Survivin (IC50 = 0.54 nM)

即使浓度为 30 μM，YM155 对存活基因启动子驱动的荧光素酶报告基因活性也不敏感。通过对生存素基因启动子的转录抑制，YM155 显着降低缺乏 p53 的人 HRPC 细胞系 PC-3 和 PPC-1 中的内源生存素表达。另一方面，100 nM 的 YM155 对 c-IAP2、XIAP、Bcl-2、Bcl-xL、Bad、α-肌动蛋白和 β-微管蛋白的蛋白表达影响不充分。随着 caspase-3 活性的相应升高，YM155 在人类癌细胞系（例如 PC-3 和 PPC-1）中显示出显着的细胞凋亡。人类癌细胞系 PC-3、PPC-1、DU145、TSU-Pr1、22Rv1、SK-MEL-5 和 A375 均被 YM155 有效抑制（IC50 值分别为 2.3 至 11 nM）。 1] YM155 增加 NSCLC 细胞对 γ 辐射的敏感性。 YM155 和 β 辐射的组合可增强 Caspase-3 活性和凋亡细胞数量。 YM155 推迟了辐射引起的核 DNA 双链断裂。[2]

在 3 和 10 mg/kg 的剂量下，YM155 完全抑制 PC-3 sc 异种移植前列腺肿瘤的生长，而不会导致体重减轻或血细胞计数下降。根据药代动力学分析，YM155 广泛分布在整个肿瘤组织中。此外，在 PC-3 原位异种移植物中，YM155 在 5 mg/kg 剂量下表现出 80% TGI。 [1] 在裸鼠中，YM155 和 γ 放射联合治疗对 H460 或 Calu6 异种移植物表现出强大的抗肿瘤活性。 [2]

使用 Pyrobest 聚合酶和以下引物，通过 PCR 从人类基因组 DNA 中分离出 2,767 bp 的人类生存素基因启动子序列：5

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γ-H2AX免疫荧光染色。[2]
细胞在两孔Lab Tec小室载玻片（Nunc）中生长至50%融合，然后在50 nmol/LYM155或载体存在下培养48小时，然后暴露于3 Gy的γ辐射。在此后的不同时间，它们在室温下用4%多聚甲醛固定10分钟，在4°C下用0.1%Triton X-100渗透10分钟，并在室温下暴露于5%脱脂奶粉中10分钟。用PBS洗涤载玻片，然后在室温下首先用1:300稀释的组蛋白γ-H2AX小鼠单克隆抗体孵育2小时，然后用1:700稀释的Alexa 488标记的小鼠IgG山羊抗体孵育1小时。将载玻片安装在荧光安装介质中，用配备LSM5 PASCAL系统的共聚焦激光扫描显微镜观察荧光信号。以×100的放大倍数检查了三个随机区域，每个区域包含=50个细胞。如前所述，将含有≥10个免疫反应灶的核计数为γ-H2AX阳性，并计算阳性细胞的百分比[2]。

将细胞以 5-40 × 103 的密度接种在 96 孔板中。将溶解有 YM155 的 DMSO 给予细胞 48 小时。然后，使用磺基罗丹明 B 测定法计算细胞计数。台盼蓝排斥染色用于测定细胞活力。[1]

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在有或没有YM155的情况下培养48小时后，通过胰蛋白酶消化和离心（0.05%胰蛋白酶-EDTA）收集PC-3和PPC-1细胞，并将其重新悬浮在DMEM中。将细胞悬浮液用等体积的台盼蓝（0.4%工作溶液）稀释。在血细胞计数器上计数活细胞（未染色）和死细胞（染色），活细胞与细胞总数的比率表示为活细胞百分比。

