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 exhibits differential effects on growth inhibition, but is not profoundly cytotoxic in a panel of human cancer cell lines. When compared to SW620 cells, MCF7 cells exhibit more potent inhibition of DNA-PK and PI-3K. In MCF7 cells, exposure to 1 mM KU-0060648 for five days significantly reduces cell proliferation by more than 95%, but only by 55% in SW620 cells. In clonogenic survival assays, KU-0060648 increases etoposide and doxorubicin's cytotoxicity across a panel of DNA-PKcs-proficient cells, but not in DNA-PKcs-deficient cells, demonstrating that the increased cytotoxicity of the topoisomerase II poisons is caused by DNA-PK inhibition. [1]

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]

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KU-0060648 浓度依赖性地抑制细胞中DNA-PK在Ser2056位点的自磷酸化，在MCF7乳腺癌细胞中的IC₅₀为0.02 µM，在SW620结肠癌细胞中的IC₅₀为0.2 µM。[1]
它也抑制PI-3K介导的AKT在Ser473位点的磷酸化，在MCF7细胞中的IC₅₀为0.04 µM。然而，其在SW620细胞中对PI-3K的抑制活性很弱 (IC₅₀ >10 µM)，表明其抑制具有细胞系依赖性。[1]
连续暴露5天可抑制一系列人乳腺癌（MCF7, T47D, MDA-MB-231）和结肠癌（LoVo, SW620）细胞系的增殖。生长抑制 (GI₅₀) 值范围从0.21 µM (LoVo) 到1 µM (MDA-MB-231 和 SW620)。[1]
KU-0060648 (1 µM，暴露16小时) 单药细胞毒性较低（在大多数细胞系中存活率 >80%，MDA-MB-231除外，为41%）。[1]
它显著增强了拓扑异构酶II毒物依托泊苷和多柔比星的细胞毒性。在DNA-PKcs正常的细胞系 (V3-YAC, M059-Fus-1) 中，该化合物将依托泊苷的细胞毒性增强了4至13倍，将多柔比星的细胞毒性增强了高达32倍。而在DNA-PKcs缺陷的细胞系 (V3, M059J) 中，这种增强作用微乎其微，证实化学增敏作用主要归因于DNA-PK的抑制。[1]
在一组人类癌细胞系中，KU-0060648 (1 µM) 将依托泊苷的细胞毒性最高增强了105倍（在SW620细胞中），将多柔比星的细胞毒性最高增强了107倍（在MCF7细胞中）。[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]

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在携带MCF7乳腺癌异种移植瘤的小鼠中，单用KU-0060648治疗 (10 mg/kg 腹腔注射，每日两次，持续14天) 导致中位肿瘤生长延迟30天。在SW620结肠癌异种移植瘤中未观察到显著的单一药物活性。[1]
KU-0060648 与依托泊苷磷酸酯联用显著增强了抗肿瘤活性。在MCF7移植瘤中，联合用药导致中位生长延迟55天，而单独使用依托泊苷为38天，单独使用KU-0060648为30天。[1]
在SW620移植瘤中，当KU-0060648与依托泊苷联用时，特别是在抑制剂每日给药两次的情况下，观察到活性增强的趋势。[1]

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。

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DNA-PK和PI-3K细胞活性测定： 细胞用一系列浓度的KU-0060648处理1小时。对于DNA-PK抑制评估，随后用X射线照射细胞（10 Gy），30分钟后裂解。对于PI-3K抑制评估，在1小时预孵育后，用胰岛素样生长因子-1 (50 ng/ml) 处理细胞30分钟。制备细胞裂解液，使用特异性抗体通过蛋白质印迹法测定磷酸化DNA-PKcs (Ser2056) 或磷酸化AKT (Ser473) 相对于总蛋白的水平。进行光密度分析以计算抑制百分比和IC₅₀值。[1]
克隆形成存活实验： 为了评估细胞毒性和化学增敏作用，将培养板中的细胞暴露于依托泊苷或多柔比星，同时使用或不使用KU-0060648 (1 µM)，持续16小时。然后收集细胞，在无药培养基中接种到培养皿中，并使其形成克隆10-14天。对克隆进行染色和计数。[1]
生长抑制 (SRB) 实验： 细胞连续暴露于KU-0060648 5天。然后使用磺酰罗丹明B (SRB) 比色法测量细胞密度以确定生长抑制 (GI₅₀)。[1]

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.

