TAK-285 is a novel dual HER2 and EGFR(HER1) inhibitor with IC50 of 17 nM and 23 nM, >10-fold selectivity for HER1/2 than HER4, less potent to MEK1/5, c-Met, Aurora B, Lck, CSK etc. Phase 1.

EGFR/HER1:23 nM, HER2:17 nM

在测试的34种激酶中，TAK-285仅显著地抑制HER4，其IC50为260 nM，同时对MEK1、MEK5、c-Met、Aurora B、Lck、CSK和Lyn B有轻微抑制作用，其IC50分别为1.1 μM、5.7 μM、4.2 μM、1.7 μM、2.4 μM、4.7 μM和5.2 μM，对其他激酶的抑制活性不明显，其IC50 >10 μM。TAK-285对BT-474细胞（过表达HER2的人乳腺癌细胞系）显示出显著的生长抑制活性，其GI50为17 nM。[1] 与强效的HER2抑制剂SYR127063相比，TAK-285对HER2和EGFR的体外抑制效力相似。与野生型蛋白的完整胞浆区域相比，用于HER2-KD和EGFR-KD结构测定的突变和缩短边界并未显著改变TAK-285的抑制活性(IC50)。TAK-285与EGFR的非活性构象结合，并显示出与lapatinib在活性位点中相似的结合模式。[2]

TAK-285的口服生物利用度在大鼠中为97.7%，在小鼠中为72.2%，剂量为50 mg/kg。以100 mg/kg的剂量口服给药TAK-285，每日两次，连续14天，在HER2高表达的BT-474肿瘤异种移植小鼠模型中显示出显著的抗肿瘤效果，肿瘤/对照（T/C）比率为29%，同时不影响体重。类似于BT-474模型，TAK-285在小鼠中对4-1ST（HER2高表达的人类胃癌肿瘤）异种移植产生剂量依赖性的肿瘤生长抑制，T/C比率分别为44%和11%，剂量为50 mg/kg和100 mg/kg，每日两次，且不显著影响小鼠体重。此外，TAK-285治疗在大鼠中对4-1ST肿瘤的生长产生剂量依赖性抑制，剂量为6.25 mg/kg和12.5 mg/kg时，T/C比率分别为38%和14%，特别值得注意的是，剂量为25 mg/kg和50 mg/kg时，肿瘤出现回归，T/C比率分别为-12%和-16%。口服给药TAK-285后，在大鼠脑中检测到大量化合物以药理学活性的非结合形式存在（约为其游离血浆水平的20%），表明TAK-285具有治疗CNS恶性肿瘤/转移的潜力。

HER2 and EGFR kinase assay: The cytoplasmic domain (amino acids 676-1255) of human HER2 and the cytoplasmic domain (amino acids 669-1210) of human EGFR are expressed as N-terminal peptide (DYKDDDD)-tagged protein using a baculovirus expression system. The expressed HER2 kinase and EGFR kinase are purified by anti-FLAG M2 affinity gel. The EGFR and HER2 kinase assays are performed using radiolabeled [γ-32P]ATP in 96-well plates. The kinase reactions are performed in 50 mM Tris-HCl (pH 7.5), 5 mM MnCl2, 0.01% Tween 20, and 2 mM DTT containing 0.9 uCi of [γ-32P]ATP per reaction, 50 μM ATP, 5 ug/mL poly(Glu)-Tyr (4:1), and each purified cytoplasmic domain (0.25 μg/mL EGFR or HER2) in a total volume of 50 μL. To measure the IC50 value for enzyme inhibition, Increasing concentrations of TAK-285 are incubated with the enzyme for 5 minutes prior to the reaction at room temperature. The kinase reactions are initiated by adding ATP. After 10 minutes at room temperature, the reactions are stopped by the addition of 10% (final concentration) trichloroacetic acid. The γ-32P phosphorylated proteins are filtrated in a harvest plate with a cell harvester and washed free of [γ-32P]ATP with 3% phosphoric acid. The plates are dried followed by the addition of 25 μL of MicroScint0. The radioactivity is counted by a TopCount scintillation counter. IC50 values are calculated by nonlinear regression analysis of the percent inhibitions.

The cells are treated continuously with various concentrations of TAK-285 for 5 days. The live cell numbers are counted with a particle analyzer.(Only for Reference)