit (activated)	KDR	Flt1	CSF-1R	c-Raf	Lck
IC50	80 nM	9 nM	8 nM	15 nM	41 nM	22 nM [1]
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 Ki 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]
Features

Protocol(Only for Reference)

Kinase Assay: ^[1]

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 Km 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 Assay: ^[1]

Cell lines	HMC-1 and COLO-205
Concentrations	0--1 μM
Incubation Time	48 hours
Method	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 Study: ^[1]

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.
Dosages	≤200 mg/kg
Administration	Administered via p.o.
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol, 5 mg/mL

1

References

Clinical Trial Information( data from http://clinicaltrials.gov)

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00513851	Completed	Advanced Solid Tumors	Astellas Pharma Inc|OSI Pharmaceuticals	2006-04	Phase 1
NCT00603356	Completed	Advanced Solid Tumors	Astellas Pharma Inc|OSI Pharmaceuticals	2007-11	Phase 1

Chemical Information

Download OSI-930 SDF

Chemical Name

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.44344	4.4344	8.8688	13.3032

Customer Reviews (2)

Click to enlarge	Rating
	Source	OSI-930 purchased from Selleck
	Method
	Cell Lines	HEK293-pcDNA3.1/HEK293-ABCG2-482-R2 cells
	Concentrations	10 µM
	Incubation Time
	Results	Table 2. shows the reversal effect of OSI-930 and Fumitremorgin C (FTC) on the cytotoxicity of mitoxantrone to HEK293/pcDNA3.1 and HEK293-ABCG2-482-R2.

Product Citations (2)

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  OSI-930 analogues as novel reversal agents for ABCG2-mediated multidrug resistance. [Kuang Y, et al. Biochem Pharmacol 2012;84(6):766-74]

  
  PubMed: 22750060


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  c-KIT signaling is targeted by pathogenic Yersinia to suppress the host immune response. [Micheva-Viteva SN, et al. BMC Microbiol 2013;13(1):249]

  
  PubMed: 24206648