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半胱氨酸天冬氨酸蛋白酶-3活性的测量。[1]
根据制造商的说明，用CPP32/SCaspase-3荧光蛋白酶检测试剂盒（MBL）测量半胱氨酸天冬氨酸蛋白酶-3活性。在用YM155孵育48小时后，PC-3和PPC-1细胞在100μL细胞裂解缓冲液（随试剂盒提供）中裂解，得到等体积（50μL）的细胞裂解液（100μg蛋白质）。加入2倍反应缓冲液后，将混合物加入黑色96孔板中。然后以5 mL/孔的速度加入DEVD-AFC底物（附有试剂盒），并将混合物在37°C下孵育30分钟。用荧光分光光度计在390 nm的激发波长和460 nm的发射波长下对荧光发射进行定量。

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体外细胞生长抑制试验。[1]
YM155的抗增殖活性通过国家癌症研究所使用的方法进行测量。用YM155处理48小时后，通过磺基罗丹明B测定细胞计数。GI50值通过逻辑分析计算，即药物浓度导致对照细胞在药物孵育过程中净蛋白质增加（通过硫罗丹明B染色测量）减少50%。该测定一式三份，平均GI50值由四次独立测定的结果得出。

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体外抗肿瘤活性的时间依赖性。[1]
A549细胞用YM155、甲氨蝶呤或阿霉素处理。通过用新鲜培养基洗涤五次，在不同长度的暴露时间后去除每种化合物。在细胞处理开始后的72小时温育时，用Alamar Blue测定法（Serotec；参考文献22）测定所选化合物对细胞增殖的影响，每种浓度（n=1）两次。通过逻辑分析计算IC50。为了了解每种化合物的体外作用模式和药效学特性，以对数标度绘制了YM155、阿霉素（作为曲线下面积依赖性药物）和甲氨蝶呤（作为时间相关药物）的对数斜率（暴露时间）和对数斜率的倒数（A549抗增殖作用的IC50），并比较了三种药物的IC50暴露时间曲线的斜率。

PC-3 s.c. (orthotopic) xenografts in male nude mice (BALB/c nu/nu)
5 mg/kg
Subcutaneous injection as a 3-day continuous infusion per week for 3 weeks by an implanted micro-osmotic pump.

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For the in vivo experiments, YM155 was dissolved and diluted in saline immediately before administration.
In vivo antitumor activities against PC-3 s.c. xenograft model.[1]
Five-week-old male nude mice (BALB/c nu/nu) were used. PC-3 cells (2 × 106–3 × 106) were injected into the flanks of the mice and allowed to reach a tumor volume of >100 mm3 in tumor volume (length × width2 × 0.5). YM155 was s.c. administered as a 3-day continuous infusion per week for 2 weeks using an implanted micro-osmotic pump (Alzet model 1003D, Durect) or i.v. administered five times a week for 2 weeks. The percentage of tumor growth inhibition 14 days after initial YM155 administration was calculated for each group using the following formula: MTV = 100 × {1 − [(MTV of the treated group on day 14) − (MTV of the treated group on day 0)] / [(MTV of the control group on day 14) − (MTV of the control group on day 0)]}, where MTV is mean tumor volume. For both the frozen tumors and plasma samples, survivin expression levels were analyzed by Western blotting and YM155 drug concentration by high-performance liquid chromatography/triple quadrupole mass spectrometry (LC/MS/MS) using validated methods.

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In vivo antitumor activities against PC-3 orthotopic xenograft model. [1]
For orthotopic implantation, a PC-3 cell suspension (1 × 106/20 μL per mouse) was injected into the prostate dorsolateral lobe (right side) of 7-week-old male nude mice (BALB/c nu/nu). Two weeks after implantation, YM155 was s.c. administered as a 3-day continuous infusion per week for 3 weeks at 5 mg/kg/d using an implanted micro-osmotic pump (Alzet model 1003D, Durect). Body weight was measured periodically starting from the first day of YM155 administration. Three weeks later, the tumors adhering to the prostate and seminal vesicle were taken and weighed as tumor weight. The antitumor activity of YM155 was expressed as a percentage of tumor growth inhibition calculated using the following formula: MTW = 100 × [1 − (MTW on day 21 of the treated group) / (MTW on day 21 of the control group)].

