Ganetespib (STA-9090)   中文名称: 伽那司匹

HSP90

在基因组特征的 NSCLC 细胞系中，genesetib 诱导细胞凋亡、抑制下游信号传导、耗尽受体酪氨酸激酶并抑制增殖，IC50 值范围为 2 至 30 nM。此外，在通过产生 EGFR 和 ERBB2 突变体而变得独立于 IL-3 的同基因 Ba/F3 pro-B 细胞中，genetecib 的效力大约高出 20 倍[1]。在体外，genetecib 在导致已知 Hsp90 客户蛋白降解方面比安沙霉素抑制剂 17-allylamino-17-demethoxygeldanamycin (17-AAG) 更有效。它还在多种实体瘤和血液肿瘤细胞系中表现出强大的细胞毒性[2]。强HSP90抑制剂genesetespib已被证明可以在体外破坏犬肿瘤细胞系[3]。在 HEL92.1.7 细胞中，与 P6 和 17-AAG 相比，genesetib 表现出更有效或更长时间的 JAK/STAT 抑制活性[4]。

在 NCI-H1975 异种移植物中，genetecib（125 mg/kg，IV）在肿瘤中的蓄积量高于在正常组织中的蓄积量，并且比 17-AAG 具有更高的体内疗效，且不会引起额外的毒性。它还会减慢增殖并促进细胞凋亡以及 EGFR 耗竭[1]。 （100、125、150 mg/kg，静脉注射）在癌基因成瘾的实体和血液异种移植模型中显着抑制发育和/或逆转肿瘤生长，表现出强大的抗癌功效[2]。

HSP90结合测定[1]
在裂解缓冲液（20 mM HEPES，pH 7.4，1 mM EDTA，5 mM MgCl2，100 mM KCl）中处理指数生长的细胞，并在4°C下与浓度增加的17-AAG或ganetespib一起孵育30分钟，然后与连接到Dynabeads MyOne Streptavidin T1磁珠的生物素GM一起在4℃下孵育1小时。珠在裂解缓冲液中洗涤三次，并在95°C的SDS–PAGE样品缓冲液中加热5分钟。样品在4-12%Bis-Tris梯度凝胶上解析，并使用抗HSP90抗体进行蛋白质印迹。

细胞增殖测定[1]
根据制造商的规范，使用CCK-8比色测定法在至少两个样品中进行细胞增殖测定。使用Kaleidagraph或Graphpad Prism计算IC50值

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蛋白质印迹[1]
如前所述（9）制备全细胞裂解物。测定蛋白质浓度，并在4-12%双-三梯度凝胶（Invitrogen）上对等量（20μg）进行SDS-PAGE。HSP27抗体来自Enzo Life Sciences。p23和HSP90α抗体来自StressMarq

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免疫沉淀[1]
将500μg全细胞裂解物与2μg与蛋白A Dynabeads偶联的小鼠抗p23单克隆抗体免疫沉淀。将与p23结合的蛋白质在4-12%双-三梯度凝胶上解析，并用抗HSP90抗体进行蛋白质印迹。

Establishment and treatment of xenografts[1]
Female 7–8-week-old C.B-17 SCID mice were maintained under pathogen-free conditions. All procedures were approved by the Synta Pharmaceutical Institutional Animal Care and Use Committee. NCI-H1975 or HCC827 cells were cultured as above and 0.5 – 1×107 cells were mixed with 50% RPMI 1640/50% Matrigel and subcutaneously injected into the flanks of SCID mice. For efficacy studies, animals with 100-200 mm3 tumors were then randomized into treatments groups of eight. Tumor volumes (V) were calculated by the equation V = 0.5236×L×W×T (Length, width, and thickness). Animals were treated by intravenous bolus tail vein injection at 10 ml/kg with ganetespib formulated in 10/18 DRD (10% DMSO, 18% Cremophor RH 40, 3.6% dextrose and 68.4% water). As a measurement of in vivo efficacy, the relative size of treated and control tumors [(%T/C) value] was determined from the change in average tumor volumes of each drug-treated group relative to the vehicle-treated group, or itself in the case of tumor regression. Body weights were monitored daily. For biomarker studies, mice bearing NCI-H1975 xenografts were treated with either a single dose of vehicle or ganetespib, or with 5 daily doses of vehicle or ganetespib, in groups of 3 or 8, and harvested at various time points. Tumors were excised and flash frozen in liquid nitrogen for preparation of protein lysates or fixed in 10% neutral buffered formalin for immunohistochemistry.

