Pelitinib (EKB-569)

Oral gavage

NHEK, A431, MCF-7, and MDA-468

A Phase II study of Pelitinib in subjects with advanced non-small cell lung cancer has been completed.

Pelitinib (EKB-569) is a potent EGFR inhibitor with IC50 of 38.5 nM.

More improved than EKI-785.

38.5 nM [1], 38.5 nM [1], 38.5 nM [1], 38.5 nM [1], 38.5 nM [1], 38.5 nM [1]

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]

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 ug protein) are incubated for 2 hours at 4 C with 15 mL of EGFR polyclonal antibody. After 2 hours, 50 uL 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.

467, 92

DMSO 13 mg/mL, Water <1 mg/mL, Ethanol <1 mg/mL