Afatinib (BIBW2992) Dimaleate

Catalog No.S7810

Afatinib (BIBW2992) Dimaleate Chemical Structure

Molecular Weight(MW): 717.18

Afatinib (BIBW2992) Dimaleate irreversibly inhibits EGFR/HER2 including EGFR(wt), EGFR(L858R), EGFR(L858R/T790M) and HER2 with IC50 of 0.5 nM, 0.4 nM, 10 nM and 14 nM, respectively; 100-fold more active against Gefitinib-resistant L858R-T790M EGFR mutant.

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Cited by 35 Publications

Purity & Quality Control

Choose Selective EGFR Inhibitors

Biological Activity

Description Afatinib (BIBW2992) Dimaleate irreversibly inhibits EGFR/HER2 including EGFR(wt), EGFR(L858R), EGFR(L858R/T790M) and HER2 with IC50 of 0.5 nM, 0.4 nM, 10 nM and 14 nM, respectively; 100-fold more active against Gefitinib-resistant L858R-T790M EGFR mutant.
Targets
EGFR (L858R) [1]
(Cell-free assay)
EGFR (wt) [1]
(Cell-free assay)
EGFR (L858R/T790M) [1]
(Cell-free assay)
HER2 [1]
(Cell-free assay)
0.4 nM 0.5 nM 10 nM 14 nM
In vitro

BIBW2992 is more effective than erlotinib, gefitinib, or lapatinib in inhibiting survival of lung cancer cell lines harboring wild-type (H1666) or L858R/T790M (NCI-H1975) EGFR. BIBW2992 is similarly effective against NSCLC lines expressing HER2 776insV (NCI-H1781) or EGFR E746_A750del (HCC827), but shows no activity toward A549 cells, which express wild-type EGFR and HER2.[1] Afatinib enhances cytotoxicity of topotecan and mitoxantrone to SP cells, and increases the apoptosis induced by topotecan and mitoxantrone in SP cells.[2]

