Src HuH7 (GI50 = 9 nM); Src PLC/PRF/5 (IC50 = 13 nM); Src Hep3B (IC50 = 26 nM); Src HepG2 (IC50 = 60 nM)

一种针对 Src 底物袋的 Src 抑制剂是替巴尼布林 (KX2-391)。与四种肝细胞癌 (HCC) 细胞系相比，即 Huh7 (GI50=9 nM)、PLC/PRF/5 (GI50=13 nM)、Hep3B (GI50=26 nM) 和 HepG2 (GI50=60 nM) ，tirbanibulin (KX2-391) 表现出陡峭的剂量反应曲线[1]。研究发现，tirbanibulin (KX2-391) 可抑制某些白血病细胞，例如源自具有 T3151 突变的慢性白血病细胞的细胞，这些细胞对目前上市的药物具有耐药性。在 SYF/c-Src527F 和 NHH3T3/c-Src527F 细胞中，在 Src 驱动的细胞生长测定中评估替巴尼布林 (KX2-391)，显示 GI50 值分别为 39 nM 和 23 nM[2]。

临床前癌症动物模型已证明口服替巴尼布林 (KX2-391) 可减少原发肿瘤生长并抑制转移[2]。

Src 抑制剂替巴尼布林 (KX2-391) 靶向 Src 底物袋。肝细胞癌 (HCC) 细胞系 Huh7 (GI50=9 nM)、PLC/PRF/5 (GI50=13 nM)、Hep3B (GI50=26 nM) 和 HepG2 (GI50=60 nM) 表现出陡峭的剂量反应用替巴尼布林 (KX2-391) 治疗时的曲线。

Hep3B、HepG2、PLC/PRF/5、Huh7 和其他肝细胞系经常在 37°C 和 5% CO2 的条件下在含有 2% 胎牛血清 (FBS) 的基础培养基中生长和保存。在 96 孔板的每个孔中，将细胞以 4.0×103/190 μL 和 8.0×103/190 μL 接种在含有 1.5% FBS 的基础培养基中。添加浓度范围为 6,564 至 0.012 nM 的替巴尼布林 (KX2-391)（一式三份）之前，将其在 37°C 和 5% CO2 下再培养一晚。孵育处理过的细胞需要三天时间。第三天，向每个孔中添加 10 μL 5 mg/mL 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四唑 (MTT) 溶液，并将细胞孵育 4 小时。小时。将 10% SDS 添加到稀 HCl 中以溶解甲臜产物。光密度在 570 nm 处测量。使用替巴尼布林 (KX2-391) 进行平行实验以比较其效力和活性。使用GraphPad Prism 5统计软件，计算生长抑制曲线、50%抑制浓度（GI50）、80%抑制浓度（GI80）。报告了 570 nm 波长 (OD570) 信号格式的光密度和代表最大响应百分比的归一化数据。

Mouse bearing MDA-MB-231 tumors; Oral gavage; 1, 5mg/kg dose
Xenograft procedures and KX-01 oral dosing were as described (11). Briefly, mammary fat pad tumors were established by injecting 5×106 MDA-MB-231 cells in 150μl of PBS-Matrigel mixture (1:2) orthotopically and bilaterally into the mammary fat pads of female NUDE mice (two tumors/mouse). Treatments were started when tumors reached ∼80-100mm3. The first study used MDA-MB-231 xenografts and was performed using vehicle (ultra-pure water) and two doses of KX-01 (1, 5mg/kg) administered twice/day (BID) by oral gavage (using metal 22g feeding needle) for 28 days. A similar experiment was performed with MDA-MB-157 xenografts (another ER/PR/HER2 negative model) to assess KX-01 response. A second study was performed to test combination of KX-01 with paclitaxel on tumor growth. MDA-MD-231 tumor xenograft bearing mice were treated with vehicle or KX-01 (5mg/kg) BID, paclitaxel by intraperitoneal injection (IP) once/week, or combination of KX-0+paclitaxel. Treatments were for 40 days for all groups. A third study used MDA-MB-157 xenografts with the same combination treatment. A fourth study tested the effect of KX-01 or combination with paclitaxel for 24 days on larger MDA-MB-231 tumors (∼300mm3). Tumors were allowed to reach ∼300mm3 before beginning treatments. In this experiment mice were treated with KX-01 at a higher dose of 15mg/kg, and mice were treated once/day instead of twice/day. Paclitaxel was used at a dose of 20mg/kg IP once/week. In all experiments, tumor caliper measurements were taken twice/week and tumor volume was by calculated by the formula: 0.523×LM2 (where L-large diameter, M-small diameter). At the end the experiments animals were sacrificed and tumors and mouse organs removed. Tissues were either stored in 10% neutral buffered formalin for paraffin embedding, or snap frozen for measurement of chromosome-17 by real-time PCR, and embedded for frozen sectioning for CD-31 staining. Immunohistochemistry (IHC) was performed as described on paraffin-embedded tumor tissues [3].

[1]. Lau GM, et al. Expression of Src and FAK in hepatocellular carcinoma and the effect of Src inhibitors on hepatocellular carcinoma in vitro. Dig Dis Sci, 2009, 54(7), 1465-1474.
[2]. Fallah-Tafti A, et al. Thiazolyl N-benzyl-substituted acetamide derivatives: synthesis, Src kinase inhibitory and anticancer activities. Eur J Med Chem, 2011, 46(10), 4853-4858.

[3]. Peptidomimetic Src/pretubulin inhibitor KX-01 alone and in combination with paclitaxel suppresses growth, metastasis in human ER/PR/HER2-negative tumor xenografts. Mol Cancer Ther. 2012 Sep; 11(9): 1936–1947.

C26H29N3O3

431.53

431.22089

C, 71.92; H, 6.52; N, 10.06; O, 11.50

897016-82-9

Tirbanibulin dihydrochloride;1038395-65-1;Tirbanibulin Mesylate;1080645-95-9

White to off-white solid powder

2.9

63.7Ų

O=C(C1=CC=C(/C=C/C2=NNC3=C2C=CC=C3)C=C1)N4CCNCC4

YYLKKYCXAOBSRM-JXMROGBWSA-N

InChI=1S/C20H20N4O/c25-20(24-13-11-21-12-14-24)16-8-5-15(6-9-16)7-10-19-17-3-1-2-4-18(17)22-23-19/h1-10,21H,11-14H2,(H,22,23)/b10-7+

(E)-(4-(2-(1H-indazol-3-yl)vinyl)phenyl)(piperazin-1-yl)methanone

KX 01, KX2-391; KX-01, KX-2-391; Tirbanibulin; KX2391; KX-2391; Tirbanibulin free base; KXO1; KX 2-391; KX 2391

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.82 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 4% DMSO+30% PEG 300+ddH2O:5 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。