KU-0060648

PI3Kα (IC50 = 4 nM); PI3Kβ (IC50 = 0.5 nM); PI3Kγ (IC50 = 0.594 μM);PI3Kδ (IC50 = 0.1 nM); DNA-PK (IC50 = 8.6 nM)

KU-0060648 对生长抑制表现出不同的作用，但在一组人类癌细胞系中没有明显的细胞毒性。与 SW620 细胞相比，MCF7 细胞表现出更有效的 DNA-PK 和 PI-3K 抑制作用。在 MCF7 细胞中，暴露于 1 mM KU-0060648 5 天，细胞增殖显着降低 95% 以上，但在 SW620 细胞中仅降低 55%。在克隆生存测定中，KU-0060648 在一组 DNA-PKcs 熟练的细胞中增加依托泊苷和阿霉素的细胞毒性，但在 DNA-PKcs 缺陷的细胞中则不然，这表明拓扑异构酶 II 毒物的细胞毒性增加是由 DNA-PK 引起的抑制。 [1]

KU-0060648 可增加 MCF7 和 SW620 异种移植模型中依托泊苷的抗肿瘤活性，并且在 MCF7 异种移植模型中具有单药活性。 [1]

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在体内，腹腔注射KU-0060648显著抑制了裸鼠体内HepG2异种移植物的生长。AKT-mTOR活化在异种移植肿瘤中也受到抑制。最后，我们发现DNA-PKcs在人HCC组织中的表达显著上调。[2]

KU-0060648抗DNA-PK和PI-3K细胞活性的测定[1]
在X射线照射（10 Gy）前，在暴露于不同浓度KU-0060648 1小时的细胞中测定DNA-PK自磷酸化。30分钟后，根据制造商的说明，使用磷安全提取试剂制备细胞裂解物。通过蛋白质印迹法测定Ser2056处DNA-PKcs相对于未磷酸化DNA-PKcs的自磷酸化水平。为了测定PI-3K活性，在用50ng/ml胰岛素样生长因子-1处理30分钟之前，将细胞暴露于不同浓度的KU-0060648中1小时。通过蛋白质印迹法测定PI-3K依赖性AKT磷酸化（Ser473）相对于非磷酸化AKT的水平。

细胞毒性和生长抑制研究[1]
通过克隆试验测量细胞毒性。在6孔板中生长的细胞在收获和接种到直径10cm的无药物培养基中的皮氏培养皿之前，暴露于含有或不含KU-0060648（1μM）的依托泊苷或阿霉素16小时。10至14天后，用结晶紫对菌落进行染色，并用自动菌落计数器计数。如前所述，通过SRB测定法测定连续暴露于KU-0060648 5天后的细胞生长抑制。GI50是导致50%细胞生长抑制的浓度。

human-tumor SW620 or MCF7 xenograft models
10 mg/kg, twice daily
i.p.

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KU-0060648 plasma pharmacokinetics following different routes of administration[1]
All in vivo experiments were reviewed and approved by the relevant institutional animal welfare committees and performed according to national law. We determined the plasma pharmacokinetics of KU-0060648 following administration intravenously (i.v.), intra-peritoneally (i.p.) or orally (p.o.) at 10 mg/kg to female Balb C mice. KU-0060648 was formulated in a vehicle of equimolar phosphoric acid, made up to volume with sterile saline and at final pH 5. Mice were killed at intervals up to 360 minutes after KU-0060648 administration and plasma concentrations of KU-0060648 were determined by LC-MS/MS analysis, as previously described. KU-0060648 distribution to tumour xenografts[1]
Female athymic mice were maintained and handled in isolators under specific pathogen free conditions for tissue distribution and efficacy studies. KU-0060648 (12.5 mg/kg i.v.) was administered to MCF7 or SW620 tumour-bearing mice (650 mm3), which were killed 60 or 240 minutes later. Tumours were excised and homogenised in PBS (1:3 w/v), using a stirrer mascercarator homogenizer , in 10 second bursts, on ice. Plasma and tumour KU-0060648 concentrations were determined by LC-MS/MS analysis, as previously described.

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DNA-PK ex vivo pharmacodynamic assay[1]
KU-0060648 at 2.5 or 25 mg/kg or vehicle alone was administered to SW620 tumour-bearing mice i.v.. After 1 or 4 hours, animals were killed and tumours were excised and homogenised. DNA-PK activity within tumour homogenates was determined by measuring the DNA-PK-dependent phosphorylation of a p53 peptide substrate (Ser15), using an ELISA assay, as described previously.

