2. JAK/STAT Signaling Protein Tyrosine Kinase/RTK Autophagy Apoptosis PI3K/Akt/mTOR MAPK/ERK Pathway
  3. EGFR Autophagy Apoptosis c-Met/HGFR Akt p38 MAPK
  4. Afatinib

Afatinib (BIBW 2992) is an orally active, potent and irreversible dual specificity inhibitor of ErbB family (EGFR and HER2), with IC50 values of 0.5 nM, 0.4 nM, 10 nM and 14 nM for EGFRwt, EGFRL858R, EGFRL858R/T790M and HER2, respectively. Afatinib can be used for the research of esophageal squamous cell carcinoma (ESCC), non-small cell lung cancer (NSCLC) and gastric cancer.

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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Solid + Solvent (Highly Recommended)
10 mM * 1 mL in DMSO
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Based on 64 publication(s) in Google Scholar

Other Forms of Afatinib:

Afatinib Related Antibodies

Top Publications Citing Use of Products

60 Publications Citing Use of MCE Afatinib

WB

    Afatinib purchased from MedChemExpress. Usage Cited in: Cancer Sci. 2018 Apr;109(4):1166-1176.  [Abstract]

    Influence of Afatinib or Neratinib on human epidermal growth factor receptor 2 (HER2) and the downsignal pathway in gastric cancer cell lines.

    Afatinib purchased from MedChemExpress. Usage Cited in: Mol Cancer Ther. 2018 Mar;17(3):603-613.  [Abstract]

    Immunoblot analysis of U-CH1, MUG-Chor1 and Chor-IN-1 cells treated with Afatinib for 2 h or 48 h. Protein cell extracts are resolved on SDS-PAGE gel and membranes probed with the indicated antibodies. IC50s for each cell line are reported.

    Afatinib purchased from MedChemExpress. Usage Cited in: Mol Cancer Ther. 2018 Mar;17(3):603-613.  [Abstract]

    Immunoblot analysis of U-CH1 cells treated with the indicated doses of inhibitors (Afatinib, Erlotinib and Lapatinib) for 2 h (upper panel) or 48 h (lower panel). Protein cell extracts are resolved on SDS-PAGE gel and membranes probed with the indicated antibodies. IC50s of the different inhibitors are reported.

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

    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.

    View All EGFR Isoform Specific Products:

    View All Isoforms
    EGFR/ErbB1/HER1 ErbB2/HER2 ErbB3/HER3 ErbB4/HER4

    View All Akt Isoform Specific Products:

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    Akt Akt1 Akt2 Akt3

    View All p38 MAPK Isoform Specific Products:

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    p38 MAPK p38α p38β MAPK13 p38δ p38γ

    • Biological Activity

    • Purity & Documentation

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    • Customer Review

    Description

    Afatinib (BIBW 2992) is an orally active, potent and irreversible dual specificity inhibitor of ErbB family (EGFR and HER2), with IC50 values of 0.5 nM, 0.4 nM, 10 nM and 14 nM for EGFRwt, EGFRL858R, EGFRL858R/T790M and HER2, respectively. Afatinib can be used for the research of esophageal squamous cell carcinoma (ESCC), non-small cell lung cancer (NSCLC) and gastric cancer[1][2][3][4].

    IC50 & Target[1]

    EGFRL858R

    0.4 nM (IC50)

    EGFR

    0.5 nM (IC50)

    EGFRL858R/T790M

    10 nM (IC50)

    HER2

    14 nM (IC50)

    HER3

     

    In Vitro

    Afatinib (100 nM) sufficiently prevents heregulin-stimulated HER3 phosphorylation[1].
    Afatinib (0-10000 nM) effectively inhibits anchorage-independent proliferation of NIH-3T3 cells ectopically expressing EGFR mutants, and inhibits cell proliferation of H1666, H3255, and NCI 1975 cells[1].
    Afatinib (48-72 h)shows growth inhibition in HKESC-1, HKESC-2, SLMT-1 and EC-1 cells[2].
    Afatinib (0-1 μM, 24-48 h) inhibits AKT and MAPK pathways, and inhibits EGFR and AKT phosphorylation in ESCC cell lines[2].
    Afatinib (0-1 μM, 16-48 h) induces G0/G1 cell cycle arrest in HKESC-2 and EC-1[2].
    Afatinib (0-1 μM, 24-48 h) effectively induces apoptotic cell death in HKESC-2 and EC-1[2].

    MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

    Afatinib Related Antibodies

    Cell Proliferation Assay[1]

    Cell Line: NIH-3T3 cells, H1666, H3255, and NCI 1975 cells
    Concentration: 0, 1, 10, 100, 1000, 10000 nM
    Incubation Time:
    Result: Effectively inhibited anchorage-independent proliferation of NIH-3T3 cells ectopically expressing EGFR mutants. Showed inhibition of anchorage independent cell proliferation of various lung cancer cell lines (H1666, H3255, and NCI 1975 cells), with IC50 values of 60 nM, 0.7 nM and 99 nM, respectively.

    Cell Viability Assay[2]

    Cell Line: HKESC-1, HKESC-2, SLMT-1 and EC-1 cell lines
    Concentration:
    Incubation Time: 48 and 72 hours
    Result: Observed over 95% of growth inhibition. The respective IC50 concentrations at 48 hours (HKESC-1=0.078 μM, HKESC-2=0.115 μM, KYSE510=3.182 μM, SLMT-1=4.625 μM and EC-1=1.489 μM) and 72 hours (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) were all in lower micro-molar range.

    Western Blot Analysis[2]

    Cell Line: HKESC-2 cells and EC-1 cells
    Concentration: 0, 0.01, and 0.1 μM (HKESC-2 cells), 0, 0.1 and 1 μM (EC-1 cells)
    Incubation Time: 24 and 48 hours
    Result: Reduced the phosphorylation of EGFR and the endogenous expression level of HER2 receptors in ESCC cells. Suppressed AKT phosphorylation in a dose and time dependent manner. Significantly reduced the phosphorylation level of the downstream effectors of the AKT-mTOR axis especially in HKESC-2 cells. Inhibited the two major downstream pathways of the ErbB/HER axis, namely, AKT and MAPK pathways in ESCC cell lines.

    Cell Cycle Analysis[2]

    Cell Line: HKESC-2 cells and EC-1 cells
    Concentration: 0, 0.01, and 0.1 μM (HKESC-2 cells), 0, 0.1 and 1 μM (EC-1 cells)
    Incubation Time: 16, 24, and 48 hours
    Result: Induced G0/G1 cell cycle arrest in both tested ESCC cell lines in a time and dose dependent manner. In HKESC-2 cells, the percentage of cells in G0/G1 phase was increased from 38.2% to 68.1% at 0.01 μM of afatinib and to 74.7% at 0.1 μM of afatinib, from 24 hours (82.4% G0/G1 arrest at 0.01 μM and 86.2% at 0.1 μM) to 48 hours (from 74.7% to 88.2% for 0.01 μM and 91.0% for 0.1 μM). In EC-1 cells, the percentage of cells arrested in the G0/G1 phase was increased from 59.1% to 66.6% and 72.2% at 24 and 48 hours respectively.

    Apoptosis Analysis[2]

    Cell Line: HKESC-2 cells and EC-1 cells
    Concentration: 0, 0.01, and 0.1 μM (HKESC-2 cells), 0, 0.1 and 1 μM (EC-1 cells)
    Incubation Time: 24 and 48 hours
    Result: Effectively induced cell death by triggering apoptotic mechanisms in ESCC cell lines. Showed a stronger expression level of cleaved Poly (ADP-ribose) polymerase (PARP) in these cell lines.
    In Vivo

    Afatinib (0-20 mg/kg, Orally, daily for 25 days) shows dramatic tumor regression and downregulation of EGFR, HER2, HER3 and AKT phosphorylation[1].
    Afatinib (15 mg/kg, Orally, in a schedule of 5 days on plus 2 days off, for two weeks) strongly inhibits the growth of HKESC-2 tumor[2].

    MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

    Animal Model: Athymic NMRI-nu/nu female mice (21–31 g, five to six-week-old, transgenic murine lung cancer model and xenograft models)[1]
    Dosage: 15 mg/kg, 20 mg/kg
    Administration: Orally, daily for 25 days
    Result: Resulted in dramatic tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2% in a standard xenograft model of the epidermoid carcinoma cell line A431, and downregulation of EGFR and AKT phosphorylation. Induced regression of large tumors in this HER2-driven model, effectively controlled xenograft tumor formation by the NCIH1975 cell line, expressing EGFR L858R/T790M, with a T/C value of 12% for doses of 20 mg/kg. Induced more than 50% percent tumor reduction after a 4-week treatment period. Downregulated EGFR, HER2 and HER3 phosphorylation.
    Animal Model: Six weeks old female athymic nude mice (nu/nu) (16-20 g)[2]
    Dosage: 15 mg/kg
    Administration: Oral gavage in a schedule of 5 days on plus 2 days off, for two weeks
    Result: Strongly inhibited the growth of HKESC-2 tumor. Average tumor sizes of vehicle and treatment at end point are 348 ± 24 mm3 and 108 ± 36 mm3 respectively.
    Clinical Trial
    Molecular Weight

