Afatinib (BIBW2992) Dimaleate

Catalog No.S7810

For research use only.

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. Afatinib (BIBW2992) Dimaleate induces autophagy.

Afatinib (BIBW2992) Dimaleate Chemical Structure

CAS No. 850140-73-7

Selleck's Afatinib (BIBW2992) Dimaleate has been cited by 154 publications

Purity & Quality Control

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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. Afatinib (BIBW2992) Dimaleate induces autophagy.
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 NV3GZZFDWHKxbHnm[ZJifGmxbjDhd5NigQ>? MY\BcpRqeHKxbHnm[ZJifGm4ZTDhZ5Rqfmm2eTDh[4FqdnO2IHj1cYFvKEiFQ{iyO{Bk\WyuczDh[pRmeiB7NjDodpMh[nliTWTTJIF{e2G7LDDJR|UxRThibl2= NIWyZm0zODV3MEKxNi=>
NCI-H1563 NF\nU|dIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M33DSGlvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLVixOVY{KGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:QS55NkCzNUDPxE1? NVXlfoo2W0GQR1XS
NCI-H1573 NYjGO|F6T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= MYXJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMUW3N{Bk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvPzN4MzFOwG0> M1L4PHNCVkeHUh?=
NCI-H1581 M33zRWdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 M4PCZ2lvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLVixOVgyKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OjlwM{G0OUDPxE1? NVfvcpc3W0GQR1XS
NCI-H1648 M2H0dmdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 MVPJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMU[0PEBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvPDRzOEKg{txO NIHTS2JUSU6JRWK=
NCI-H1793 M{nVbWdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 NXzRc5lxUW6qaXLpeIlwdiCxZjDoeY1idiCQQ1mtTFE4QTNiY3XscEBoem:5dHigbY4h[SClZXzsJJZq[WKrbHn0fUBie3OjeT6gTWM2OD1zND6yNFg4KM7:TR?= MlLRV2FPT0WU
NCI-H1838 MYjHdo94fGhiaX7obYJqfGmxbjDhd5NigQ>? MUnJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMUizPEBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvPjB{OUmg{txO NV\EVYM6W0GQR1XS
NCI-H1770 NWLUVmZ4T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= Mm\TTY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEG3O|Ah[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF0xNjFyMkig{txO NEjvZopUSU6JRWK=
NCI-H1651 NHrBUXpIem:5dHigbY5pcWKrdHnvckBie3OjeR?= NGGwPHVKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3INVY2OSClZXzsJIdzd3e2aDDpckBiKGOnbHygeoli[mmuaYT5JIF{e2G7LjDJR|UxRTJ3LkW5O{DPxE1? NVr6PFFjW0GQR1XS
NCI-H1975 M3vM[Gdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 MYHJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMUm3OUBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvPjF5Mkig{txO NFHR[o5USU6JRWK=
NCI-H1666 NFfETWRIem:5dHigbY5pcWKrdHnvckBie3OjeR?= MYnJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMU[2OkBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVEvQDB7NEig{txO NUjuXWZ5W0GQR1XS
NCI-H2009 NITxVmtIem:5dHigbY5pcWKrdHnvckBie3OjeR?= NELoPZZKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3INlAxQSClZXzsJIdzd3e2aDDpckBiKGOnbHygeoli[mmuaYT5JIF{e2G7LjDJR|UxRTNwNkGyNVYh|ryP NFLRTFRUSU6JRWK=
NCI-H2126 NFXZWolIem:5dHigbY5pcWKrdHnvckBie3OjeR?= MVXJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMkGyOkBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvPTF7Mk[g{txO NYfzWFNpW0GQR1XS
NCI-H1693 NGDic4tIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M2HTN2lvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLVixOlk{KGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:Oy56NEG4NUDPxE1? NIXYZnNUSU6JRWK=
NCI-H2087 MVLHdo94fGhiaX7obYJqfGmxbjDhd5NigQ>? M1XTS2lvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLViyNFg4KGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:PDlwOU[4NkDPxE1? MkLMV2FPT0WU
NCI-H2029 MmO2S5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? MVTJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMkCyPUBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVQ{NjV3OEGg{txO NGTMcnZUSU6JRWK=
NCI-H2170 NEG3WZlIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M2nvPGlvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLViyNVcxKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OC5yM{eyPEDPxE1? MnXnV2FPT0WU
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NCI-H2030 NF;ZcmxIem:5dHigbY5pcWKrdHnvckBie3OjeR?= M3fRcWlvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLViyNFMxKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OTRwNkS0PEDPxE1? NIDZSIdUSU6JRWK=
NCI-H2052 NEn6SHNIem:5dHigbY5pcWKrdHnvckBie3OjeR?= MoDrTY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEKwOVIh[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF0zOS56OU[zJO69VQ>? NHmxWmZUSU6JRWK=
NCI-H292 NW\VfVI{T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= Mn\4TY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEK5NkBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVAvODZyOTFOwG0> Ml3ZV2FPT0WU
NCI-H441 MmnaS5Jwf3SqIHnubIljcXSrb36gZZN{[Xl? MV;Jcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KNESxJINmdGxiZ4Lve5RpKGmwIHGgZ4VtdCC4aXHibYxqfHliYYPzZZkvKEmFNUC9N{42QTh2MTFOwG0> M{S4SHNCVkeHUh?=
NCI-H2291 NVfrN|ZQT3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= MVHJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMkK5NUBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVUvQTh7NEOg{txO NWXNV4dVW0GQR1XS
NCI-H358 MX;Hdo94fGhiaX7obYJqfGmxbjDhd5NigQ>? NGrmS5FKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3IN|U5KGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:OC53M{KxJO69VQ>? NF7IZ5lUSU6JRWK=
NCI-H460 NIDodmVIem:5dHigbY5pcWKrdHnvckBie3OjeR?= NGjX[FRKdmirYnn0bY9vKG:oIHj1cYFvKE6FST3IOFYxKGOnbHyg[5Jwf3SqIHnuJIEh[2WubDD2bYFjcWyrdImgZZN{[XlwIFnDOVA:PDZwNUKzJO69VQ>? MV\TRW5ITVJ?
NCI-H661 M{Dw[Gdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 NYX5OoljUW6qaXLpeIlwdiCxZjDoeY1idiCQQ1mtTFY3OSClZXzsJIdzd3e2aDDpckBiKGOnbHygeoli[mmuaYT5JIF{e2G7LjDJR|UxRTF4Lkm2PVQh|ryP MlP4V2FPT0WU
NCI-H23 M2G2UWdzd3e2aDDpcohq[mm2aX;uJIF{e2G7 M2LRUmlvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLViyN{Bk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVM4NjRzOU[g{txO NGjVelhUSU6JRWK=
NCI-H630 NWK5XXZDT3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= M1rVfmlvcGmkaYTpc44hd2ZiaIXtZY4hVkOLLVi2N|Ah[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF0yNjR3NUiyJO69VQ>? M1\QU3NCVkeHUh?=
NCI-H2342 NXP1OFB1T3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= MYrJcohq[mm2aX;uJI9nKGi3bXHuJG5EUS2KMkO0NkBk\WyuIHfyc5d1cCCrbjDhJINmdGxidnnhZoltcXS7IHHzd4F6NiCLQ{WwQVE4NjV3NzFOwG0> NYrvOlhrW0GQR1XS
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NCI-H2405 NIG4[Y5Iem:5dHigbY5pcWKrdHnvckBie3OjeR?= Mni5TY5pcWKrdHnvckBw\iCqdX3hckBPS0lvSEK0NFUh[2WubDDndo94fGhiaX6gZUBk\WyuII\pZYJqdGm2eTDhd5NigS5iSVO1NF0yPy5zOUe0JO69VQ>? NHrBVmhUSU6JRWK=
NCI-H520 NV\OSW9zT3Kxd4ToJIlvcGmkaYTpc44h[XO|YYm= NYHsVHB4UW6qaXLpeIlwdiCxZjDoeY1idiCQQ1mtTFUzOCClZXzsJIdzd3e2aDDpckBiKGOnbHygeoli[mmuaYT5JIF{e2G7LjDJR|UxRTJ2Lki4OlEh|ryP MVfTRW5ITVJ?
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NSCLC NHO1RlhEgXSxdH;4bYNqfHliYYPzZZk> MlvXO|IhcA>? NHzZc4hEgXSxdH;4bYNqfHliYXfhbY5{fCCqdX3hckBPW0OOQzDj[YxteyxiSVO1NF0xNjVibl2= M{S1UlIxOThyOUC1
Assay
Methods Test Index PMID
Growth inhibition assay Cell apoptosis ; Cell viability 25537503 26515464
Western blot CIP2A / p-AKT / AKT / PARP ; p-EGFR / p-IGF1R / p-ERK / p-AKT 25537503 26052929
Immunofluorescence p65 ; vacuoles 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 (from reference)

