Tesevatinib (XL-647)

Cell experiment:

Growth inhibition of H1975 and A431 cells by increasing concentrations of Tesevatinib (XL-647), gefitinib, or erlotinib is determined by seeding 5000 cells per well in 96-well plates. The following day, cells are washed once with low-serum RPMI 1640 (0.1% fetal bovine serum, 1% nonessential amino acids, and 1% penicillin/streptomycin), after which 90 μL of the low-serum RPMI 1640 are added. Test compounds (Tesevatinib (XL-647)) are diluted to 10 times the test concentrations and 10 μL are added to triplicate wells for a 72-h incubation. Cell viability is determined[1].

Animal experiment:

Mice: Tumor-bearing mice are given either Tesevatinib (XL-647), erlotinib, or gefitinib at 100 mg/kg and tumors are harvested 1 to 72 h later. Half an hour before respective time point, EGF (50 μg/mouse) is given via i.v. bolus injection with tumors dissected 30 min later and tumor extracts are prepared by homogenization in 10 volumes of ice-cold lysis buffer. Lysates are clarified by centrifugation and EGFR tyrosine phosphorylation levels are determined by ELISA[1].

References:

[1]. Gendreau SB, et al. Inhibition of the T790M gatekeeper mutant of the epidermal growth factor receptor by EXEL-7647. Clin Cancer Res. 2007 Jun 15;13(12):3713-23.