product name Afatinib (BIBW2992) Dimaleate
Description: Afatinib dimaleate, the salt form of Afatinib, also know as BIBW 2992, is an orally bioavailable dual receptor tyrosine kinase (RTK) inhibitor. It 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 in cell-free assays, respectively; and is100-fold more active against Gefitinib-resistant L858R-T790M EGFR mutant.
References: Oncogene. 2008 Aug 7;27(34):4702-11; Br J Cancer. 2008 Jan 15;98(1):80-5.
717.18
Formula
C24H25ClFN5O3.2C4H4O4
CAS No.
850140-73-7
Storage
-20℃ for 3 years in powder form
-80℃ for 2 years in solvent
Solubility (In vitro)
DMSO: 100 mg/mL (139.4 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL
Solubility (In vivo)
5% DMSO+30% PEG 300+ddH2O: 28 mg/mL
Synonyms
BIBW 2992
other peoduct :References PubMed ID：:http://www.ncbi.nlm.nih.gov/pubmed/19416386/
| In Vitro |
In vitro activity: 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. Kinase 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 Assay: 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. |
|---|---|
| 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. |
| Animal model | Athymic NMRI-nu/nu female mice |
| Formulation & Dosage | Dissolved in 0.5% methocellulose-0.4% polysorbate-80 (Tween 80); 20 mg/kg; Oral gavage |
| References | Oncogene. 2008 Aug 7;27(34):4702-11; Br J Cancer. 2008 Jan 15;98(1):80-5. |