Cell Data
Cell Lines Assay Type Concentration Incubation Time Formulation Activity Description PMID
HCC827 cell MXvQdo9tcW[ncnH0bY9vKGG|c3H5 MlPvRY51cXC{b3zp[oVz[XSrdnWgZYN1cX[rdImgZYdicW6|dDDoeY1idiCKQ1O4Nlch[2WubIOgZYZ1\XJiOU[gbJJ{KGK7IF3UV{Bie3OjeTygTWM2OD16IH7N NGLreoczODV3MEKxNi=>
NCI-H1563 NYfIb4VnT3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= MkTUTY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEG1OlMh[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF06Njd4MEOxJO69VQ>? M1LicHNCVkeHUh?=
NCI-H1573 M3PIS2dzd3e2aDDpcohq[mm2aX;uJIF{e2G7 MXPJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMUW3N{Bk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvPzN4MzFOwG0> MYTTRW5ITVJ?
NCI-H1581 NVn3coI2T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= NH[4XXZKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3INVU5OSClZXzsJIdzd3e2aDDpckBiKGOnbHygeoli[mmuaYT5JIF{e2G7LjDJR|UxRTJ7LkOxOFUh|ryP M2L0d3NCVkeHUh?=
NCI-H1648 MmTDS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? M1rLWWlvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLVixOlQ5KGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OC52NEG4NkDPxE1? NHjKWW1USU6JRWK=
NCI-H1793 M1XQPGdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 MkTFTY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEG3PVMh[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF0yPC5{MEi3JO69VQ>? M1HTPXNCVkeHUh?=
NCI-H1838 MlXJS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? NH6wSppKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3INVg{QCClZXzsJIdzd3e2aDDpckBiKGOnbHygeoli[mmuaYT5JIF{e2G7LjDJR|UxRTBwNkCyPVkh|ryP MVnTRW5ITVJ?
NCI-H1770 NYq1Vnl1T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= MXTJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMUe3NEBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvOTB{ODFOwG0> NVr6NYxlW0GQR1XS
NCI-H1651 MmOzS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? MV7Jcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMU[1NUBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVI2NjV7NzFOwG0> M3rZRXNCVkeHUh?=
NCI-H1975 NWnybnZNT3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= M33Ed2lvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLVixPVc2KGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OC54MUeyPEDPxE1? M2X2cHNCVkeHUh?=
NCI-H1666 MofWS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? NV7v[3JtUW6qaXLpeIlwdiCxZjDoeY1idiCQQ1mtTFE3PjZiY3XscEBoem:5dHigbY4h[SClZXzsJJZq[WKrbHn0fUBie3OjeT6gTWM2OD1zLkiwPVQ5KM7:TR?= MX7TRW5ITVJ?
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NCI-H630 NIHr[WJIem:5dHigbY5pcWKrdHnvckBie3OjeR?= NI\qblFKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3IOlMxKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OS52NUW4NkDPxE1? NFXMW4RUSU6JRWK=
NCI-H2342 MkHlS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? MnP2TY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEKzOFIh[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF0yPy53NUeg{txO Mnm5V2FPT0WU
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SHP-77 NHTKd4lIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M3\iOmlvcGmkaYTpc44hd2ZiaIXtZY4hW0iSLUe3JINmdGxiZ4Lve5RpKGmwIHGgZ4VtdCC4aXHibYxqfHliYYPzZZkvKEmFNUC9NlkvPjF6MjFOwG0> NI\1b4JUSU6JRWK=
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OVCAR-8 NV;MXYw6T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= M4\sRWlvcGmkaYTpc44hd2ZiaIXtZY4hV1[FQWKtPEBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVE2Njh6NkGg{txO MVPTRW5ITVJ?
SK-LU-1 NHK1dYhIem:5dHigbY5pcWKrdHnvckBie3OjeR?= NYHrN25iUW6qaXLpeIlwdiCxZjDoeY1idiCVSz3MWU0yKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:PDBwNkW2PEDPxE1? MW\TRW5ITVJ?
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PANC-10-05 Mke4S5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? NXT0RWVvUW6qaXLpeIlwdiCxZjDoeY1idiCSQV7DMVExNTB3IHPlcIwh\3Kxd4ToJIlvKGFiY3XscEB3cWGkaXzpeJkh[XO|YYmuJGlEPTB;MUSuOlI1PyEQvF2= M4rrXnNCVkeHUh?=
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MDA-MB-231 MljIVJJwdGmoZYLheIlwdiCjc4PhfS=> MV63NkBp M4jsRWFvfGmycn;sbYZmemG2aY\lJIFkfGm4aYT5JIFo[Wmwc4SgbJVu[W5iTVTBMW1DNTJ|MTDj[YxteyCjZoTldkA4OiCqcoOgZpkhVVSWIHHzd4F6NCCHQ{WwQVEhdk1? NUjnS3FyOjBzOEC5NFY>
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A549 NWLyR3c6S3m2b4TvfIlkcXS7IHHzd4F6 NF:5PWI4OiCq NFrNRlREgXSxdH;4bYNqfHliYXfhbY5{fCCqdX3hckBCPTR7IHPlcIx{KGGodHXyJFczKGi{czDifUBOXFRiYYPzZZktKEmFNUC9NE4xPSEQvF2= M17XTlIxOThyOUC1
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HeLa NV\ud4ROS3m2b4TvfIlkcXS7IHHzd4F6 NYT5N3pMPzJiaB?= M{TGXGN6fG:2b4jpZ4l1gSCjZ3HpcpN1KGi3bXHuJGhmVGFiY3XscJMh[W[2ZYKgO|IhcHK|IHL5JG1VXCCjc4PhfUwhUUN3ME22MlgyKM7:TR?= Mn;FNlAyQDB7MEW=
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NSCLC NXnEXJZQS3m2b4TvfIlkcXS7IHHzd4F6 NIS5T3Q4OiCq NEizOJhEgXSxdH;4bYNqfHliYXfhbY5{fCCqdX3hckBPW0OOQzDj[YxteyxiSVO1NF0xNjVibl2= MoTKNlAyQDB7MEW=

... Click to View More Cell Line Experimental Data

Assay
Methods Test Index PMID
Growth inhibition assay
Cell apoptosis; 

PubMed: 25537503     


Dose-dependent effects of afatinib on apoptosis. Data are mean ± SD. n = 3 for each concentration. *, p < 0.05, **, p < 0.01, vs. no afatinib. 

Cell viability; 

PubMed: 26515464     


A. MTS assay for Ba/F3 cells harboring EGFR exon 19 deletion and L858R. The proportional cell viability is shown. B. MTS assay for Ba/F3 cells harboring EGFR exon 19 deletion+T790M and L858R+T790M. The proportional cell viability is shown. C. MTS assay for Ba/F3 cells harboring wild type EGFR. The proportional cell viability is shown. Erlotinib, afatinib, osimertinib, and rociletinib were used as EGFR-TKIs. Error bars indicate standard deviation.

