TAK-659 hydrochloride (TAK-659) is a potent and selective inhibitor of spleen tyrosine kinase (SYK) with an IC50 value of 3.2 nM. It is selective against most other kinases, but potent toward both SYK and FLT3.

Syk:3.2 nM

在广泛的激酶检测中，TAK-659对SYK和FLT-3的选择性超过290种其他蛋白激酶50倍以上。TAK-659的处理抑制了初级CLL细胞和Burkitt's淋巴瘤细胞共培养中的Syk激活和BCR信号。在悬浮培养的初级CLL细胞中，TAK-659处理后，随着剂量增加，BCR刺激后SykTyr525、Btk、NFκB、ERK1/2和STAT3的磷酸化水平降低。TAK-659抑制Syk可诱导CLL细胞凋亡，并消除BCR和共培养衍生的存活信号。TAK-659抑制初级CLL细胞向BMSC、CXCL12和CXCL13的趋化性，并消除微环境诱导的化学抗性。TAK-659不抑制患有CLL的患者初级T细胞中的TCR信号和T细胞激活的分子特征。在细胞增殖测定中，TAK-659显示对依赖SYK的细胞系（OCI-LY10）有抑制作用；而对含有影响SYK活性突变的B细胞淋巴瘤敏感，对黏附的初级或固体肿瘤细胞系则无细胞毒性。在细胞活性测定中，TAK-659对FLT3-ITD依赖的细胞系（MV4-11和MOLM-13）敏感，但WT FLT3的RS4-11（ALL细胞系）和RA1（Burkitt's淋巴瘤细胞系）对TAK-659不敏感。在培养的人类肿瘤细胞中，TAK-659强效地抑制了血液来源的细胞系增长，对敏感细胞系统（例如，弥散性大B细胞淋巴瘤和AML）的半最大响应浓度（EC50）范围从11到775 nM。

在FLT3依赖的MV4-11异种移植模型中，TAK-659在每天60 mg/kg的剂量下，经过20天的用药后显示出肿瘤退化。初步的血浆和尿液药代动力学数据表明，TAK-659被迅速吸收(中位Tmax 2-3小时)，在稳态暴露度中表现出中等的变异性(DN-AUCtau的CV为40-50%)，平均峰谷比率为3.2–4.2，且在连续每日给药15天后平均积累量为2.1-至2.6倍。未改变药物的肾脏清除率(CLr)占表观口服清除率的30–34%，这表明CLr对TAK-659系统清除率的贡献≥30–34%。TAK-659在体内阻断了针对IgD（免疫球蛋白D抗体）激活的小鼠外周B细胞中CD86表达。

Kinases are prepared in Base Reaction Buffer (20 mM Hepes pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) and substrate is added with 1.5 mM CaCl2, 16 μg/mL Calmodulin, and 2 mM MnCl2. Varying concentrations of SAR-20347 in DMSO are added to the kinase reaction along with 10 μM 33P-ATP (activity 0.01 μCi/μL final) for IC50 determination[1].

Cell lines: FLT3-dependent cell lines (MV4-11 and MOLM-13)Cells are maintained at 37°C in a humidified atmosphere containing 5-8% CO2. In a panel of hematological and solid tumor cell lines, inhibition of cell viability is determined using the soluble tetrazolium salt, MTS. Cells are seeded in 96-well tissue culture plates and are incubated at 37°C/5% CO2 for 24 hours prior to addition of compounds or DMSO vehicle. After 72 or 96 hours of incubation with compounds, MTS conversion by metabolically active cells is determined by measuring the OD490 nm of the wells using a Thermomax microplate reader. To generate concentration-response curves, cells are treated in duplicate with a range of serial compound dilutions. Prior to addition to cells, compound dilutions are prepared in DMSO. Equal amounts of DMSO are added to cells (final concentration is 0.5%). After background correction and normalization against DMSO-treated cells, EC50 values are calculated by curve-fitting these cell viability results using nonlinear regression analysis.

Animal Models: Athymic nude mice. Formulation: 0.5% carboxymethylcellulose (CMC). Dosages: 10,30,60 mg/kg QD. Administration: by oral gavage