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.

EGFR:92 nM (cell free), HER2:59 nM (cell free)

方法：36 种乳腺癌细胞系用 Neratinib 处理 5 天，使用 cell counting and acid phosphatase assay 检测细胞增殖。
结果：HER2 阳性细胞系对 Neratinib 的敏感性明显高于三阴性或管腔细胞系。然而，一种管腔细胞系 MDA-MB-175 对 Neratinib 表现出敏感性 (IC50<0.001 µM)，而其中一种 HER2 阳性细胞系 UACC-732 对 Neratinib 相对不敏感 (IC50=0.65 µM)。[1]
方法：人乳腺癌细胞 BT474 和人表皮癌细胞 A431 用 Neratinib (2-100 nM) 处理 3 h，使用 Western Blot 检测靶点蛋白表达水平。
结果：Neratinib 在 BT474 细胞中的 5 nM 处将配体非依赖性受体磷酸化降低了50% (IC50)。在相当剂量 (IC50=3 nM) 下，它还抑制 A431 细胞中 EGFR 的 EGF 依赖性磷酸化。[2]

方法：为检测体内抗肿瘤活性，将 Neratinib (20-80 mg/kg in 0.5% methocellulose-0.4% Tween 80) 口服给药给携带 3T3/neu 异种移植瘤的 nude 小鼠，每天一次，持续十天。
结果：Neratinib 以剂量依赖性方式减少肿瘤生长。[2]

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].

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].

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].