| 名稱 | Neratinib |
| 描述 | Neratinib (HKI-272) is a tyrosine kinase receptor inhibitor that inhibits HER2 and EGFR (IC50=59/92 nM) with irreversible and oral activity. Neratinib has antitumor activity and can be used to treat breast cancer. |
| 細胞實驗 | Cells were plated in 96-well tissue culture plates (3T3, 3T3/neu, 5000 cells/well; A431, SK-Br-3, BT474, MDA-MB-435, and SW620, 10,000 cells/well). The following day, dilutions of compound (0.5 ng/ml–5 μg/ml) were added, and cells were cultured for 2 days (6 days for BT474). Cell proliferation was determined using sulforhodamine B, a protein binding dye. Briefly, cells were fixed with 10% trichloroacetic acid and washed extensively with water. Cells were then stained with 0.1% sulforhodamine B and washed in 5% acetic acid. Protein-associated dye was solubilized in 10 mm Tris, and absorbance was measured at 450 nm (Victor^2). Inhibition of cell proliferation was calculated using the formula: percentage of inhibition = 100 ? 100 (Td ? To/Tc ? To), where Td is the absorbance of drug-treated cells, Tc is the absorbance of untreated cells, and To is the absorbance at the time of drug addition. To values were determined by plating cells separately and fixing them at the time of drug addition. The concentration of compound which inhibits cell proliferation by 50% (IC50) was determined from inhibition curves [1]. |
| 激酶實驗 | Activity of HER-2 and EGFR cytoplasmic domains was measured by an autophosphorylation assay using time-resolved fluorometry. Compounds were prepared as 10 mg/ml stocks in DMSO and diluted in 25 mm HEPES (pH 7.5; 0.002 ng/ml–20 μg/ml). Enzyme [diluted in 100 mm HEPES (pH 7.5) and 50% glycerol] was incubated with inhibitor in 4 mm HEPES (pH 7.5), 0.4 mm MnCl2, 20 μm sodium vanadate, and 0.2 mm DTT for 15 min at room temperature in 96-well ELISA plates. The kinase reaction was initiated by the addition of 40 μm ATP and 20 mm MgCl2 and allowed to proceed for 1 h at room temperature. Plates were washed, and phosphorylation was detected using Europium-labeled anti-phospho-tyrosine antibodies (15 ng/well). After washing and enhancement steps according to the manufacturer's recommendations, signal was detected using a Victor^2 fluorescence reader (excitation wavelength 340 nm, emission wavelength 615 nm). The concentration of compound that inhibited receptor phosphorylation by 50% (IC50) was calculated from inhibition curves [1]. |
| 動物實驗 | Athymic female nude mice (5 animals/group) were implanted s.c. with BT474 tumor fragments (～30 mm^3). When tumors reached 200–300 mg, animals were given a single oral dose (40 mg/kg) of HKI-272 in pH 2.0 water. Tumors from control and treated animals were excised at 1, 3, 6, and 24 h and minced. Tumor fragments were suspended in 10 mm Tris (pH 7.5), 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 2 mm sodium vanadate, and 100 mm sodium fluoride and lysed by homogenization on ice with a polytron. After clarification by centrifugation, protein concentration in lysates was estimated using the Bio-Rad DC protein assay. Sixty μg of lysate pooled from each group were analyzed by SDS-PAGE and immunoblotting with phospho-tyrosine-specific antibodies. Pooled extracts were also immunoprecipitated using 4 μg of anti-HER-2 antibodies for 1 h at 4°C. Immune complexes were collected on protein A-agarose, washed, and analyzed by immunoblotting using phospho-tyrosine-specific antibodies. Extracts from individual tumors were analyzed to determine variability between animals [1]. |
| 體外活性 | 方法：36 種乳腺癌細胞系用 Neratinib 處理 5 天，使用 cell counting and acid phosphatase assay 檢測細胞增殖。
結果：HER2 陽性細胞系對 Neratinib 的敏感性明顯高于三陰性或管腔細胞系。然而，一種管腔細胞系 MDA-MB-175 對 Neratinib 表現出敏感性 (IC50<0.001 μM)，而其中一種 HER2 陽性細胞系 UACC-732 對 Neratinib 相對不敏感 (IC50=0.65 μM)。[1]
方法：人乳腺癌細胞 BT474 和人表皮癌細胞 A431 用 Neratinib (2-100 nM) 處理 3 h，使用 Western Blot 檢測靶點蛋白表達水平。
結果：Neratinib 在 BT474 細胞中的 5 nM 處將配體非依賴性受體磷酸化降低了50% (IC50)。在相當劑量 (IC50=3 nM) 下，它還抑制 A431 細胞中 EGFR 的 EGF 依賴性磷酸化。[2] |
| 體內活性 | 方法：為檢測體內抗腫瘤活性，將 Neratinib (20-80 mg/kg in 0.5% methocellulose-0.4% Tween 80) 口服給藥給攜帶 3T3/neu 異種移植瘤的 nude 小鼠，每天一次，持續(xù)十天。
結果：Neratinib 以劑量依賴性方式減少腫瘤生長。[2] |
| 存儲條件 | Powder: -20°C for 3 years | In solvent: -80°C for 1 year | Shipping with blue ice/Shipping at ambient temperature. |
| 溶解度 | H2O : < 1 mg/mL (insoluble or slightly soluble)
Ethanol : < 1 mg/mL (insoluble or slightly soluble)
DMSO : 5 mg/mL (8.9 mM), Sonication is recommended.
10% DMSO+40% PEG300+5% Tween 80+45% Saline : 0.25 mg/mL (0.45 mM), Solution.
|
| 關鍵字 | solid tumors | non–small-cell lung cancer | Neratinib | Inhibitor | inhibit | HKI272 | HKI 272 | HER2 | HER1 | gastric cancer | ErbB-1 | Epidermal growth factor receptor | EGFR | BT474 | breast cancer | A431 |
| 相關產品 | Lapatinib | Chalcone | Gefitinib | Erlotinib | Osimertinib | Erlotinib hydrochloride | Afatinib Dimaleate | Tannic acid | Lidocaine Hydrochloride hydrate | Genistein | Khellin | Osimertinib mesylate |
| 相關庫 | 抑制劑庫 | 抗癌活性化合物庫 | 經典已知活性庫 | 激酶抑制劑庫 | EMA 上市藥物庫 | 臨床失敗化合物庫 | FDA 上市藥物庫 | 免疫/炎癥分子化合物庫 | 藥物功能重定位化合物庫 | 酪氨酸激酶分子庫 | 抗癌臨床化合物庫 | 抗癌藥物庫 |