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  3. Temsirolimus

Temsirolimus  (Synonyms: 替西罗莫司; CCI-779)

目录号: HY-50910 纯度: 98.57%
COA 产品使用指南

Temsirolimus 是 mTOR 抑制剂,IC50 值为 1.76 μM。Temsirolimus 能激活自噬 (autophagy),在动物模型中防止心脏功能恶化。

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Temsirolimus Chemical Structure

Temsirolimus Chemical Structure

CAS No. : 162635-04-3

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Top Publications Citing Use of Products

    Temsirolimus purchased from MCE. Usage Cited in: Autophagy. 2019 Jun;15(6):998-1016.  [Abstract]

    The p-MTOR level is significantly suppressed in the experimental group after the mice are treated with CCI-779.

    Temsirolimus purchased from MCE. Usage Cited in: Sci Transl Med. 2018 Jul 18;10(450). pii: eaaq1093.  [Abstract]

    Western blot analysis of selected MAPK and AKT/mTOR pathway components in Trametinib- and Temsirolimus-treated cells.

    Temsirolimus purchased from MCE. Usage Cited in: Genome Med. 2016 Oct 31;8(1):116.  [Abstract]

    Phosphorylation level of RPS6 upon Temsirolimus treatment. The effective target engagement is confirmed in Temsirolimus-treated tumors by showing reduced phosphorylation of downstream mTOR targets, RPS6 and 4EBP1, and an associated increase in autophagy (LC3A/B).

    查看 mTOR 亚型特异性产品:

    • 生物活性

    • 实验参考方法

    • 纯度 & 产品资料

    • 参考文献

    生物活性

    Temsirolimus is an inhibitor of mTOR with an IC50 of 1.76 μM. Temsirolimus activates autophagy and prevents deterioration of cardiac function in animal model[8].

    IC50 & Target[1]

    mTOR

    1.76 μM (IC50)

    体外研究
    (In Vitro)

    Temsirolimus 有效抑制 mTOR 激酶活性,IC50 为 1.76 μM,类似于 Rapamycin 在没有 FKBP12 的情况下 IC50 为 1.74 μM。Temsirolimus (10 nM 至 <5 μM) 通过 FKBP12 依赖性机制显示适度和选择性的抗增殖活性,但可以在低微摩尔浓度 (5-15 μM) 下完全抑制大量肿瘤细胞的增殖,涉及 FKBP12 非依赖性抑制 mTOR 信号。微摩尔而不是纳摩尔浓度 (20 μM) 的 Temsirolimus 处理导致整体蛋白质合成和多核糖体分解的显著下降,同时翻译延伸因子 eEF2 和翻译起始因子 eIF2A 的磷酸化迅速增加[1]
    Temsirolimus 抑制核糖体蛋白 S6 的磷酸化,在 PTEN 阳性 DU145 细胞中比在 PTEN 阴性 PC-3 细胞中更有效,并以浓度依赖性方式抑制两种细胞的细胞生长和克隆形成存活[2]
    Temsirolimus (100 ng/mL) 有效抑制原代人淋巴细胞白血病 (ALL) 细胞的增殖并诱导细胞凋亡[3]

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    体内研究
    (In Vivo)

