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Afatinib dimaleate (Synonyms: Afatinib dimaleate, BIBW 2992, BIBW 2992MA2)

カタログ番号GC10707

EGFRとErbB2の阻害剤

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Afatinib dimaleate 化学構造

Cas No.: 850140-73-7

サイズ 価格 在庫数 個数
10mM (in 1mL DMSO)
$40.00
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10mg
$46.00
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50mg
$71.00
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100mg
$98.00
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200mg
$147.00
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Sample solution is provided at 25 µL, 10mM.

Product Documents

Quality Control & SDS

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Protocol

Kinase experiment:

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 supplied by Transduction Laboratories) 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].

Cell experiment:

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].

Animal experiment:

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.

References:

[1]. Li D, et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008 Aug 7;27(34):4702-11.
[2]. Wong CH, et al. Preclinical evaluation of afatinib (BIBW2992) in esophageal squamous cell carcinoma (ESCC). Am J Cancer Res. 2015 Nov 15;5(12):3588-99

Background

アファチニブジマレートは、EGFRファミリー阻害剤であり、EGFRwt、EGFRL858R、EGFRL858R/T790MおよびHER2に対するIC50がそれぞれ0.5 nM、0.4 nM、10 nMおよび14 nMです。

細胞外キナーゼアッセイにおいて、Afatinib(BIBW2992)ジマレートは、EGFRとHER2の野生型および変異型に対して強力な活性を示し、L858R EGFRに対するGefitinibと同等のポテンシーを持ちますが、Gefitinib耐性のL858R-T790M EGFR二重変異体に対しては100倍以上も活性が高く、IC50値は10 nMです。 BIBW2992はまた、HER2に対するin vitroポテンシーでLapatinibおよびCanertinibと比較可能であり、IC50値は14 nMです。この評価で最も感度が高かったキナーゼはlynであり、IC50値は736 nMです[1]。 AfatinibはこれらErbBファミリーレセプターの不可逆的阻害剤です。食道扁平上皮癌(ESCC)細胞株ではAfatinibが低微モル濃度範囲で感受性を示し(48時間培養時:HKESC-1=78nM, HKESC-2=115nM, KYSE510=3.182μm, SLMT-1=4.625μm and EC-1=1.489μm; 72時間培養時:HKESC-1=2nM, HKESC-2=2nM, KYSE510=1.090μm, SLMT-1=1.161μm and EC-1=109nM)、最大の成長抑制率は95%以上です。 Afatinibは、HKESC-2およびEC-1において、投与量と時間依存性を持ってG0/G1細胞周期停止を強力に誘導することができます[2]。

アファチニブ(15 mg/kg)は、治療が開始されるとHKESC-2腫瘍の成長を強力に抑制します。終点での車両および治療の平均腫瘍サイズはそれぞれ348±24 mm3および108±36 mm3であり、明らかな差があります。そして、アファチニブ投与終了後も腫瘍サイズが短期間で反発しないことがわかりました。治療中の体重変化の急激な変化がなく、アファチニブの毒性は最小限であり、この薬剤は良好に耐容されています[2]。

参考文献:
[1]. Li D, et al. BIBW2992、不可逆的EGFR/HER2抑制剂,在临床前肺癌模型中高效。Oncogene. 2008年8月7日;27(34):4702-11。
[2]. Wong CH, et al. Afatinib(BIBW2992)在食道鳞状细胞癌(ESCC)中的临床前评估。Am J Cancer Res. 2015年11月15日;5(12):3588-99

Chemical Properties

Cas No. 850140-73-7 SDF
同義語 Afatinib dimaleate, BIBW 2992, BIBW 2992MA2
Chemical Name (Z)-but-2-enedioic acid;(E)-N-[4-(3-chloro-4-fluoroanilino)-7-[(3S)-oxolan-3-yl]oxyquinazolin-6-yl]-4-(dimethylamino)but-2-enamide
Canonical 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
Formula C32H33ClFN5O11 M.Wt 718.08
溶解度 ≥ 35.9 mg/mL in DMSO, ≥ 6.39 mg/mL in EtOH with ultrasonic and warming, ≥ 147.2 mg/mL in Water Storage Store at -20°C
General tips Please select the appropriate solvent to prepare the stock solution according to the solubility of the product in different solvents; once the solution is prepared, please store it in separate packages to avoid product failure caused by repeated freezing and thawing.Storage method and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored at -20°C, please use it within 1 month.
To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time.
Shipping Condition Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request.

Complete Stock Solution Preparation Table

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1 mg 5 mg 10 mg
1 mM 1.3926 mL 6.963 mL 13.926 mL
5 mM 0.2785 mL 1.3926 mL 2.7852 mL
10 mM 0.1393 mL 0.6963 mL 1.3926 mL
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