Afatinib

SKU: orb1305744

Description

Afatinib

Research Area

Cardiovascular Research, Cell Biology, Pharmacology & Drug Discovery, Signal Transduction

Key Properties

• CAS Number: 439081-18-2
• MW: 485.94
• Purity: 99.96% (May vary between batches)
• Formula: C₂₄H₂₅ClFN₅O₃
• SMILES: CN(C)CC=CC(=O)Nc1cc2c(Nc3ccc(F)c(Cl)c3)ncnc2cc1O[C@H]1CCOC1
• Target: HER|||EGFR
• Solubility: DMSO:250 mg/mL (514.47 mM);H2O:< 1 mg/mL (insoluble or slightly soluble);Ethanol:12 mg/mL (24.69 mM);10% DMSO+40% PEG300+5% Tween 80+45% Saline:9 mg/mL (18.52 mM)

Bioactivity

• Target IC50:
  ERB4:1 nM (cell free)|EGFR (L858R/T790M):10 nM (cell free)|HER2:14 nM (cell free)|EGFR (WT):0.5 nM (cell free)|EGFR (L858R):0.4 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 .
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• In Vitro:
  (S)-Afatinib shows potent activity against wild-type and mutant forms of EGFR and HER2 (IC50s: 0.5, 0.4, 10, 14 nM for EGFRwt, EGFR L858R, EGFR L858R/T790M, and HER2, respectively). In the human breast cancer cell line, treatment with 100 nM (S)-Afatinib was sufficient to prevent heregulin-stimulated HER3 phosphorylation . Esophageal squamous cell carcinoma (ESCC) cell lines were sensitive to afatinib with IC50 concentrations at lower micromolar range (at 72 hour incubation: HKESC-1 = 0.002 μM, HKESC-2 = 0.002 μM, KYSE510 = 1.090 μM, SLMT-1 = 1.161 μM and EC-1 = 0.109 μM). The phosphorylation of ErbB family downstream effectors such as pAKT, pS6 and pMAPK were significantly inhibited in HKESC-2 and EC-1. Apoptosis was observed in both cell lines at 24 hours after exposure to afatinib .
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• Cell Research:
  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) .
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• Animal Research:
  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 .
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Storage & Handling

• Storage: -20°C
• Expiration Date: 12 months from date of receipt.
• Disclaimer: For research use only

Alternative Names

Afatinib, BIBW2992, BIBW 2992, BIBW-2992, HER2, HER4, EGFR (wt), EGFR (L858R/T790M), EGFR (L858R)

Compound Identifiers

PubChem CID

57519523