Afatinib (BIBW 2992) is an irreversible EGFR family inhibitor with IC₅₀s of 0.5 nM, 0.4 nM, 10 nM and 14 nM for EGFR^wt, EGFR^L858R, EGFR^L858R/T790M and HER2, respectively.

In cell-free in vitro kinase assays, Afatinib (BIBW2992) dimaleate shows potent activity against wild-type and mutant forms of EGFR and HER2, similar to Gefitinib in potency for L858R EGFR, but about 100-fold more active against the Gefitinib-resistant L858R-T790M EGFR double mutant, with an IC₅₀ of 10 nM. BIBW2992 is furthermore comparable to Lapatinib and Canertinib for in vitro potency against HER2, with an IC₅₀ of 14 nM. The most sensitive kinase in this evaluation is lyn with an IC₅₀ of 736 nM^[1]. Afatinib is an irreversible inhibitor of these ErbB family receptors. Esophageal squamous cell carcinoma (ESCC) cell lines are sensitive to Afatinib with IC₅₀ concentrations at lower micro-molar range (at 48 hour incubation: HKESC-1=78 nM, HKESC-2=115 nM, KYSE510=3.182 μM, SLMT-1=4.625 μM and EC-1=1.489 μM; and at 72 hour incubation: HKESC-1=2 nM, HKESC-2=2 nM, KYSE510=1.090 μM, SLMT-1=1.161 μM and EC-1=109 nM) with a maximum growth inhibition over 95%. Afatinib can strongly induce G₀/G₁ cell cycle arrest in HKESC-2 and EC-1 in a dose- and time-dependent manner^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Afatinib (15 mg/kg) strongly inhibits the growth of HKESC-2 tumor once the treatment began. Average tumor sizes of vehicle and treatment at end point are 348±24 mm³ and 108±36 mm³ respectively, showing significantly difference between them. And apparently tumor size does not bounce back in a short period of time after the end of Afatinib administration. Without rapid change of body weight throughout the treatment shows that the toxicity of Afatinib is minimal and this drug is well tolerated^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

485.94

C₂₄H₂₅ClFN₅O₃

850140-72-6

阿法替尼

Room temperature in continental US; may vary elsewhere.

DMSO : 100 mg/mL (205.79 mM; Need ultrasonic)

请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。
储备液的保存方式和期限：-80°C, 6 months; -20°C, 1 month。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

请根据您的实验动物和给药方式选择适当的溶解方案。以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液，再依次添加助溶剂：

——为保证实验结果的可靠性，澄清的储备液可以根据储存条件，适当保存；体内实验的工作液，建议您现用现配，当天使用； 以下溶剂前显示的百
分比是指该溶剂在您配制终溶液中的体积占比；如在配制过程中出现沉淀、析出现象，可以通过加热和/或超声的方式助溶

• 1.

  请依序添加每种溶剂： 0.5% Methylcellulose/saline water

  Solubility: 5 mg/mL (10.29 mM); Suspended solution; Need ultrasonic
• 2.

  请依序添加每种溶剂： 10% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.5 mg/mL (5.14 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.14 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (5.14 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (5.14 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• 4.

  请依序添加每种溶剂： 5% DMSO    40% PEG300    5% Tween-80    50% saline

  Solubility: ≥ 2.5 mg/mL (5.14 mM); Clear solution

• [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.

  [3]. Wang XK, et al. Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo. Oncotarget. 2014 Dec 15;5(23):11971-85.

  [4]. Yoshioka T, et al. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer. Cancer Sci. 2018 Apr;109(4):1166-1176.

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) 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 OD_450nm. All data points are performed in triplicates^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [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.

  [3]. Wang XK, et al. Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo. Oncotarget. 2014 Dec 15;5(23):11971-85.

  [4]. Yoshioka T, et al. Antitumor activity of pan-HER inhibitors in HER2-positive gastric cancer. Cancer Sci. 2018 Apr;109(4):1166-1176.