    CCI-779 (20 mg/kg,腹腔注射) 抑制两种前列腺癌异种移植物的生长,PC-3 肿瘤的生长以剂量依赖性方式受到抑制,并且生长抑制作用大于 DU145 肿瘤[2]。在具有人 ALL 的 NOD/SCID 异种移植模型中,10 mg/kg/天的 Temsirolimus 处理导致外周血母细胞减少和脾肿大[3]。与对照组相比,Temsirolimus (20 mg/kg,ip 5 天/周) 给药后 1 周后 DAOY 异种移植物的生长显著延迟 160%,2 周后延迟 240%。单次大剂量 Temsirolimus (100 mg/kg,ip) 处理可在 1 周内诱导 37% 的肿瘤体积消退。Temsirolimus 处理 2 周还将耐雷帕霉素 U251 异种移植物的生长延迟了 148%[4]。在亨廷顿病小鼠模型中,Temsirolimus 对 mTOR 的抑制可提高四种不同行为任务的表现并减少聚集体形成[5]。给药 Temsirolimus 可诱导显著的剂量依赖性抗肿瘤反应,对抗 8226、OPM-2 和 U266 异种移植物的皮下生长,8226 和 OPM-2 的 ED50 分别为 20 mg/kg 和 2 mg/kg,它们分别与抑制增殖和血管生成、诱导细胞凋亡和缩小肿瘤细胞大小有关[6]

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Clinical Trial
    分子量

    1030.29

    Formula

    C56H87NO16

    CAS 号
    性状

    固体

    颜色

    White to off-white

    运输条件

    Room temperature in continental US; may vary elsewhere.

    储存方式

    4°C, protect from light, stored under nitrogen

    *In solvent : -80°C, 1 years; -20°C, 6 months (protect from light, stored under nitrogen)

    溶解性数据
    In Vitro: 

    Ethanol 中的溶解度 : 200 mg/mL (194.12 mM; 超声助溶)

    DMSO 中的溶解度 : ≥ 100 mg/mL (97.06 mM; 吸湿的 DMSO 对产品的溶解度有显著影响,请使用新开封的 DMSO)

    H2O 中的溶解度 : < 0.1 mg/mL (insoluble)

    * "≥" means soluble, but saturation unknown.

    配制储备液
    浓度 溶剂体积 质量 1 mg 5 mg 10 mg
    1 mM 0.9706 mL 4.8530 mL 9.7060 mL
    5 mM 0.1941 mL 0.9706 mL 1.9412 mL
    查看完整储备液配制表

    * 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液;一旦配成溶液,请分装保存,避免反复冻融造成的产品失效
    储备液的保存方式和期限:-80°C, 1 years; -20°C, 6 months (protect from light, stored under nitrogen)。-80°C储存时,请在1年内使用, -20°C储存时,请在6个月内使用。

    • 摩尔计算器

    • 稀释计算器

    Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

    质量
    =
    浓度
    ×
    体积
    ×
    分子量 *

    Concentration (start) × Volume (start) = Concentration (final) × Volume (final)

    This equation is commonly abbreviated as: C1V1 = C2V2

    浓度 (start)

    C1

    ×
    体积 (start)

    V1

    =
    浓度 (final)

    C2

    ×
    体积 (final)

    V2

    In Vivo:

    请根据您的 实验动物和给药方式 选择适当的溶解方案。

    以下溶解方案都请先按照 In Vitro 方式配制澄清的储备液,再依次添加助溶剂:
    ——为保证实验结果的可靠性,澄清的储备液可以根据储存条件,适当保存;体内实验的工作液,建议您现用现配,当天使用
    以下溶剂前显示的百分比是指该溶剂在您配制终溶液中的体积占比;如在配制过程中出现沉淀、析出现象,可以通过加热和/或超声的方式助溶

    • 方案 一

      请依序添加每种溶剂: 10% EtOH    40% PEG300    5% Tween-80    45% Saline

      Solubility: ≥ 5 mg/mL (4.85 mM); 澄清溶液

      此方案可获得 ≥ 5 mg/mL(饱和度未知)的澄清溶液。

      1 mL 工作液为例,取 100 μL 50.0 mg/mL 的澄清 EtOH 储备液加到 400 μL PEG300 中,混合均匀;再向上述体系中加入 50 μL Tween-80,混合均匀;然后再继续加入 450 μL 生理盐水 定容至 1 mL

