Nilotinib (AMN107) is a Bcr-Abl tyrosine kinase inhibitor with oral activity. Nilotinib has antitumor activity and may be used for the treatment of Imatinib-resistant chronic myelogenous leukemia (CML).

Abl (WT):15 nM (cell free)

方法: 表达 Bcr-Abl 的野生型或突变体的 Ba/F3 细胞用 Nilotinib 处理 72 h，使用 methanethiosulfonate-based viability assay 检测细胞活力。
结果: Nilotinib 以比 imatinib 高 20 倍的效力抑制表达野生型 Bcr-Abl 的细胞的生长 (IC50:13 对 260 nmol/L)。除了T315I外，所有测试的 imatinib 抗性突变体都保持了类似的改善。[1]
方法: 黑色素瘤细胞系 D04 用 Nilotinib (0.1-10 µM) 处理 3 h，使用 Western Blot 方法检测靶点蛋白表达水平。
结果: Nilotinib 在低至 100 nM 的浓度下刺激了强大的 MEK 和 ERK 磷酸化。[2]

方法: 为检测体内抗肿瘤活性，将 Nilotinib (25 mg/kg) 和 PD184352 (25 mg/kg) 灌胃给药给携带 BCR-ABL 或 BCR-ABLT315I 的 Ba/F3 同种异体移植物的 Balb/cJ 小鼠，每天一次，持续二十天。
结果: Nilotinib 强烈抑制 BCR-ABL 肿瘤的生长，但 PD184352 没有抑制，PD184352 也没有增强 Nilotinib 的生长抑制活性。相反，BCR-ABLT315I 肿瘤对 Nilotinib 和 PD184352 都不敏感，但这些药物协同作用抑制了肿瘤的生长。[2]

Kinase assays using wild-type and mutant glutathione S-transferase (GST)–Abl fusion proteins (c-Abl amino acids 220-498) were done as described, with minor alterations. GST-Abl fusion proteins were released from glutathione-Sepharose beads before use; the concentration of ATP was 5 μmol/L. Immediately before use in kinase autophosphorylation and in vitro peptide substrate phosphorylation assays, GST-Abl kinase domain fusion proteins were treated with LAR tyrosine phosphatase according to the manufacturer's instructions. After 1-hour incubation at 30°C, LAR phosphatase was inactivated by addition of sodium vanadate (1 mmol/L). Immunoblot analysis comparing untreated GST-Abl kinase to dephosphorylated GST-Abl kinase was routinely done using phosphotyrosine-specific antibody 4G10 to confirm complete (>95%) dephosphorylation of tyrosine residues and c-Abl antibody CST 2862 to confirm equal loading of GST-Abl kinase. The inhibitor concentration ranges for IC50 determinations were 0 to 5,000 nmol/L (imatinib and AMN107) or 0 to 32 nmol/L (BMS-354825). The BMS-354825 concentration range was extended to 1,000 nmol/L for mutant T315I. These same inhibitor concentrations were used for the in vitro peptide substrate phosphorylation assays. The three inhibitors were tested over these same concentration ranges against GST-Src kinase and GST-Lyn kinase [1].

Ba/F3 cell lines were plated in triplicate and incubated with escalating concentrations of imatinib, AMN107, or BMS-354825 for 72 hours. Proliferation was measured using a methanethiosulfonate-based viability assay. IC50 and IC90 values are reported as the mean of three independent experiments done in quadruplicate. The inhibitor concentration ranges for IC50 and IC90 determinations were 0 to 2,000 nmol/L (imatinib and AMN107) or 0 to 32 nmol/L (BMS-354825). The imatinib concentration range was extended to 6,400 nmol/L for mutants with IC50 >2,000 nmol/L. The BMS-354825 concentration range was extended to 200 nmol/L for mutant T315I [1].

The GIST xenograft lines GK1X, GK2X and GK3X in nude mice were established from GIST patients as described in our previous study [10]. These xenograft lines were maintained by continual passage in BALB/cSLc-nu/nu mice. Mice bearing GK1X, GK2X and GK3X tumors (6–8 mice per group) were treated daily with vehicle or 40 mg/kg imatinib or nilotinib for 4 weeks. Tumor volume (TV) was determined from caliper measurements of tumor length (L) and width (w) according to the formula LW2/2. TV was determined every two to three days and on the day of evaluation. Mice were sacrificed and the percentage of tumor growth inhibition (TGI) was calculated as follows: TGI (%) ?=? [1– (mean of treatment group tumor volume on evaluation day – mean of treatment group tumor volume on day 1)/(mean of control group tumor volume on evaluation day – mean of control group tumor volume on day 1)]×100 [2].