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Evaluation of tumor growth in vivo. [2]
Tumor cells (2 × 106) were injected s.c. into the right hind leg of 6-week-old female athymic nude mice (BALB/c nu/nu). Tumor volume was determined from caliper measurement of tumor length (L) and width (W) according to the formula LW2/2. Treatment was initiated when the tumors in each group of animals achieved an average volume of ∼200 to 250 mm3. Treatment groups (each containing eight mice) consisted of vehicle control (physiologic saline), YM155 alone, vehicle plus radiation, and YM155 plus radiation. Vehicle or YM155 at a dose of 5 mg/kg of body mass was administered over 7 consecutive days (days 1-7) with the use of an implanted micro-osmotic pump (Alzet model 1003D; Durect). Mice in the radiation groups received 10 Gy of γ-radiation from a cobalt irradiator either as a single fraction on day 3 of drug treatment or fractionated over 5 consecutive days (days 3 to 7); the radiation was targeted to the tumor, with the remainder of the body shielded with lead. Growth delay (GD) was calculated as the time required to achieve a 5-fold increase in volume for treated tumors minus that for control tumors. The enhancement factor was then determined as: (GDcombination − GDYM155)/GDradiation.[2]

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Luciferase-expressing RENCA cells were implanted to the subrenal capsule of the left kidney and tail vein, respectively, to produce a mouse RCC model with an orthotopic tumor and metastatic lung tumors. The mice were randomly divided into four groups with an even distribution of IVIS values. Group 1 received intraperitoneal (IP) injection of 100 μL of phosphate-buffered saline (PBS) as a vehicle control; group 2 received YM155 alone (1 mg/kg body weight per day for 1 week by IP injection); group 3 received IL-2 alone (6000 U of recombinant IL-2 by IP injection on days 0, 4, and 8 of treatment); and group 4, the combination therapy group, received YM155 and IL-2 (dose and dosing schedule the same as in group 2 plus group 3). Tumor imaging was performed and tumor volume was analyzed in all groups on day 14 post-treatment. The mice were sacrificed, and the weights of the orthotopic tumor in the left kidney and sections of bilateral lung tissues with metastatic growth were measured.[3]

[1]. YM155, a novel small-molecule survivin suppressant, induces regression of established human hormone-refractory prostate tumor xenografts. Cancer Res. 2007 Sep 1;67(17):8014-21.

[2]. Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines. Clin Cancer Res. 2008 Oct 15;14(20):6496-504.

[3]. A combination of YM-155, a small molecule survivin inhibitor, and IL-2 potently suppresses renal cell carcinoma in murine model. Oncotarget. 2015 Aug 28;6(25):21137-47.

Sepantronium bromide is an organic bromide salt consisting of sepantronium cations and bromide anions. It has been found to selectively inhibit survivin (BIRC5) gene promoter activity and to down-regulate survivin in vitro, so leading to induction of apoptosis. It has a role as an antineoplastic agent, a survivin suppressant and an apoptosis inducer. It contains a sepantronium.
Sepantronium Bromide is a small-molecule proapoptotic agent with potential antineoplastic activity. Sepantronium bromide selectively inhibits survivin expression in tumor cells, resulting in inhibition of survivin antiapoptotic activity (via the extrinsic or intrinsic apoptotic pathways) and tumor cell apoptosis. Survivin, a member of the inhibitor of apoptosis (IAP) gene family, is expressed during embryonal development and is absent in most normal, terminally differentiated tissues; upregulated in a variety of human cancers, its expression in tumors is associated with a more aggressive phenotype, shorter survival times, and a decreased response to chemotherapy.