25537503 26515464
Western blot
CIP2A / p-AKT / AKT / PARP; 

PubMed: 25537503     


Time-dependent effects of afatinib on CIP2A, p-AKT and apoptosis-related proteins in the sensitive H358 and resistant H460 cells. Cells were exposed to 10 μM afatinib for up to 48 hours. Immunoblots were scanned and quantitated to determine the ratio of the level of CIP2A to actin. Data are mean ± SD. n = 3 for the different time intervals.

p-EGFR / p-IGF1R / p-ERK / p-AKT; 

PubMed: 26052929     


Effects of afatinib on EGFR and IGF1R Signaling Pathways. H1975 cells were treated with afatinib with various doses and cell lysates were subjected to Western blot analysis. Afatinib effectively inhibits p-EGFR and p-ERK while p-AKT was partially inhibited. IGF1R phosphorylation was increased upon afatinib treatment in H1975 cells.

25537503 26052929
Immunofluorescence
p65 ; 

PubMed: 26317651     


Effects of different treatments on the nuclear translocation of the NF-κB subunit p65. A2780T cells were treated with 2.5 μM afatinib for 48 hours, or 10 μM PDTC for 2 hours. Subsequently, the NF-κB subunit p65 (red) was localized by immunofluorescence and a confocal microscopy. Nuclei were stained with DAPI (blue). Scale bar = 10 μm.

vacuoles; 

PubMed: 29713246     


Autophagy activation upon treatment with Afatinib and Sorafenib was validated using immunofluorescence in order to visualize a creation of autophagosomes; ×60 magnification. DMSO treatment was used as control. Green—autophagy vacuoles; Blue—Hoechst, nuclear staining. 

26317651 29713246
In vivo In the MDA-MB-453 xenograft model, BIBW2992 (20 mg/kg, p.o.) results in dramatic tumor regression with a cumulative treated/control tumor volume ratio (T/C ratio) of 2%, and downregulation of EGFR and AKT phosphorylation.[1] In A7, A431, FaDu, UT-SCC-14 and UT-SCC-15 xenograft models, application of BIBW 2992 (30 mg/kg, p.o.) leads to a significant prolongation of tumour growth time.[3] In HER2-amplified xenograft medel, Afatinib (30 mg/kg, p.o.) results a markedly inhibition in tumor growth and a significant improvement in the duration of overall survival.[4] In HER2-positive gastric cancer NCI-N87 xenograft, afatinib (25 mg/kg, p.o.) leads to dramatic tumor volume regression within 4 d and near-complete tumor resolution after 21 d of treatment.[5]

Protocol

Kinase Assay:[1]
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In vitro kinase activity assay:

EGFR kinase: Each 100 µL enzyme reaction contained 10 μL of inhibitor 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 min 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 100µL 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. HER2-IC enzyme: Enzyme activity is assayed in the presence or absence of serial inhibitor dilutions performed in 50 % Me2SO. Each 100 µL reaction contains similar components as described for EGFR kinase assay with addition of 1000 µM Na3VO4. The enzymatic reaction is started by addition of 50µL of 500 µM ATP solution made in 10 mM Mg-acetate. The dilution of the enzyme is set so that incorporation of phosphate into bio-pEY is linear with respect to time and amount of enzyme. The enzyme preparation is diluted in 20 mM HEPES pH 7.4, 130 mM NaCl, 0.05% Triton X-100, 1 mM DTT and 10% glycerol. Assays are carried out at room temperature for 30 min and terminated by the addition of 50 µL of stop solution. Src kinase assays: Each 100 µL reaction contained 10 µL of inhibitor in 50 % Me2SO, 20µL of enzyme preparation, 20 µL of substrate solution supplemented with 1000 µM Na3VO4. The enzymatic reaction is started by addition of 50 µL of a 1000 µM ATP solution made in 10 mM Mg-acetate. BIRK kinase assay: 250 mM Tris pH 7.4, 10mM DTT, 2.5 mg/mL poly(EY), 5 mg/mL bio-pEY is used as substrate solution and enzymatic reaction is started by addition of 50 µL of a 2 mM ATP solution made in 8 mM MnCl2, 20 mM Mg-acetate. VEGF2 and HGFR kinase assays: Assays are carried out at room temperature for 20 minutes and terminated by the addition of 10 µL of 5 % H3PO4. The precipitate is then trapped onto GF/B filters using a 96 well filter mate universal harvester. After extensive washing the filter plate is dried for 1 h at 50°C, sealed and incorporated radioactivity is determined by scintillation counting using a TopCount™ or a Microbeta b counter™.
Cell Research:[2]
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  • Cell lines: SP and NSP cell lines
  • Concentrations: 1 μM
  • Incubation Time: 48 h
  • Method: Cytotoxicity is determined using MTT assay. The IC 50 value is defined as the drug concentration resulting in 50% cell death. Both the fitted sigmoidal dose response curve and IC50 are calculated by Bliss method.
    (Only for Reference)
Animal Research:[4]
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  • Animal Models: SCID mice harbouring ARK2 xenografts
  • Formulation: DMSO
  • Dosages: 25 mg/kg
  • Administration: p.o.
    (Only for Reference)

