BMS-599626 (AC480) has been used in trials studying the treatment of Cancer, Metastases, and HER2 or EGFR Expressing Advanced Solid Malignancies.

HER2:30 nM, HER4:190 nM, HER1:20 nM

在体内,口服60 mg/kg到240 mg/kg范围的BMS-599626,导致剂量依赖性抑制 Sal2肿瘤生长,在其人乳腺肿瘤KPL-4异种移植物中产生有效的抗肿瘤活性最大耐受剂量为180 mg/kg,并且在其他HER2扩增异种移植模型以及其他HER1过表达异种移植模型中也具有类似的抗肿瘤活性.

MS-599626抑制表达高水平HER1和/或HER2的肿瘤细胞增殖,包括Sal2,BT474,N87,KPL-4,HCC202,HCC1954,HCC1419,AU565,ZR-75-30,MDA-MB-175,GEO和PC9细胞,IC50分别为0.24 μM,0.31 μM,0.45 μM,0.38 μM,0.94 μM,0.34 μM,0.75 μM,0.63 μM,0.51 μM,0.84 μM,0.90 μM和0.34 μM。BMS-599626选择性抑制重组HER1和HER2激酶的酶活性,IC50分别为20 nM和30 nM。BMS-599626通过促进周期重新分布和抑制DNA修复,显著增强了表达EGFR和Her2细胞的HN-5细胞的放射敏感性。此外,BMS-599626也抑制相关受体HER4,但是效力较低,IC50为190 nM。BBMS-599626不会明显抑制不表达HER1或HER2的卵巢肿瘤细胞系A2780和MRC5成纤维细胞的增殖。

Protein kinase assays: The entire cytoplasmic sequences of HER1, HER2, and HER4 are expressed as recombinant proteins in Sf9 insect cells. HER1 and HER4 are expressed as fusion proteins with glutathione-S-transferase and are purified by affinity chromatography on glutathione-S-Sepharose. HER2 is subcloned into the pBlueBac4 vector and expressed as an untagged protein using an internal methionine codon (M687) for translation initiation. The truncated HER2 protein is isolated by chromatography on a column of DEAE-Sepharose equilibrated in a buffer that contains 0.1 M NaCl, and the recombinant protein is eluted with a buffer containing 0.3 M NaCl. For the HER kinase assays, reaction volumes are 50 μL and contains 10 ng of glutathione-S-transferase fusion protein or 150 ng of partially purified HER2. The mixtures also contains 1.5 μM poly(Glu/Tyr) (4:1), 1 μM ATP, 0.15 μCi [γ-33P]ATP, 50 mM Tris-HCl (pH 7.7), 2 mM DTT, 0.1 mg/mL bovine serum albumin, and 10 mM MnCl2. Reactions are allowed to proceed at 27°C for 1 hour and are terminated by the addition of 10 μL of a stop buffer (2.5 mg/mL bovine serum albumin and 0.3 M EDTA), followed by a 108-μL mixture of 3.5 mM ATP and 5% trichloroacetic acid. Acid-insoluble proteins are recovered on GF/C Unifilter plates with a Filtermate harvester. Incorporation of radioactive phosphate into the poly(Glu/Tyr) substrate is determined by liquid scintillation counting. Percent inhibition of kinase activity is determined by nonlinear regression analyses and data are reported as the inhibitory concentration required to achieve 50% inhibition relative to control reactions (IC50). Data are the averages of triplicate determinations. All other tyrosine kinases are also assayed using poly(Glu/Tyr) as a substrate. Kinetics of HER1 and HER2 inhibition are determined in reaction mixtures that contains varying concentrations of ATP and BMS-599626.

All cell lines are maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells are plated at 1,000 per well in 96-well plates and are cultured for 24 hours before BMS-599626 is added. BMS-599626 is diluted in culture medium such that the final concentrations of DMSO are ≤ 1%. Following the addition of BMS-599626, the cells are cultured for an additional 72 hours before cell viability is determined by measuring the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye with the CellTiter96 kit. For some cell lines, there is a lack of a correlation between 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye metabolism and cell number, and a thymidine uptake assay is used to measure proliferation of these cell lines. Cells are plated in 96-well plates and treated with compounds as above. At the end of the 72-hour incubation, cells are pulsed with [3H]thymidine (0.4 μCi/well) for 3 hours before they are harvested. Cells are digested with 2.5% trypsin for 10 minutes at 37 °C and are harvested by filtration using a Packard Filtermate Harvester and GF/C Unifilter plates. Incorporation of radioactive thymidine into nucleic acids is determined by liquid scintillation counting.(Only for Reference)