Ispinesib is a specific inhibitor of kinesin spindle protein (KSP), with a K_{i app} of 1.7 nM.

Ispinesib is a potent, highly specific inhibitor of KSP, with a K_{i app} of 1.7 nM^[1]. Ispinesib (150 nM) inhibits BT-474 and MDA-MB-468 cell lines, with GI₅₀s of 45 and 19 nM, respectively^[2]. Ispinesib (SB715992, 15 and 30 nM) suppresses the proliferation of PC-3 prostate cancer cell by 48.65% and 52.16%, and induces apoptosis of prostate cancer cell by 1094.88% and 1516.70%, respectively. Ispinesib up regulates genes responsible for apoptosis and cell cycle arrest, and down regulates genes responsible for cell proliferation and survival. The anti-proliferation and pro-apoptotic activities of Ispinesib can be enhanced by genistein^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Ispinesib (SCID, 8 mg/kg; nude, 10 mg/kg, q4d × 3) reduces tumor volume in mice bearing tumor xenografts of ER-positive (MCF7), HER2-positive (KPL4, HCC1954, and BT-474), and triple-negative (MDA-MB-468) breast cancer cells via i.p. one dose every 4 days repeated three times^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

517.06

C₃₀H₃₃ClN₄O₂

336113-53-2

伊斯平斯；伊匹尼塞

Room temperature in continental US; may vary elsewhere.

4°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

DMSO : ≥ 125 mg/mL (241.75 mM)

* “≥” means soluble, but saturation unknown.

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, 6 months; -20°C, 1 month (protect from light)。-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。

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

• 1.

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

  Solubility: ≥ 2.5 mg/mL (4.84 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.84 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀；向上述体系中加入50 μL Tween-80，混合均匀；然后继续加入 450 μL生理盐水定容至 1 mL。

  将 0.9 g 氯化钠，完全溶解于 100 mL ddH₂O 中，得到澄清透明的生理盐水溶液

• 2.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (4.84 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.84 mM，饱和度未知) 的澄清溶液，此方案不适用于实验周期在半个月以上的实验。

  以 1 mL 工作液为例，取 100 μL 25.0 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Lad L, et al. Mechanism of inhibition of human KSP by ispinesib. Biochemistry. 2008 Mar 18;47(11):3576-85.

  [2]. Purcell JW, et al. Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer. Clin Cancer Res. 2010 Jan 15;16(2):566-76.

  [3]. Davis DA, et al. Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line. BMC Cancer. 2006 Jan 24;6:22.

Kinesin specificity analysis is carried out using a pyruvate kinase−lactate dehydrogenase detection system that couples the production of ADP to oxidation of NADH. Absorbance changes are monitored at 340 nm. Steady-state studies using nanomolar concentrations of KSP are performed using a sensitive fluorescence-based assay utilizing a pyruvate kinase, pyruvate oxidase, and horseradish peroxidase coupled detection system that couples the generation of ADP to oxidation of Amplex Red to fluorescent resorufin. Generation of resorufin is monitored by fluorescence (λexcitation = 520 nm and λemission= 580 nm). Steady-state biochemical experiments are performed in PEM25 buffer [25 mM Pipes-K⁺ (pH 6.8), 2 mM MgCl₂, 1 mM EGTA] supplemented with 10 µM paclitaxel for experiments involving microtubules. The IC₅₀ for steady-state inhibition is determined at 500 µM ATP, 5 µM MTs, and 1 nM KSP in PEM25 buffer. K_{i app} (apparent inhibitor dissociation constant) estimates of Ispinesib are extracted from the concentration-response curves, with explicit correction for enzyme concentration^[1].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

PC-3 prostate cancer cells are seeded in 96 well plates at a density of 4 × 10³ cells/well. PC-3 cells are incubated for 24 hours to allow attachment to the surface of each well of the tissue culture plate. Then, the cells are treated with varying concentration of reagents and incubated for 1 to 3 days. First, PC-3 cells are treated with 15 and 30 nM of Ispinesib, respectively. Second, PC-3 cells are subjected to combinational treatments with 7.5 or 10 nM of Ispinesib plus 30 μM of genistein. Finally, PC-3 cells are pre-treated with 30 μM of genistein for 24 hours followed by treatment with 15 nM of Ispinesib. Control cells are treated with 0.3 mM Na₂CO₃ (vehicle control). After treatment, PC3 cells are incubated at 37°C with MTT (0.5 mg/mL) for 2 hours and isopropyl alcohol at room temperature for 1 hour. The spectrophotometric absorbance of each sample is then determined by using ULTRA Multifunctional Micro Plate Reader at 595 nm^[3].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

Mice with a tumor volume of ∼250 mm³ receive a single dose of Ispinesib (10 mg/kg). Tumors are dissected, fixed in 10% buffered formalin, and embedded in paraffin, and 5-μm tissue sections are prepared. Antigen retrieval is done by boiling in 50 mM citrate buffer (pH 5.5), and sections are then incubated in 3% hydrogen peroxide, washed in PBS-0.1% Tween, and blocked in 10% goat serum. Phospho-histone H3 (PH3) antibody is detected using Alexa Fluor 488 secondary antibody. Images are taken with a microscope at ×10 magnification and captured using MetaMorph software to quantify PH3 expression by computing the area ratio of PH3-positive cells per total cells. Ki67/cleaved caspase-3 staining is done. Nonfluorescent images are taken on an Olympus BX41 microscope at ×20 magnification^[2].

上海金畔生物科技有限公司 has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Lad L, et al. Mechanism of inhibition of human KSP by ispinesib. Biochemistry. 2008 Mar 18;47(11):3576-85.

  [2]. Purcell JW, et al. Activity of the kinesin spindle protein inhibitor ispinesib (SB-715992) in models of breast cancer. Clin Cancer Res. 2010 Jan 15;16(2):566-76.

  [3]. Davis DA, et al. Increased therapeutic potential of an experimental anti-mitotic inhibitor SB715992 by genistein in PC-3 human prostate cancer cell line. BMC Cancer. 2006 Jan 24;6:22.