Kinase Assay:[1]
  • 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]
  • 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.
Animal Research:[4]
  • Animal Models: SCID mice harbouring ARK2 xenografts
  • Dosages: 25 mg/kg
  • Administration: p.o.

Solubility (25°C)

In vitro

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

Chemical Information

Molecular Weight 717.18
Formula

C24H25ClFN5O3.2C4H4O4

CAS No. 850140-73-7
Storage 3 years -20°C powder
2 years -80°C in solvent
Smiles CN(C)CC=CC(=O)NC1=C(C=C2C(=C1)C(=NC=N2)NC3=CC(=C(C=C3)F)Cl)OC4CCOC4.C(=CC(=O)O)C(=O)O.C(=CC(=O)O)C(=O)O

In vivo Formulation Calculator (Clear solution)

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mg/kg g μL

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%DMSO %

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Working concentration: mg/ml;

Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )

Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O, mix and clarify.

Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.

Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such
as vortex, ultrasound or hot water bath can be used to aid dissolving.

Molarity Calculator

Mass Concentration Volume Molecular Weight

Clinical Trial Information

NCT Number Recruitment Interventions Conditions Sponsor/Collaborators Start Date Phases
NCT04795245 Active not recruiting Drug: afatinib Non-squamous Non-Small Cell Lung Cancer Boehringer Ingelheim August 26 2021 --
NCT04497584 Recruiting Drug: Afatinib + Prednisone Advanced Squamous Non Small Cell Lung Cancer University of Texas Southwestern Medical Center August 4 2021 Phase 2
NCT04413201 Recruiting Drug: Afatinib|Drug: Osimertinib Non-squamous NSCLC Michael Hopp|Boehringer Ingelheim|Johannes Gutenberg University Mainz September 11 2020 Phase 4
NCT04206787 Recruiting Drug: Afatinib Non-squamous Non-Small Cell Lung Cancer Boehringer Ingelheim May 20 2020 --
NCT04158947 Recruiting Drug: Afatinib|Drug: T-DM1 HER2-positive Breast Cancer|Brain Metastases xuexin he|Second Affiliated Hospital School of Medicine Zhejiang University May 10 2020 Phase 2

(data from https://clinicaltrials.gov, updated on 2022-01-17)

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

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