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Afatinib Dimaleate

別名：BIBW2992 Dimaleate

Afatinib 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 Dimaleate化学構造

CAS No. 850140-73-7

サイズ 価格(税別) 在庫状況
10mM (1mL in DMSO) JPY 29500 国内在庫あり
JPY 22000 国内在庫あり
JPY 37000 国内在庫あり
JPY 55500 国内在庫あり
JPY 118500 国内在庫なし(納期7~10日)

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Afatinib Dimaleate関連製品

シグナル伝達経路

EGFR Signaling Pathways

EGFRシグナル伝達経路

EGFR阻害剤の選択性比較

Cell Data

Cell Lines Assay Type Concentration Incubation Time 活性情報 PMID
NCI-H1975 Proliferation assay 72 h Antiproliferative activity against human NCI-H1975 cells expressing EGFR T790M mutant after 72 hrs by SRB assay, IC50=0.148 μM 24183742
A431 Proliferation assay 72 h Antiproliferative activity against human A431 cells after 72 hrs by SRB assay, IC50=0.023 nM 24183742
MDA-MB-231 Proliferation assay 72 h Antiproliferative activity against human MDA-MB-231 cells after 72 hrs by MTT assay, EC50=1 nM 20180906
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生物活性

製品説明 Afatinib 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
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]
Kinase Assay 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™.
細胞実験 細胞株 SP and NSP cell lines
濃度 1 μM
反応時間 48 h
実験の流れ 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.
実験結果図 Methods Biomarkers 結果図 PMID
Growth inhibition assay Cell apoptosis Cell viability 25537503
Western blot CIP2A / p-AKT / AKT / PARP p-EGFR / p-IGF1R / p-ERK / p-AKT 25537503
Immunofluorescence p65 vacuoles 26317651
In Vivo
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]
動物実験 動物モデル SCID mice harbouring ARK2 xenografts
投与量 25 mg/kg
投与経路 p.o.
NCT Number Recruitment Conditions Sponsor/Collaborators Start Date Phases
NCT06302621 Recruiting
Advanced Solid Tumor|Unresectable Solid Tumor|Metastatic Solid Tumor|Cholangiocarcinoma
Massachusetts General Hospital|Boehringer Ingelheim|Incyte Corporation
 July 1 2024 Phase 1
NCT05298176 Recruiting
Non-Small Cell Lung Cancer
Amsterdam UMC location VUmc
 January 4 2022 Phase 2
NCT04795245 Completed
Non-squamous Non-Small Cell Lung Cancer
Boehringer Ingelheim
 August 26 2021 --
NCT04497584 Recruiting
Advanced Squamous Non Small Cell Lung Cancer
University of Texas Southwestern Medical Center
 August 4 2021 Phase 2
NCT04413201 Active not recruiting
Non-squamous NSCLC
Michael Hopp|Boehringer Ingelheim|Johannes Gutenberg University Mainz
 September 11 2020 Phase 4

化学情報

分子量 717.18 化学式

C24H25ClFN5O3.2C4H4O4
CAS No. 850140-73-7 SDF Download Afatinib Dimaleate SDFをダウンロードする
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
保管

In vitro
Batch:

DMSO : 100 mg/mL ( (139.43 mM)； 吸湿したDMSOは溶解度を減少させます。新しいDMSOをご使用ください。)

Water : Insoluble

Ethanol : Insoluble

モル濃度計算器

実験計算

モル濃度計算器

質量 濃度 体積 分子量

投与溶液組成計算機(クリア溶液)

ステップ１：実験データを入力してください。（実験操作によるロスを考慮し、動物数を1匹分多くして計算・調製することを推奨します）

mg/kg g μL 匹

ステップ２：投与溶媒の組成を入力してください。（ロット毎に適した溶解組成が異なる場合があります。詳細については弊社までお問い合わせください）

% DMSO % % Tween 80 % ddH2O
%DMSO %

計算結果：

投与溶媒濃度： mg/ml;

DMSOストック溶液調製方法： mg 試薬を μL DMSOに溶解する（濃度 mg/mL, 注：濃度が当該ロットのDMSO溶解度を超える場合はご連絡ください。 )

投与溶媒調製方法：Take μL DMSOストック溶液に μL PEG300，を加え、完全溶解後μL Tween 80，を加えて完全溶解させた後 μL ddH2O，を加え完全に溶解させます。

投与溶媒調製方法：μL DMSOストック溶液に μL Corn oil，を加え、完全溶解。

注意：１.ストック溶液に沈殿、混濁などがないことをご確認ください；
２.順番通りに溶剤を加えてください。次のステップに進む前に溶液に沈殿、混濁などがないことを確認してから加えてください。ボルテックス、ソニケーション、水浴加熱など物理的な方法で溶解を早めることは可能です。

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