    485.94

    Formula

    C24H25ClFN5O3
    CAS No.
    Appearance

    Solid

    Color

    White to yellow

    SMILES

    O=C(NC1=C(C=C2C(C(NC3=CC(Cl)=C(C=C3)F)=NC=N2)=C1)O[C@H]4CCOC4)/C=C/CN(C)C
    Shipping

    Room temperature in continental US; may vary elsewhere.
    Storage
    Powder -20°C 3 years
    4°C 2 years
    In solvent -80°C 2 years
    -20°C 1 year
    Solvent & Solubility
    In Vitro: 

    DMSO : 100 mg/mL (205.79 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

    Preparing
    Stock Solutions
    Concentration Solvent 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
    View the Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

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    Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

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    In Vivo:

    Select the appropriate dissolution method based on your experimental animal and administration route.

    For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
    To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
    The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

    • Protocol 1

      Add each solvent one by one:  10% DMSO    40% PEG300    5% Tween-80    45% Saline

      Solubility: ≥ 2.5 mg/mL (5.14 mM); Clear solution

      This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown).

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.

      Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
    • Protocol 2

      Add each solvent one by one:  10% DMSO    90% Corn Oil

      Solubility: ≥ 2.5 mg/mL (5.14 mM); Clear solution

      This protocol yields a clear solution of ≥ 2.5 mg/mL (saturation unknown). If the continuous dosing period exceeds half a month, please choose this protocol carefully.

      Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (25.0 mg/mL) to 900 μL Corn oil, and mix evenly.

    For the following dissolution methods, please prepare the working solution directly. It is recommended to prepare fresh solutions and use them promptly within a short period of time.
    The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

    • Protocol 1

      Add each solvent one by one:  0.5% Methylcellulose/saline water

      Solubility: 5 mg/mL (10.29 mM); Suspended solution; Need ultrasonic

    In Vivo Dissolution Calculator
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    Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.
    Please enter your animal formula composition:
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    Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.
    The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).
    Calculation results:
    Working solution concentration: mg/mL
    Method for preparing stock solution: mg drug dissolved in μL  DMSO (Stock solution concentration: mg/mL).
    The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).
    Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.
     If the continuous dosing period exceeds half a month, please choose this protocol carefully.
    Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.
    Purity & Documentation

    Purity: 99.92%

    COA (254 KB) LCMS (103 KB)

    Handling Instructions (2659 KB)
    References

    Complete Stock Solution Preparation Table

    * Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
    Storage method and period of stock solution: -80°C, 2 years; -20°C, 1 year. When stored at -80°C, please use it within 2 years. When stored at -20°C, please use it within 1 year.

    Optional Solvent Concentration Solvent Mass 1 mg 5 mg 10 mg 25 mg
    DMSO 1 mM 2.0579 mL 10.2893 mL 20.5787 mL 51.4467 mL
    5 mM 0.4116 mL 2.0579 mL 4.1157 mL 10.2893 mL
    10 mM 0.2058 mL 1.0289 mL 2.0579 mL 5.1447 mL
    15 mM 0.1372 mL 0.6860 mL 1.3719 mL 3.4298 mL
    20 mM 0.1029 mL 0.5145 mL 1.0289 mL 2.5723 mL
    25 mM 0.0823 mL 0.4116 mL 0.8231 mL 2.0579 mL
    30 mM 0.0686 mL 0.3430 mL 0.6860 mL 1.7149 mL
    40 mM 0.0514 mL 0.2572 mL 0.5145 mL 1.2862 mL
    50 mM 0.0412 mL 0.2058 mL 0.4116 mL 1.0289 mL
    60 mM 0.0343 mL 0.1715 mL 0.3430 mL 0.8574 mL
    80 mM 0.0257 mL 0.1286 mL 0.2572 mL 0.6431 mL
    100 mM 0.0206 mL 0.1029 mL 0.2058 mL 0.5145 mL
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    Afatinib Related Classifications

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      Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

    Keywords:

    Afatinib850140-72-6BIBW 2992BIBW2992BIBW-2992EGFRAutophagyApoptosisc-Met/HGFRAktp38 MAPKEpidermal growth factor receptorErbB-1HER1PKBProtein kinase BNSCLCH1666H3255NCI-H1975NIH-3T3 cellsA431ESCCHER2AKTHKESC-1HKESC-2SLMT-1EC-1anticancerorally activelung cancerInhibitorinhibitorinhibit

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