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Pharmacokinetic Study: KU-0060648 was formulated in a vehicle of equimolar phosphoric acid, adjusted to pH 5 with sterile saline. Female Balb C mice received a single dose of 10 mg/kg via intravenous (i.v.), intraperitoneal (i.p.), or oral (p.o.) routes. Blood was collected at various time points up to 360 minutes for plasma concentration analysis.[1]
Tumor Distribution Study: Female athymic mice bearing MCF7 or SW620 subcutaneous xenografts (~650 mm³) received a single i.v. dose of KU-0060648 (12.5 mg/kg). Mice were sacrificed at 60 or 240 minutes post-dose. Tumors were excised, homogenized in PBS, and analyzed for drug concentration alongside plasma.[1]
Ex vivo DNA-PK Activity Assay: SW620 tumor-bearing mice received a single i.v. dose of KU-0060648 (2.5 or 25 mg/kg) or vehicle. Mice were sacrificed at 1 or 4 hours post-dose. Tumors were excised and homogenized. DNA-PK activity in the homogenate was determined by measuring the DNA-PK-dependent phosphorylation of a p53 peptide substrate (Ser15) using an ELISA-based assay.[1]
Efficacy Study in Xenografts: Mice bearing established SW620 or MCF7 subcutaneous xenografts were treated when tumors reached approximately 5x5 mm.
For SW620 model: Animals received vehicle (control), KU-0060648 alone (10 mg/kg i.p., once or twice daily for 5 days), etoposide phosphate alone (11.35 mg/kg i.p., once daily for 5 days), or the combination.
For MCF7 model: Animals received vehicle, KU-0060648 alone (10 mg/kg i.p., twice daily for 14 days), etoposide phosphate alone (11.35 mg/kg i.p., once daily for 5 days), or the combination, with the first dose of KU-0060648 given immediately prior to etoposide.[1]

表2列出了通过不同途径向Balb/C小鼠给予10 mg/kg KU-0060648后测定的血浆药代动力学参数。口服给药后KU-0060648的生物利用度≥100%。腹腔注射给药后KU-0060648的药代动力学参数与静脉注射给药相似，生物利用度为78%。[1]

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静脉注射给药后KU-0060648在MCF7和SW620荷瘤小鼠体内的组织分布[1]
向携带MCF7或SW620异种移植瘤的小鼠静脉注射KU-0060648（12.5 mg/kg）后，KU-0060648广泛分布于肿瘤组织，并在血浆清除后仍保留在肿瘤组织中（表2）。 KU-0060648 在肿瘤中的浓度维持在 1 μM 以上（该浓度在体外可产生化疗增敏作用），且至少持续 4 小时。小鼠单次静脉注射 10 mg/kg 剂量后，KU-0060648 的血浆峰浓度 (Cmax) 为 19 µg/ml（5 分钟），消除半衰期 (T₁/₂) 为 102 分钟，清除率 (CL) 为 41 ml/kg/min。[1] 口服给药 (10 mg/kg po) 的生物利用度接近 100% (119%)，Tmax 为 120 分钟，T₁/₂ 为 142 分钟。[1] 腹腔注射给药 (10 mg/kg ip) 的生物利用度为 78%，Tmax 为 30 分钟。半衰期为 106 分钟。[1]
在荷瘤小鼠中静脉注射 12.5 mg/kg 剂量后，KU-0060648 广泛分布于 MCF7 和 SW620 异种移植瘤中。血浆清除后，肿瘤浓度至少维持在 1 µM 以上（体外化疗增敏的有效浓度）。[1]

在MCF7异种移植疗效研究中，KU-0060648单药治疗的毒性可忽略不计（最大体重下降3%）。与依托泊苷磷酸酯联合用药时，最大体重下降7%，该结果被认为是可接受的。[1]
在SW620异种移植研究中，KU-0060648单药治疗或依托泊苷磷酸酯单药治疗均未引起显著的体重下降。二者联合用药时，最大体重下降11%。[1]

[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-乙基-1-哌嗪基)-N-[4-[2-(4-吗啉基)-4-氧代-1-苯并吡喃-8-基]-1-二苯并噻吩基]乙酰胺是二苯并噻吩类化合物。

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KU-0060648 是一种双重 ATP 竞争性 DNA-PK 和 PI-3K 抑制剂，由 LY294002 药效团开发而来。它对其他PIKK家族成员（如ATM、ATR和mTOR）具有选择性。[1]
它对拓扑异构酶II抑制剂（依托泊苷、阿霉素）的化疗增敏作用主要归因于DNA-PK的抑制，从而削弱了DNA双链断裂的非同源末端连接（NHEJ）修复途径。[1]
在MCF7异种移植瘤（携带PIK3CA突变）中观察到了单药抗肿瘤活性，但在SW620异种移植瘤中未观察到，这表明PI-3K通路激活的肿瘤可能对其敏感。[1]
该研究为DNA-PK和PI-3K双重抑制的治疗策略提供了原理验证。[1]

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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。