-20℃ 3 years powder
-80℃ 2 years in solvent
Afatinib (BIBW 2992) is an irreversible EGFR family inhibitor with IC50s of 0.5/0.4/10/14/1 nM for EGFRwt, EGFR (L858R), EGFR (L858R/T790M), HER2, and HER4, respectively.
|2 mg||In stock||50.00|
|5 mg||In stock||72.00|
|10 mg||In stock||139.00|
|25 mg||In stock||231.00|
|50 mg||In stock||381.00|
|100 mg||In stock||605.00|
|1 mL * 10 mM (in DMSO)||In stock||142.00|
|Description||Afatinib (BIBW 2992) is an irreversible EGFR family inhibitor with IC50s of 0.5/0.4/10/14/1 nM for EGFRwt, EGFR (L858R), EGFR (L858R/T790M), HER2, and HER4, respectively.|
|Targets&IC50||EGFR (L858R) :ic50 0.4 nM (cell free), EGFR (L858R/T790M) :ic50 10 nM (cell free), EGFR (wt) :ic50 0.5 nM (cell free), HER2 :ic50 14 nM (cell free), ErbB4 :ic50 1 nM (cell free),|
|In vivo||Daily oral treatment with BIBW2992 at 20 mg/kg for 25 days resulted in dramatic tumor regression and downregulation of EGFR and AKT phosphorylation. Xenograft tumor formation by the NCIH1975 cell line, expressing EGFR L858R/T790M, was effectively controlled by BIBW2992, with a T/C value of 12% for doses of 20 mg/kg . Afatinib could effectively inhibit HKESC-2 tumor growth in mice without obvious toxicity. Afatinib alone has shown excellent growth inhibitory effect on ESCC in in vivo models .|
|Kinase Assay||The wild type tyrosine kinase domain of the human EGFR, as well as the EGFR L858R/T790M double mutant, were fused to Glutathione-S-transferase (GST) and extracted as described in Supplementary methods. The L858R mutant was purchased from Upstate. Enzyme activity was then assayed in the presence or absence of serial inhibitor dilutions performed in 50% Me2SO. A random polymer pEY (4:1) from Sigma was used as substrate. Biotinylated pEY was added as a tracer substrate. The kinase domain of HER2 was cloned using baculovirus system and extracted similarly to that of EGFR kinase domain. Detailed procedures for EGFR, HER2, SRC, BIRK and VEGFR2 kinase activity assays are included in Supplementary information .|
Cells (1×10^4) were transferred into each well of a 96-well plate and cultured over night in serum-free media for EGFR phosphorylation assay. After addition of test compounds on the next day, the plates were then incubated at 37°C for 1 hour. EGF-stimulation was done at 100 ng/ml for 10 min at room temperature. Cells were washed with ice cold PBS before extraction with 120 μl per well HEPEX buffer and shaken for 1 h at room temperature. In all 2×10^4 cells per well was used for HER2 phosphorylation assay. Streptavidin precoated plates were coated with anti-EGFR-biotin at 1:100 dilution with blocking buffer and c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin. Extracts from above steps were then transferred to the antibody-coated wells and incubated for 1 h at room temperature. Assessment of color development is described in Supplementary information. Extinction was measured at 450 nm. The data generated were analysed by the program PRISM. Normalized values were used to calculate the IC50 by a nonlinear regression curve fit (variable slope) .
Cell lines: NSCLC cells
Six weeks old female athymic nude mice (nu/nu) weighing about 16-20 gram were housed by Laboratory Animal Services Centre of The Chinese University of Hong Kong. The experiment was conducted by researchers under license from the Hong Kong Government Department of Health and according to approval given by Animal Experimentation Ethics Committee of the Chinese University of Hong Kong. ESCC xenografts were established by inoculating HKESC-2 (0.6 × 10^5 cells re-suspended in 50 μl of HBSS-buffer) subcutaneously into both flanks of the nude mice. When tumor size reached to 4-6 mm diameter, they were randomized in either treatment (15 mg/kg) or vehicle control group. Afatinib for treatment was prepared by dissolving in 0.5% methylcellulose before administration. Either drug or vehicle was administered to mouse by oral gavage in a schedule of 5 days on plus 2 days off for two weeks. Drug efficacy was evaluated by monitoring the change in tumor size with caliper. Tumor volume was calculated with the formula Tumor Volume = (width2 × length)/2 .
Animal Model: Athymic NMRI-nu/nu femice
-20℃ 3 years powder
-80℃ 2 years in solvent
DMSO: 90 mg/mL (185.2 mM)
Ethanol: 12 mg/mL (24.7 mM)
Water: <1 mg/mL
( < 1 mg/ml refers to the product slightly soluble or insoluble )
0.5% methylcellulose+0.2% Tween 80: 30 mg/mL
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