Afatinib

Catalog No.S1011 Batch:S101114

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Technical Data

Formula

C24H25ClFN5O3
Molecular Weight 485.94 CAS No. 850140-72-6
Solubility (25°C)* In vitro DMSO 97 mg/mL (199.61 mM)
Ethanol 97 mg/mL (199.61 mM)
Water Insoluble
* <1 mg/ml means slightly soluble or insoluble.
* Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations.
* Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

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Biological Activity

Description Afatinib inhibits EGFR/ErbB irreversibly in vitro with IC50 of 0.5, 0.4, 10, 14, 1 nM for EGFRwt, EGFR L858R , EGFR L858R/T790M ErbB2 (HER2) and ErbB4 (HER4), respectively. Afatinib induces autophagy.
Targets
EGFR (L858R) [1]
(Cell-free assay)		 EGFR (wt) [1]
(Cell-free assay)		 ErbB4 [2]
(Cell-free assay)		 EGFR (L858R/T790M) [1]
(Cell-free assay)		 HER2 [1]
(Cell-free assay)
0.4 nM 0.5 nM 1 nM 10 nM 14 nM
In vitro BIBW2992 shows potent activity against both wild-type and mutant forms of EGFR and HER2. It is similar to Gefitinib in potency against L858R EGFR, but about 100-fold more active against the Gefitinib resistant L858R-T790M EGFR double mutant. BIBW2992 exhibits potent effects on both EGFR and HER2 phosphorylation in vivo. It compares favorably to reference compounds (such as Lapatinib et al.) in all cell types tested, such as human epidermoid carcinoma cell line A431 expressing wt EGFR, murine NIH-3T3 cells transfected with wt HER2, as well as breast cancer cell line BT-474 and gastric cancer cell line NCI-N87, which express endogenous HER2. [1]
In vivo Daily oral administration of BIBW2992 at 20 mg/kg for 25 days results in dramatic tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2%. Reduced phosphorylation of EGFR and AKT is confirmed by immunohistochemical staining of tissue sections. Therefore, like lapatinib and neratinib, BIBW2992 is a next generation tyrosine kinase inhibitor (TKI) that inhibits human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) kinases irreversibly. BIBW2992 is not only active against EGFR mutations targeted by first generation TKIs like Erlotinib or Gefitinib, but also against those insensitive to these standard therapies. [1]

Protocol (from reference)

Kinase Assay:

[1]
  • In vitro kinase activity assays

    The wild type tyrosine kinase domain of the human EGFR as well as that of the EGFR L858R/T790M double mutant are fused to Glutathione-S-transferase (GST), and extracted. Enzyme activity is then assayed in the presence of the inhibitor BIBW2992, serially diluted in 50% DMSO. A random polymer pEY (4:1) is used as substrate and biotinylated pEY (bio-pEY) is added as a tracer substrate. The kinase domain of HER2 is cloned using the baculovirus system and extracted similarly to that of EGFR kinase. Details of assays for EGFR, HER2, SRC, BIRK and VEGFR2 kinase activity are available in Supplementary information.
Cell Assay:

[1]
  • Cell lines

    NSCLC cells

  • Concentrations

    0-10 μM

  • Incubation Time

    1 hour

  • Method

    1 × 104 NSCLC cells are transferred into each well of a 96-well plate and cultured overnight in serum-free media for the EGFR phosphorylation assay. After addition of BIBW2992 on the next day, the plates are incubated at 37 °C for 1 hour. EGF-stimulation is done using 100 ng/mL for 10 min at room temperature. Cells are washed with ice cold PBS, extracted with 120 μL HEPEX buffer per well and shaken at room temperature for 1 hour. In all 2 × 104 cells per well is used for the HER2 phosphorylation assay. Streptavidin precoated plates are coated with anti-EGFR-biotin at 1:100 dilution in blocking buffer and c-erb2/HER2 oncoprotein Ab-5(Clone N24)-Biotin. Cell extracts is then transferred to the antibody-coated wells and incubated at room temperature for 1 hour. Extinction is measured at 450 nm.
Animal Study:

[1]
  • Animal Models

    Athymic NMRI-nu/nu female mice

  • Dosages

    20 mg/kg

  • Administration

    Oral administration

Customer Product Validation

Data from [Int J Proteomics, 2011, 2011, 215496]

Data from [Int J Proteomics, 2011, 2011, 215496]

, , Dr. Zhang of Tianjin Medical University

Data from [Data independently produced by , , Nature, 2016, 534(7609):647-51]

Selleck's Afatinib has been cited by 440 publications

Human fetal brain self-organizes into long-term expanding organoids [ Cell, 2024, 187(3):712-732.e38] PubMed: 38194967
Dynamic regulation of tissue fluidity controls skin repair during wound healing [ Cell, 2024, S0092-8674(24)00825-0] PubMed: 39168124
Tet methylcytosine dioxygenase 2 (TET2) deficiency elicits EGFR-TKI (tyrosine kinase inhibitors) resistance in non-small cell lung cancer [ Signal Transduct Target Ther, 2024, 9(1):65] PubMed: 38461173
Comprehensive mutational scanning of EGFR reveals TKI sensitivities of extracellular domain mutants [ Nat Commun, 2024, 15(1):2742] PubMed: 38548752
FGFR inhibition blocks NF-ĸB-dependent glucose metabolism and confers metabolic vulnerabilities in cholangiocarcinoma [ Nat Commun, 2024, 15(1):3805] PubMed: 38714664
Single cell lineage tracing reveals clonal dynamics of anti-EGFR therapy resistance in triple negative breast cancer [ Genome Med, 2024, 16(1):55] PubMed: 38605363
Epidermal growth factor receptor (EGFR) is a target of the tumor-suppressor E3 ligase FBXW7 [ Proc Natl Acad Sci U S A, 2024, 121(12):e2309902121] PubMed: 38483988
Tubuloid differentiation to model the human distal nephron and collecting duct in health and disease [ Cell Rep, 2024, 43(1):113614] PubMed: 38159278
The mTOR pathway controls phosphorylation of BRAF at T401 [ Cell Commun Signal, 2024, 22(1):428] PubMed: 39223665
A destabilizing Y891D mutation in activated EGFR impairs sensitivity to kinase inhibition [ NPJ Precis Oncol, 2024, 8(1):3] PubMed: 38182677

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SHIPPING AND STORAGE
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