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Various accumulating evidence suggests that survivin, a member of the inhibitor of apoptosis (IAP) family, plays an important role in drug resistance and cancer cell survival in many types of cancer, including hormone-refractory prostate cancer (HRPC). Here, we characterized YM155, a novel small-molecule survivin suppressant, using a survivin gene promoter activity assay. YM155 suppressed expression of survivin and induced apoptosis in PC-3 and PPC-1 human HRPC cell lines at 10 nmol/L. In contrast, YM155 up to 100 nmol/L showed little effect on expression levels of other IAP- or Bcl-2-related proteins. In a s.c. xenografted PC-3 tumor model in mice, 3-day continuous infusions of YM155 at 3 to 10 mg/kg induced massive tumor regression accompanied by suppression of intratumoral survivin. YM155 also completely inhibited the growth of orthotopically xenografted PC-3 tumors. No significant decreases in body weight were observed in mice treated with YM155 during the experimental period. Pharmacokinetic analyses indicated that YM155 is highly distributed to tumors and at concentrations approximately 20-fold higher than those in plasma. Our findings represent the first attempt to show tumor regression and suppression of survivin in p53-deficient human HRPC cells by a single small molecular compound treatment. Further extensive investigation of YM155 in many types of cancer, including HRPC, seems to be worthwhile to develop this novel therapeutic approach.[1]

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Purpose: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non-small cell lung cancer (NSCLC) cell lines to gamma-radiation. Experimental design: The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. Results: YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to gamma-radiation in vitro. The combination of YM155 and gamma-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone gamma-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and gamma-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. Conclusions: These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.[2]

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YM155, a small molecule inhibitor of the antiapoptotic protein survivin, has been developed as a potential anti-cancer drug. We investigated a combination therapy of YM155 and interleukin-2 (IL-2) in a mouse model of renal cell carcinoma (RCC). YM155 caused cell cycle arrest and apoptosis in renal cancer (RENCA) cells. Next, luciferase-expressing RENCA cells were implanted in the left kidney and the lung of BALB/c mice to develop RCC metastatic model. In this orthotopic renal and metastatic lung tumors models, YM155 and IL-2 additively decreased tumor weight, lung metastasis, and luciferin-stained tumor images. Also, the combination significantly suppressed regulatory T cells and myeloid-derived suppressor cells compared with single agent treatment. We suggest that a combination of YM155 and IL-2 can be tested as a potential therapeutic modality in patients with RCC.[3]

C20H19BRN4O3

443.29

442.064

C, 54.19; H, 4.32; Br, 18.03; N, 12.64; O, 10.83

781661-94-7

Sepantronium hydrochloride;355406-09-6

11178236

White Solid powder

77.96

0

6

5

28

571

0

[Br-].O=C1C2=C([H])C([H])=C([H])C([H])=C2C(C2=C1N(C([H])([H])C([H])([H])OC([H])([H])[H])C(C([H])([H])[H])=[N+]2C([H])([H])C1C([H])=NC([H])=C([H])N=1)=O

QBIYUDDJPRGKNJ-UHFFFAOYSA-M

InChI=1S/C20H19N4O3.BrH/c1-13-23(9-10-27-2)17-18(24(13)12-14-11-21-7-8-22-14)20(26)16-6-4-3-5-15(16)19(17)25;/h3-8,11H,9-10,12H2,1-2H3;1H/q+1;/p-1

1-(2-methoxyethyl)-2-methyl-3-(pyrazin-2-ylmethyl)benzo[f]benzimidazol-3-ium-4,9-dione;bromide

YM-155; Sepantronium bromide; YM155; Sepantronium bromide; 781661-94-7; YM155; YM-155; Ym 155; YM155 (Sepantronium Bromide); Sepantronium (bromide); 4,9-Dihydro-1-(2-methoxyethyl)-2-methyl-4,9-dioxo-3-(2-pyrazinylmethyl)-1H-naphth[2,3-d]imidazolium bromide; YM 155

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

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

配方 1 中的溶解度: ≥ 2 mg/mL (4.51 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 20.0 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: 2 mg/mL (4.51 mM) 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; 超声助溶.
例如，若需制备1 mL的工作液，可将 100 μL 20.0 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 3 中的溶解度: Saline: 30mg/mL .

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配方 4 中的溶解度: 50 mg/mL (112.79 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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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。