Pelitinib (EKB-569)

 

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

Biological Activity

Description	Pelitinib (EKB-569) is a potent irreversible EGFR inhibitor with IC50 of 38.5 nM. Phase2.
Targets	EGFR
IC50	38.5 nM [1]
In vitro	Pelitinib displays much higher inhibitory activity against EGFR, compared with the closely related c-erbB-2, as well as other kinases such as Src, Cdk4, c-Met, Raf, and MEK/ERK, with IC50 ranging from 282 nM for Src to >20 μM for Cdk4. Consistently, Pelitinib treatment significantly inhibits the autophosphorylation of EGFR but not c-Met in A431 cells. [1] Pelitinib potently inhibits the proliferation of normal human keratinocytes (NHEK), as well as A431 and MDA-468 tumor cells with IC50 of 61 nM, 125 nM, and 260 nM, respectively, while displaying little activity against MCF-7 cells with IC50 of 3.6 μM. Pelitinib inhibits EGF-induced phosphorylation of EGFR in A431 and NHEK cells with IC50 of 20-80 nM, as well as the phosphorylation of STAT3 with IC50 of 30-70 nM. Pelitinib at 75-500 nM also specifically inhibits the activation of AKT and ERK1/2, without affecting NF-κB pathway. In NHEK cells, Pelitinib also potently inhibits TGF-α mediated EGFR activation with IC50 of 56 nM, as well as activation of STAT3 and ERK1/2 with IC50 of 60 nM and 62 nM, respectively. [2]
In vivo	A single oral dose of 10 mg/kg Pelitinib potently inhibits the EGFR phosphorylation in A431 xenografts with over-expressed EGFR, by 90% within 1 hour, and by >50% after 24 hours. Administration of Pelitinib at 20 mg/kg/day inhibits tumorigenesis in APCMin/+ mice by 87%, equivalent to the effect of used with 2 times doses of EKI-785 (40 mg/kg/day), consistent with greater in vivo potency. [1] Pelitinib selectively inhibits EGFR signaling in airway epithelial cells in vivo. In the mouse model of airway epithelial remodeling that is inducible by viral infection and features a delayed but permanent switch to goblet cell metaplasia, Pelitinib treatment at 20 mg/kg/day corrects all 3 aspects of epithelial remodeling, by completely blocking the increase of ciliated cells and decrease of Clara cells, and significantly inhibiting the metaplasia of goblet cells. [3]
Features	An improved version of EKI-785.

Protocol(Only for Reference)

Kinase Assay: ^[1]

Autophosphorylation of EGFR in cells

For experiments using cells in culture, A431 cells are treated with various concentrations of Pelitinib for 2.75 hours before co-incubation with 100 ng/mL EGF for 0.25 hour. Cells are washed twice with cold phosphate-buffered saline (PBS) before adding to lysis buffer (10 mM Tris, pH 7.5, 5 mM ethylenediamine tetra-acetic acid (EDTA), 150 mM NaCl, 1% Triton X-100, 1% Sodium deoxycolate, 0.1 % SDS, 1 mM PMSF, 10 mg/mL pepstatin A, 10 mg/mL leupeptin, 20 KIU/mL aprotinin, 2 mM sodium orthovanadate, and 100 mM sodium fluoride) for 20 minutes on ice, before immunoprecipitation and SDS-PAGE-immunoblotting. For immunoprecipitation, cultured cells are placed in cold lysis buffer and immediately homogenized on ice with a polytron with several pulses. The homogenate is first centrifuged at 2500 rpm (20 minutes, 4 °C) and then again at 14,000 rpm in a microcentrifuge (10 minutes, 4 °C). Supernatants (1000 μg protein) are incubated for 2 hours at 4 °C with 15 mL of EGFR polyclonal antibody. After 2 hours, 50 μL of protein G plus/protein A agarose beads is added and incubated with constant rotation for 2 hours at 4 °C. After washing with lysis buffer, beads are boiled for 2 minutes in Laemmli sample buffer. Proteins are then resolved by SDS-PAGE, transferred to immobilon membrane and probed overnight with an anti-phosphotyrosine antibody conjugated with horseradish peroxidase (HRP). Membranes are developed using the ECL reagent. Total EGFR protein is determined by stripping membranes and re-probing with receptor-specific antibodies. Quantitation of bands is done by densitometry, using ImageQuant software with a Molecular Dynamics laser transmittance scanner.

Cell Assay: ^[2]

Cell lines	NHEK, A431, MCF-7, and MDA-468
Concentrations	Dissolved in DMSO, final concentrations ~10 μM
Incubation Time	5 days
Method	Cells are seeded in 96-well dishes, and after 2 hours, Pelitinib is added and incubated for 5 days. After incubation, the medium is removed from each well and fresh medium (150 μL) + 1 mg/mL MTT solution (50 μL) is added. After incubation for 2 hours at 37 °C, the medium is replaced with 150 μL DMSO, and absorbance at 540 nm in each well is determined. The IC50 is calculated by linear regression of the data.

Animal Study: ^[1]

Animal Models	Athymic nu/nu female mice bearing subcutaneous A431 tumors, or APCMin/+ male mice, a murine model of human familial adenomatous polyposis (FAP)
Dosages	10, or 20 mg/kg/day
Administration	Oral gavage
Solubility	30% PEG400/0.5% Tween80/5% propylene glycol, 30 mg/mL

1

References

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

NCT Number	Recruitment	Conditions	Sponsor /Collaborators	Start Date	Phases
NCT00067548	Completed	Non-Small-Cell Lung Carcinoma|Carcinoma, Non-Small Cell Lung|Lung Neoplasms	Wyeth is now a wholly owned subsidiary of Pfizer		Phase 2
NCT00072748	Completed	Colorectal Neoplasms|Colonic Neoplasms|Rectal Neoplasms	Wyeth is now a wholly owned subsidiary of Pfizer		Phase 2
NCT00098501	Completed	Unspecified Adult Solid Tumor, Protocol Specific	National Cancer Institute (NCI)	2004-10	Phase 1

Chemical Information

Download Pelitinib (EKB-569) SDF

Chemical Name	(E)-N-(4-(3-chloro-4-fluorophenylamino)-3-cyano-7-ethoxyquinolin-6-yl)-4-(dimethylamino)but-2-enamide

Preparing Stock Solutions

Stock Solution (1ml DMSO)	1mM	10mM	20mM	30mM
Mass(mg)	0.46792	4.6792	9.3584	-

Customer Reviews (1)

Click to enlarge	Rating
	Source	J Immunol, 2012, 188(9), 4581-4589. Pelitinib (EKB-569) purchased from Selleck
	Method	Western blot
	Cell Lines	RCH-ACV cell
	Concentrations	1 μM
	Incubation Time
	Results	Pretreatment with EGFR kinase inhibitors, PD153035 and EKB-569, a potent irreversible inhibitor, abolished the IGF-1- and IL-1β-mediated increase in Bcl-2mRNA levels.

Product Citations (1)

• 
  

  Intracellular Insulin-like Growth Factor-1 Induces Bcl-2 Expression in Airway Epithelial Cells. [Chand HS, et al. J Immunol 2012;188(9):4581-9]

  
  PubMed: 22461702