      生理盐水的配制:将 0.9 g 氯化钠,溶解于 ddH₂O 并定容至 100 mL,可以得到澄清透明的生理盐水溶液。
    • 方案 二

      请依序添加每种溶剂: 10% EtOH    90% Corn Oil

      Solubility: ≥ 5 mg/mL (4.85 mM); 澄清溶液

      此方案可获得 ≥ 5 mg/mL(饱和度未知)的澄清溶液,此方案实验周期在半个月以上的动物实验酌情使用。

      1 mL 工作液为例,取 100 μL 50.0 mg/mL 的澄清 EtOH 储备液加到 900 μL 玉米油中,混合均匀。

    动物溶解方案计算器
    请输入动物实验的基本信息:

    给药剂量

    mg/kg

    动物的平均体重

    g

    每只动物的给药体积

    μL

    动物数量

    由于实验过程有损耗,建议您多配一只动物的量
    请输入您的动物体内配方组成:
    %
    DMSO +
    +
    %
    Tween-80 +
    %
    Saline
    如果您的动物是免疫缺陷鼠或者体弱鼠,建议 DMSO 中的在最后工作液体系中的占比尽量不超过 2%。
    方案所需 助溶剂 包括:DMSO ,均可在 MCE 网站选购。 Tween 80,均可在 MCE 网站选购。
    计算结果
    工作液所需浓度 : mg/mL
    储备液配制方法 : mg 药物溶于 μL  DMSO(母液浓度为 mg/mL)。

    *In solvent : -80°C, 1 years; -20°C, 6 months (protect from light, stored under nitrogen)

    您所需的储备液浓度超过该产品的实测溶解度,以下方案仅供参考,如有需要,请与 MCE 中国技术支持联系。
    动物实验体内工作液的配制方法 : 取 μL DMSO 储备液,加入 μL  μL ,混合均匀至澄清,再加 μL Tween 80,混合均匀至澄清,再加 μL 生理盐水
    连续给药周期超过半月以上,请谨慎选择该方案。
    请确保第一步储备液溶解至澄清状态,从左到右依次添加助溶剂。您可采用超声加热 (超声清洗仪,建议频次 20-40 kHz),涡旋吹打等方式辅助溶解。
    纯度 & 产品资料

    纯度: 99.56%

    参考文献
    Kinase Assay
    [1]

    The Flag-tagged wild-type human mTOR (Flag-mTOR) DNA constructs are transiently transfected into HEK293 cells. Protein extraction and purification of Flag-mTOR are carried out 48 hours later. In vitro kinase assays of purified Flag-mTOR in the presence of various concentrations of Temsirolimus without FKBP12 are performed in 96-well plate and detected by dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) using His6-S6K1 as the substrate. Enzymes is first diluted in kinase assay buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 50 mM β-glycerophosphate, 10 mM MnCl2, 0.5 mM DTT, 0.25 μM microcystin LR, and 100 μg/mL BSA). To each well, 12 μL of the diluted enzyme is mixed briefly with 0.5 μL Temsirolimus. The kinase reaction is initiated by adding 12.5 μL kinase assay buffer containing ATP and His6-S6K to give a final reaction volume of 25 μL containing 800 ng/mL FLAG-mTOR, 100 μM ATP, and 1.25 μM His6-S6K. The reaction plate is incubated for 2 hours (linear at 1-6 hours) at room temperature with gentle shaking and then terminated by adding 25 μL Stop buffer (20 mM Hepes (pH 7.4), 20 mM EDTA, and 20 mM EGTA). The DELFIA detection of the phosphorylated (Thr-389) His6-S6K is performed at room temperature using a monoclonal anti-P(T389)-p70S6K antibody labeled with Europium-N1-ITC (Eu) (10.4 Eu per antibody). 45 μL of the terminated kinase reaction mixture is transferred to a MaxiSorp plate containing 55 μL PBS. The His6-S6K is allowed to attach for 2 hours after which the wells are aspirated and washed once with PBS. 100 μL of DELFIA buffer with 40 ng/mL Eu-P(T389)-S6K antibody is added. The antibody binding is continued for 1 hour with gentle agitation. The wells are then aspirated and washed four times with PBS containing 0.05% Tween 20 (PBST). 100 μL of DELFIA Enhancement solution is added to each well and the plates are read in a PerkinElmer Victor model plate reader.

