Tozasertib (VX 680; MK-0457) is an inhibitor of Aurora A/B/C kinases with K_is of 0.6, 18, 4.6 nM, respectively.

Tozasertib induces similar cytotoxicity with IC₅₀ of approximately 300 nM and exhibits an AUR B-like inhibitory phenotype of G2/M arrest, endoreduplication and apoptosis in BaF3 cells transfected with ABL or FLT-3 (mutant and wild type) kinases. Tozasertib prevents the CAL-62 proliferation in a time-dependent manner. Tozasertib treatment for 14 days significantly decreases the number and size of colonies by approximately 70% in the 8305C and 90% in the CAL-62, 8505C and BHT-101. Treatment of the different ATC cells with Tozasertib inhibits proliferation with the IC₅₀ between 25 and 150 nM. The Tozasertib significantly impairs the ability of the different cell lines to form colonies in soft agar. Analysis of caspase-3 activity indicates that Tozasertib induces apoptosis in the different cell lines. CAL-62 cells exposed for 12 hours to Tozasertib show an accumulation of cells with ≥ 4N DNA content. Time-lapse analysis demonstrates that Tozasertib-treated CAL-62 cells exit metaphase without dividing. Moreover, histone H3 phosphorylation is abrogated following Tozasertib treatment^[2]. Tozasertib has significant inhibitory activity against BCR-Abl bearing the T315I mutation in patient-derived samples^[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.

464.59

C₂₃H₂₈N₈OS

639089-54-6

陶扎色替

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 : ≥ 106.67 mg/mL (229.60 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: ≥ 2.08 mg/mL (4.48 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.48 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 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% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.08 mg/mL (4.48 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.48 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL 20% 的 SBE-β-CD 生理盐水水溶液中，混合均匀。

  将 2 g 磺丁基醚 β-环糊精加入 5 mL 生理盐水中，再用生理盐水定容至 10 mL，完全溶解，澄清透明
• 3.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.08 mg/mL (4.48 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.48 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 20.8 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Harrington EA, et al. VX-680, a potent and selective smallmolecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med. 2004; 10:262-7.

  [2]. Salah E, et al. Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class.J Med Chem. 2011 Apr 14;54(7):2359-67. Epub 2011 Mar 18.

  [3]. Arlot-Bonnemains Y, et al. Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines. Endocr Relat Cancer. 2008 Jun;15(2):559-68

The consumption of ATP is coupled via the pyruvate kinase/lactic dehydrogenase enzyme pair to the oxidation of NADH, which can be monitored through the decrease in absorption at 340 nm. Reactions contains 100 mM Tris (pH 8), 10 mM MgCl₂, 2.2 mM ATP, 1 mM phosphoenolpyruvate, 0.6 mg/mL NADH, 75 units/mL pyruvate kinase, 105 units/mL lactate dehydrogenase, and 0.5 mM substrate peptide (sequence: EAIYAAPFAKKK). Reactions (75 μL) are started by adding sufficient kinase to bring the reactions to 30 nM kinase concentration and the decrease in absorbance is monitored over 30 minutes at 30°C in a microtiter plate spectrophotometer. Inhibitory constants are obtained through addition of 3.75 μL Tozasertib in 100% DMSO or DMSO alone. Ki values are calculated as follows, K_i=IC₅₀/(1+[S]/K_d), where [S]=[ATP]=2.2 mM, and K_d (of ATP to Abl)=70 μM. These values are calculated assuming a K_d (ATP) of 70 μM for wild type and H396P Abl kinase domain.

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.

The CAL-62 cells are cultured in the absence (dimethyl sulfoxide, DMSO) or the presence of 500  nM Tozasertib for different periods of time (1-5 days). The dose-dependent effects of Tozasertib on cell proliferation are evaluated by treating the different ATC cells for 4 days with different concentrations of the Aurora inhibitor (5-500 nM). The cells are pulse labeled with 30 mM BrdU for 2  hours before the end of the incubation time. The BrdU incorporation is analyzed by means of a colorimetric immunoassay using the cell proliferation ELISA kit. The results from Tozasertib-treated cells are compared with those observed in control cells and expressed as a fold of variation versus control.

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.

For the HL-60 study, female athymic NCr-nu mice are inoculated subcutaneously with 107 HL-60(TB) leukemia cells into the right axillary area. Treatment is administered i.p. b.i.d. after tumors reached 150−200 mm³. Tozasertib is prepared in a vehicle of 50% PEG 300 in 50 mM phosphate buffer. Cisplatin, formulated in saline, is administered i.p. q.4.d. for a total of three injections, at a dose of 5.4 mg/kg. For the MIA PaCa-2 studies, female MF1 nude mice are inoculated with 10⁷ MIA PaCa-2 cells into the dorsal flank. Treatment is administered i.p. b.i.d. after tumors reach 175 mm³. Tozasertib is prepared in a vehicle of 50% PEG 300 in 50 mM phosphate buffer. 5-fluorouracil, formulated in saline, is administered i.v. q.4.d. at a dose of 50 mg/kg. For the HCT116 study, female Hsd RH rnu/nu rats are inoculated with 10⁷ HCT116 cells into the right flank. Treatment is administered once the tumors reached 700−950 mm³. Tozasertib is administered continuously through an indwelling femoral catheter, followed by a saline infusion for 4 d before repeating the dose cycle. For all studies, tumor volume is determined by caliper measurements three times a week.

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]. Harrington EA, et al. VX-680, a potent and selective smallmolecule inhibitor of the Aurora kinases, suppresses tumor growth in vivo. Nat Med. 2004; 10:262-7.

  [2]. Salah E, et al. Crystal structures of ABL-related gene (ABL2) in complex with imatinib, tozasertib (VX-680), and a type I inhibitor of the triazole carbothioamide class.J Med Chem. 2011 Apr 14;54(7):2359-67. Epub 2011 Mar 18.

  [3]. Arlot-Bonnemains Y, et al. Effects of the Aurora kinase inhibitor VX-680 on anaplastic thyroid cancer-derived cell lines. Endocr Relat Cancer. 2008 Jun;15(2):559-68