1. JAK/STAT Signaling
    Protein Tyrosine Kinase/RTK
    Autophagy
  2. EGFR
    Autophagy

Afatinib (Synonyms: BIBW-2992; BIBW2992; BIBW 2992)

Cat. No.: HY-10261 Purity: 99.58%
Data Sheet SDS Handling Instructions

Afatinib is an irreversible, dual EGFR/HER2 inhibitor, shows potent activity against wild-type and mutant forms of EGFR and HER2, with IC50 of 0.5 nM, 0.4 nM, 10 nM and 14 nM for EGFRwt, EGFRL858R, EGFRL858R/T790M and HER2, respectively.

For research use only. We do not sell to patients.
Afatinib Chemical Structure

Afatinib Chemical Structure

CAS No. : 850140-72-6

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Free Sample (0.5-1 mg)   Apply now  
10 mM * 1 mL in DMSO $55 In-stock
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Other Forms of Afatinib:

Top Publications Citing Use of Products

    Afatinib purchased from MCE. Usage Cited in: Oncotarget. 2015 Oct 13;6(31):31313-22.

    Effect of Ibrutinib on EGFR wt/mutant NSCLCs. Ibrutinib effects on wt EGFR and mutant EGFR- mediated signaling pathways. The results demonstrate that Ibrutinib potently inhibits both EGFR wt/mutant auto-phosphorylation at Y1068.

    Afatinib purchased from MCE. Usage Cited in: Oncotarget. 2015 Oct 13;6(31):31313-22.

    Effect of Ibrutinib, WZ4002, AZD9291 and CO1686 on EGFR phosphorylation of tyrosines 1068 and 1173 in EGFR-dependent cancer cell lines.

    Afatinib purchased from MCE. Usage Cited in: Oncotarget. 2014 Dec 15;5(23):11971-85.

    H460/MX20 cells are treated with varying concentrations (0–2.0 μM) of Afatinib for 48 h, or with 1.0 μM Afatinib for 24 h, 48 h and 72 h, respectively. ABCG2 protein levels are analyzed by Western blot. GAPDH is used as a loading control.
    • Biological Activity

    • Protocol

    • Technical Information

    • Purity & Documentation

    • References

    Description

    Afatinib is an irreversible, dual EGFR/HER2 inhibitor, shows potent activity against wild-type and mutant forms of EGFR and HER2, with IC50 of 0.5 nM, 0.4 nM, 10 nM and 14 nM for EGFRwt, EGFRL858R, EGFRL858R/T790M and HER2, respectively.

    IC50 & Target

    IC50: 0.5 nM (EGFRwt), 0.4 nM (EGFRL858R), 10 nM (EGFRL858R/T790M), 14 nM (HER2)[1]

    In Vitro

    In cell-free in vitro kinase assays, Afatinib (BIBW2992) dimaleate shows potent activity against wild-type and mutant forms of EGFR and HER2, similar to Gefitinib in potency for L858R EGFR, but about 100-fold more active against the Gefitinib-resistant L858R-T790M EGFR double mutant, with an IC50 of 10 nM. BIBW2992 is furthermore comparable to Lapatinib and Canertinib for in vitro potency against HER2, with an IC50 of 14 nM. The most sensitive kinase in this evaluation is lyn with an IC50 of 736 nM[1]. Afatinib is an irreversible inhibitor of these ErbB family receptors. Esophageal squamous cell carcinoma (ESCC) cell lines are sensitive to Afatinib with IC50 concentrations at lower micro-molar range (at 48 hour incubation: HKESC-1=78 nM, HKESC-2=115 nM, KYSE510=3.182 μM, SLMT-1=4.625 μM and EC-1=1.489 μM; and at 72 hour incubation: HKESC-1=2 nM, HKESC-2=2 nM, KYSE510=1.090 μM, SLMT-1=1.161 μM and EC-1=109 nM) with a maximum growth inhibition over 95%. Afatinib can strongly induce G0/G1 cell cycle arrest in HKESC-2 and EC-1 in a dose- and time-dependent manner[2].

    In Vivo

    Afatinib (15 mg/kg) strongly inhibits the growth of HKESC-2 tumor once the treatment began. Average tumor sizes of vehicle and treatment at end point are 348±24 mm3 and 108±36 mm3 respectively, showing significantly difference between them. And apparently tumor size does not bounce back in a short period of time after the end of Afatinib administration. Without rapid change of body weight throughout the treatment shows that the toxicity of Afatinib is minimal and this drug is well tolerated[2].