Solubility (25°C)

In vitro DMSO 100 mg/mL (139.43 mM)
Water Insoluble
Ethanol Insoluble
In vivo Add solvents to the product individually and in order(Data is from Selleck tests instead of citations):
5% DMSO+30% PEG 300+ddH2O
For best results, use promptly after mixing.
28mg/mL

* 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.

Chemical Information

Molecular Weight 717.18
Formula

C24H25ClFN5O3.2C4H4O4

CAS No. 850140-73-7
Storage powder
in solvent
Synonyms N/A

Bio Calculators

Molarity Calculator

Molarity Calculator

Calculate the mass, volume or concentration required for a solution. The Selleck molarity calculator is based on the following equation:

Mass (mg) = Concentration (mM) × Volume (mL) × Molecular Weight (g/mol)

  • Mass
    Concentration
    Volume
    Molecular Weight

*When preparing stock solutions, please always use the batch-specific molecular weight of the product found on the via label and MSDS / COA (available on product pages).

Dilution Calculator

Dilution Calculator

Calculate the dilution required to prepare a stock solution. The Selleck dilution calculator is based on the following equation:

Concentration (start) x Volume (start) = Concentration (final) x Volume (final)

This equation is commonly abbreviated as: C1V1 = C2V2 ( Input Output )

  • C1
    V1
    C2
    V2

* When preparing stock solutions always use the batch-specific molecular weight of the product found on the vial label and MSDS / COA (available online).

The Serial Dilution Calculator Equation

  • Serial Dilutions

  • Computed Result

  • C1=C0/X C1: LOG(C1):
    C2=C1/X C2: LOG(C2):
    C3=C2/X C3: LOG(C3):
    C4=C3/X C4: LOG(C4):
    C5=C4/X C5: LOG(C5):
    C6=C5/X C6: LOG(C6):
    C7=C6/X C7: LOG(C7):
    C8=C7/X C8: LOG(C8):
Molecular Weight Calculator

Molecular Weight Calculator

Enter the chemical formula of a compound to calculate its molar mass and elemental composition:

Total Molecular Weight: g/mol

Tip: Chemical formula is case sensitive. C10H16N2O2 c10h16n2o2

Instructions to calculate molar mass (molecular weight) of a chemical compound:

To calculate molar mass of a chemical compound, please enter its chemical formula and click 'Calculate'.

Definitions of molecular mass, molecular weight, molar mass and molar weight:

Molecular mass (molecular weight) is the mass of one molecule of a substance and is expressed in the unified atomic mass units (u). (1 u is equal to 1/12 the mass of one atom of carbon-12)
Molar mass (molar weight) is the mass of one mole of a substance and is expressed in g/mol.

Molarity Calculator

Mass Concentration Volume Molecular Weight

Clinical Trial Information

NCT Number Recruitment interventions Conditions Sponsor/Collaborators Start Date Phases
NCT03711422 Not yet recruiting Drug: Afatinib Non-small Cell Lung Cancer|Leptomeningeal Disease|Central Nervous System Metastases National Cancer Centre Singapore October 2018 Phase 1
NCT03083678 Recruiting Drug: Afatinib Chordoma Leiden University Medical Center|Chordoma Foundation|Boehringer Ingelheim June 21 2018 Phase 2
NCT03370770 Active not recruiting Drug: Afatinib|Drug: Osimertinib Carcinoma Non-Small-Cell Lung Boehringer Ingelheim December 28 2017 --
NCT02423525 Recruiting Drug: Afatinib Brain Cancer Santosh Kesari|Boehringer Ingelheim|John Wayne Cancer Institute December 2016 Phase 1
NCT02695290 Terminated Drug: Afatinib Carcinoma Non-Small-Cell Lung|ErbB Receptors Boehringer Ingelheim March 17 2016 Phase 4

Tech Support

Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

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