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Anti-tumour efficacy study[1]
Mice bearing SW620 or MCF7 xenografts s.c. (n = 5 per group) were treated when tumours were palpable (approx 5 mm × 5 mm, 8-10 days post implantation). Animals received normal saline i.p. once daily (control), single agent KU-0060648 10 mg/kg i.p. twice daily for either 5 days (2 × d × 5) in SW620 tumour-bearing, or 14 days (2 × d × 14) in MCF7 tumour bearing mice with doses on each day 8 hours apart, or etoposide phosphate once daily i.p. (11.35 mg/kg in saline, equivalent to 10 mg/kg free etoposide, i.p., d × 5). For combinations, KU-0060648 was administered i.p. once or twice daily for 5 days (SW620) or once daily for 14 days (MCF7), with the first dose immediately prior to etoposide phosphate.

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In vivo anti-tumor efficiency assay[2]
A significant amount of HepG2 cells (5 millions/mice) were injected subcutaneously into the right flanks of female nude mice (6-8 weeks old). When tumors reached around 100 mm3, mice were randomized into three groups with 12 mice per group: vehicle control (saline), 10 mg/kg of KU-0060648 (intraperitoneal injection or i.p., daily, for 21 days), and 50 mg/kg of KU-0060648 (i.p., daily, for 21 days). The injection was started when the tumors were established (volumes around 100 mm3). Tumor volumes, recorded every week, were calculated through the established formula: Volume (mm3) = (d2 × D)/2, in which d and D were the shortest and the longest diameter, respectively. Two weeks after initial KU-0060648 administration, xenografted tumors of two mice per group were isolated, and were subjected to Western blotting and immunohistochemistry (IHC) staining assays. Humane endpoints were applied to minimize suffering. Five weeks after initial KU-0060648 administration, HepG2 xenografts were separated through surgery and weighted.

The plasma pharmacokinetic parameters determined after administration of 10 mg/kg KU-0060648 to Balb/C mice by various routes are given in Table 2. The percentage bioavailability of KU-0060648 following p.o administration was found to be ≥100% The pharmacokinetic parameters of KU-0060648 following i.p. administration were found to be similar to that when given i.v., with 78% bioavailability.[1]

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Tissue distribution of KU-0060648 in MCF7 and SW620 tumour-bearing mice following i.v. administration[1]
Following administration of KU-0060648 (12.5 mg/kg i.v.) to mice bearing either MCF7 or SW620 xenografts, KU-0060648 distributed extensively to the tumour and was retained after clearance from the plasma (Table 2). Concentrations of KU-0060648 of over 1 μM (a level resulting in chemosensitisation in vitro) were maintained in the tumour for at least 4 hours.

[1]. Chemosensitization of cancer cells by KU-0060648, a dual inhibitor of DNA-PK and PI-3K. Mol Cancer Ther. 2012 Aug;11(8):1789-98.

[2]. KU-0060648 inhibits hepatocellular carcinoma cells through DNA-PKcs-dependent and DNA-PKcs-independent mechanisms. Oncotarget. 2016 Mar 29;7(13):17047-59.

2-(4-ethyl-1-piperazinyl)-N-[4-[2-(4-morpholinyl)-4-oxo-1-benzopyran-8-yl]-1-dibenzothiophenyl]acetamide is a member of dibenzothiophenes.

C33H34N4O4S

582.7125

582.23

C, 68.02; H, 5.88; N, 9.61; O, 10.98; S, 5.50

881375-00-4

881375-00-4

11964036

Off-white to brown solid powder

1.3±0.1 g/cm3

819.9±65.0 °C at 760 mmHg

449.7±34.3 °C

0.0±3.0 mmHg at 25°C

1.694

5.56

106.5

1

8

6

42

1010

0

O=C1C2C=CC=C(C=2OC(N2CCOCC2)=C1)C1C2SC3C(C=2C(NC(CN2CCN(CC)CC2)=O)=CC=1)=CC=CC=3

AATCBLYHOUOCTO-UHFFFAOYSA-N

InChI=1S/C33H34N4O4S/c1-2-35-12-14-36(15-13-35)21-29(39)34-26-11-10-23(33-31(26)25-6-3-4-9-28(25)42-33)22-7-5-8-24-27(38)20-30(41-32(22)24)37-16-18-40-19-17-37/h3-11,20H,2,12-19,21H2,1H3,(H,34,39)

2-(4-ethylpiperazin-1-yl)-N-[4-(2-morpholin-4-yl-4-oxochromen-8-yl)dibenzothiophen-1-yl]acetamide

KU0060648; KU 0060648; LM6DZS6PYA; 2-(4-ethylpiperazin-1-yl)-N-[4-(2-morpholin-4-yl-4-oxochromen-8-yl)dibenzothiophen-1-yl]acetamide; CHEMBL1086377; 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thiophen-1-yl)acetamide; KU-0060648

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)

DMSO: 2~2.8 mg/mL (3.4~4.8 mM)

配方 1 中的溶解度: ≥ 0.28 mg/mL (0.48 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 2.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.28 mg/mL (0.48 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 2.8mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 0.28 mg/mL (0.48 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 2.8 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 30% propylene glycol, 5% Tween 80, 65% D5W: 20 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。