    References
    Preparing Stock Solutions
    Concentration Volume (DMSO) Mass 1 mg 5 mg 10 mg
    1 mM 2.0579 mL 10.2893 mL 20.5787 mL
    5 mM 0.4116 mL 2.0579 mL 4.1157 mL
    10 mM 0.2058 mL 1.0289 mL 2.0579 mL
    Kinase Assay
    [1]

    The EGFR kinase domain-GST fusion proteins are extracted from Sf9 biomasses, 72 hours post infection, with HEPEX (20 mM HEPES pH 7.4, 100 mM NaCl, 10 mM β-glycerophosphate, 10 mM para-nitro-phenylphosphate, 30 mM NaF, 5 mM EDTA, 5% glycerol, 1% Triton X-100, 1 mM Na3VO4, 0.1% SDS, 0.5 µg/mL pepstatin A, aprotinin 20 KIU/mL, Leupeptin 2 µg/mL, Benzamidine 1 mM, 2.5 μg/mL 3,4-dichloroisocoumarin, 2.5 μg/mL trans-epoxysuccinyl-L-leucyl-L-amido butane and 0.002% PMSF) and used for the determination of the IC50 values. Each 100 µL enzyme reaction contains 10 μL of Afatinib (BIBW2992) in 50 % Me2SO, 20 μL of substrate solution (200 mM HEPES pH 7.4, 50 mM Mg-acetate, 2.5 mg/mL poly (EY), 5 μg/mL bio-pEY) and 20 µL enzyme preparation. The enzymatic reaction is started by addition of 50 µL of a 100 µM ATP solution made in 10 mM MgCl2. Assays are carried out at room temperature for 30 minutes and terminated by the addition of 50 µL of stop solution (250 mM EDTA in 20 mM HEPES pH 7.4). 100 µL are transferred to a streptavidin coated microtiterplate, after an incubation time of 60 min at room temperature the plate is washed with 200 µL of wash solution (50 mM Tris, 0.05% Tween20). A 100 µL aliquot of a HRPO- labeled anti-PY antibody (PY20H Anti-Ptyr:HRP) 250 ng/mL are added to the wells. After 60 min of incubation, the plate is washed three times with a 200 µL wash solution. The samples are then developed with a 100 µL TMB Peroxidase Solution (A:B=1:1). The reaction is stopped after 10 min. The plate is transferred to an ELISA reader and extinction is measured at OD450nm. All data points are performed in triplicates[1]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Cell Assay
    [2]

    Afatinib is dissolved in DMSO (100 mM) and stored (-80°C), and then diluted in corresponding medium just before addition to cell cultures[2].

    Human ESCC cell lines, EC-1, HKESC1 and HKESC2, SLMT1, and KYSE510 are cultured in RPMI with 10% fetal bovine serum (FBS). Cytotoxicity is assessed by a colorimetric assay using MTT. Tumour cells are cultured in 48-well plates (3000-8000 cells per well) in respective culture medium. Afatinib in complete medium is added at 24 hr after cell plating and incubated at 37°C with 5% CO2 for 48 and 72 hr. Cell growth inhibition is expressed as the percentage of absorbance of control cultures measured at 570 nm with a microplate reader and 50% of the maximum growth inhibition (IC50) is calculated by GraphPad PRISM. In each experiment, triplicate wells are performed for each drug concentration (n=3), and assay is repeated in three independent experiments[2]. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Animal Administration
    [2]

    Afatinib is dissolved in 0.5% methylcellulose (Mice)[2].

    Mice[2]
    Six weeks old female athymic nude mice (nu/nu) weighing about 16-20 gram are used. ESCC xenografts are established by inoculating HKESC-2 (6×104 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 are randomized in either treatment (15 mg/kg) or vehicle control group. Afatinib for treatment is prepared by dissolving in 0.5% methylcellulose before administration. Either drug or vehicle is administered to mouse by oral gavage in a schedule of 5 days on plus 2 days off for two weeks. Drug efficacy is evaluated by monitoring the change in tumor size with caliper. Tumor volume is calculated with the formula Tumor Volume=(width2×length)/2. MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    References
    M.Wt

    485.94

    Formula

    C₂₄H₂₅ClFN₅O₃

    CAS No.

    850140-72-6

    Storage
    Powder -20°C 3 years
      4°C 2 years
    In solvent -80°C 6 months
      -20°C 1 month
    Shipping

    Room temperature in continental US; may vary elsewhere

    Solvent & Solubility

    10 mM in DMSO

    * "<1 mg/mL" means slightly soluble or insoluble. "≥" means soluble, but saturation unknown.

    Purity: 99.58%

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    Afatinib
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