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Pharmacokinetic Analysis[1]
Female 7–8-week-old C.B-17 SCID mice bearing NCI-H1975 xenografts received a single intravenous (i.v.) dose slightly below the highest non-severely toxic dose (HNSTD, 150 mg/kg). At time points indicated, mice (n = 3/time point) were sacrificed and plasma and tissues (tumor, liver, and lung) were harvested. Concentrations of ganetespib in plasma and tissues were determined by isocratic reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometric (HPLC/MS-MS) detection.

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Dissolved in DMSO and diluted 1:10 with 20% Cremophor RH 40; 25 mg/kg; Tail vein injection

Female severe combined immune-deficient (SCID) mice

[1]. Ganetespib (STA-9090), a Non-Geldanamycin HSP90 Inhibitor, has Potent Antitumor Activity in In Vitro and In Vivo Models of Non-Small Cell Lung Cancer. Clin Cancer Res. 2012 Jul 17.

[2]. Ganetespib, a unique triazolone-containing Hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy. Mol Cancer Ther. 2012 Feb;11(2):475-84.

[3]. Phase I evaluation of STA-1474, a prodrug of the novel HSP90 inhibitor ganetespib, in dogs with spontaneous cancer. PLoS One. 2011;6(11):e27018.

[4]. Multifaceted intervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activated JAK/STAT signaling. PLoS One. 2011 Apr 14;6(4):e18552.

[5]. Identification of Therapeutic Targets in Rhabdomyosarcoma through Integrated Genomic, Epigenomic, and Proteomic Analyses. Cancer Cell. 2018 Sep 10;34(3):411-426.e19.

Ganetespib is a member of triazoles and a ring assembly.
Ganetespib is under investigation for the treatment of BREAST CANCER, Small Cell Lung Cancer, Acute Myeloid Leukaemia, and Myelodysplastic Syndrome.
Ganetespib is a synthetic small-molecule inhibitor of heat shock protein 90 (Hsp90) with potential antineoplastic activity. Ganetespib binds to and inhibits Hsp90, resulting in the proteasomal degradation of oncogenic client proteins, the inhibition of cell proliferation and the elevation of heat shock protein 72 (Hsp72); it may inhibit the activity of multiple kinases, such as c-Kit, EGFR, and Bcr-Abl, which as client proteins depend on functional HsP90 for maintenance. Hsp90, a 90 kDa molecular chaperone upregulated in a variety of tumor cells, plays a key role in the conformational maturation, stability and function of "client" proteins within the cell, many of which are involved in signal transduction, cell cycle regulation and apoptosis, including kinases, transcription factors and hormone receptors. Hsp72 exhibits anti-apoptotic functions; its up-regulation may be used as a surrogate marker for Hsp90 inhibition.

C20H20N4O3

364.4

364.153

C, 65.92; H, 5.53; N, 15.38; O, 13.17

888216-25-9

135564985

White to yellow solid powder

1.4±0.1 g/cm3

685.8±57.0 °C at 760 mmHg

368.5±32.1 °C

0.0±2.2 mmHg at 25°C

1.698

5.47

96.33

3

4

3

27

610

0

RVAQIUULWULRNW-UHFFFAOYSA-N

InChI=1S/C20H20N4O3/c1-11(2)14-9-15(18(26)10-17(14)25)19-21-22-20(27)24(19)13-4-5-16-12(8-13)6-7-23(16)3/h4-11,25-26H,1-3H3,(H,22,27)

5-[2,4-dihydroxy-5-(1-methylethyl)phenyl]-4-(1-methyl-1H-indol-5-yl)-2,4-dihydro-3H- 1,2,4-triazol-3-one

STA-9090; Ganetespib; STA 9090; STA9090

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)

配方 1 中的溶解度: 10 mg/mL (27.44 mM) in 15% Cremophor EL + 85% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*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: ≥ 2.5 mg/mL (6.86 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 25.0 mg/mL澄清DMSO储备液加入到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 3 中的溶解度: ≥ 2.5 mg/mL (6.86 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 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，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 4 中的溶解度: ≥ 2.5 mg/mL (6.86 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，您可以将 100 µL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 µL 玉米油中并混合均匀。

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配方 5 中的溶解度： 1% DMSO+30% 聚乙二醇+1% 吐温 80：30 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。