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Cell Assay
    [2]

    Survival of prostate cancer cells following various treatments is also determined in a colony-forming assay. Exponentially growing cells are exposed to varying doses of mitoxantrone or docetaxel for 24 hours, or to CCI-779 for 3 days. Following this treatment, the cells are washed and trypsinized. Serial dilutions are plated in 6-well plates in 5 mL medium. The plates are incubated for 10 days at 37°C in an atmosphere containing 5% CO2 at 90% humidity. The plates are then stained with methylene blue and colonies containing >50 cells are counted.

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    Animal Administration
    [2]

    For generation of xenografts, cells are implanted in matrigel; matrigel is stored at −20°C and then thawed on ice at 4°C for 3 hours before use. Cells are gently resuspended in 1 mL of PBS and incubated on ice for 5 minutes. A prechilled pipette is used to transfer cells to the tube containing 1 mL of matrigel, and the cell concentration is adjusted to 3×107/mL. The cells (3×106 in 0.1 mL) are injected s.c. into both flanks of mice using a 25-gauge needle. When xenografts grew to a size of about 5 mm in diameter, animals are assorted randomLy into groups of 10 mice. The following experiments are conducted: Mice bearing PC-3 tumors are treated with CCI-779 (1, 5, 10, and 20 mg per kg per day), or vehicle solution for 3 or 5 days per week for 3 weeks. Mice bearing DU145 tumors are only treated with CCI-779 (20 mg per kg per day) or vehicle solution for 3 weeks. Mice bearing PC-3 tumors receive the following treatments: (a) control, vehicle solution for CCI-779; (b) chemotherapy alone, mitoxantrone 1.5 mg/kg or docetaxel 10 mg/kg is injected i.p. weekly for 3 doses; (c) CCI-779 alone, 5 or 10 mg/kg is injected i.p. daily, three times a week for 3 weeks; (4) chemotherapy followed by CCI-779.

    MCE has not independently confirmed the accuracy of these methods. They are for reference only.

    参考文献

    完整储备液配制表

    * 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液;一旦配成溶液,请分装保存,避免反复冻融造成的产品失效
    储备液的保存方式和期限:-80°C, 1 years; -20°C, 6 months (protect from light, stored under nitrogen)。-80°C储存时,请在1年内使用, -20°C储存时,请在6个月内使用。

    可选溶剂 浓度 溶剂体积 质量 1 mg 5 mg 10 mg 25 mg
    DMSO / Ethanol 1 mM 0.9706 mL 4.8530 mL 9.7060 mL 24.2650 mL
    5 mM 0.1941 mL 0.9706 mL 1.9412 mL 4.8530 mL
    10 mM 0.0971 mL 0.4853 mL 0.9706 mL 2.4265 mL
    15 mM 0.0647 mL 0.3235 mL 0.6471 mL 1.6177 mL
    20 mM 0.0485 mL 0.2427 mL 0.4853 mL 1.2133 mL
    25 mM 0.0388 mL 0.1941 mL 0.3882 mL 0.9706 mL
    30 mM 0.0324 mL 0.1618 mL 0.3235 mL 0.8088 mL
    40 mM 0.0243 mL 0.1213 mL 0.2427 mL 0.6066 mL
    50 mM 0.0194 mL 0.0971 mL 0.1941 mL 0.4853 mL
    60 mM 0.0162 mL 0.0809 mL 0.1618 mL 0.4044 mL
    80 mM 0.0121 mL 0.0607 mL 0.1213 mL 0.3033 mL
    Ethanol 100 mM 0.0097 mL 0.0485 mL 0.0971 mL 0.2427 mL
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