Nutlin-3 is a commercial available p53-MDM2 inhibitor, with K_i of 90 nM.

Ki: 90 nM (p53-MDM2)^[1]

Nutlin-3 is an inhibitor of the MDM2-p53 interaction. In particular, co-treatment of p53-positive HCT116 cells with 1 μM of Inauhzin and 2 μM of Nutlin-3 more significantly activated p53 as measured by its protein level as well as the level of its target p21, PUMA or cleaved PARP as indication of apoptosis^[2]. Nutlin-3 is a small-molecule inhibitor that acts to inhibit MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. As a single agent, Nutlin-3 (2-10 μM) stabilizes p53 and p21^WAF levels and is toxic to WTp53-22RV1 cells (IC₅₀, 4.3 μM) but has minimal toxicity toward p53-deficient cells (IC₅₀, >10 μM). Nutlin-3 induces p53 and p21^WAF expression in a dose-dependent manner in 22RV1 cells. Short-term cell cycle assays show that, at a dose of 10 μM, Nutlin-3 increasea slightly the G₁-phase fraction and decreasea S-phase fraction of all three cell lines^[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.

Nutlin-3 can suppress the growth of xenograft tumors derived from human osteosarcoma or leukemia cells, the anti-tumor activity of Nutlin-3 even at the dose of 200 mg/kg per oral administration is marginal in an HCT116-derived xenograft tumor model^[2]. Nutlin-3 may be a useful adjunct to improve the therapeutic ratio using precision radiotherapy targeted to hypoxic cells and warrants further study in vivo^[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.

581.49

C₃₀H₃₀Cl₂N₄O₄

548472-68-0

Room temperature in continental US; may vary elsewhere.

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

In Vitro: 

Ethanol : 100 mg/mL (171.97 mM; Need ultrasonic)

DMSO : ≥ 50 mg/mL (85.99 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。-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。

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

• 1.

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

  Solubility: 5 mg/mL (8.60 mM); Clear solution; Need ultrasonic

  此方案可获得 5 mg/mL (8.60 mM) 的澄清溶液。

  以 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；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% EtOH    90% corn oil

  Solubility: 5 mg/mL (8.60 mM); Clear solution; Need ultrasonic

  此方案可获得 5 mg/mL (8.60 mM) 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 50.0 mg/mL 的澄清 EtOH 储备液加到 900 μL玉米油中，混合均匀。

• 3.

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

  Solubility: ≥ 2.5 mg/mL (4.30 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.30 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

• 4.

  请依序添加每种溶剂： 10% DMSO    90% (20% SBE-β-CD in saline)

  Solubility: 2.5 mg/mL (4.30 mM); Suspended solution; Need ultrasonic

  此方案可获得 2.5 mg/mL (4.30 mM) 的均匀悬浊液，悬浊液可用于口服和腹腔注射。

  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 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
• 5.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.5 mg/mL (4.30 mM); Clear solution

  此方案可获得 ≥ 2.5 mg/mL (4.30 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]. Yu Z, et al. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors. Int J Mol Sci. 2014 Sep 5;15(9):15741-53.

  [2]. Zhang Y, et al. Inauhzin and Nutlin3 synergistically activate p53 and suppress tumor growth.Cancer Biol Ther. Cancer Biol Ther. 2012 Aug;13(10):915-24.

  [3]. Supiot S, et al. Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53. Mol Cancer Ther. 2008 Apr;7(4):993-9.

Human non-small-cell lung carcinoma wild type p53-containing H460 and A549, human non-small-cell lung carcinoma p53-null H1299, and human colon cancer HCT116 (p53^+/+ and p53^-/-) cells are used. Cells (1.5×10⁵) are plated into 6-well plates, and incubated at 37°C overnight. After treatment of Inauhzin and Nutlin 3 at the indicated concentrations for 48 h, cells are harvested, fixed in 70% ice-cold ethanol overnight at -20°C, resuspended in propidium iodide-solution (50 µg/mL PI, 0.1 mg/mL RNase A, 0.05% Tritin X-100 in PBS) for 40 min at 37°C, then analyzed for DNA content using a flow cytometer and proprietary software^[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.

Mice^[2]
Five-week-old female SCID mice are used. Mice are subcutaneously inoculated with 3×10⁶ HCT116^p53+/+ cells in the right flank and tumor growth is monitored with calipers. After the mean tumor volume reaches 50-100 mm³, animals are administered Inauhzin intraperitoneally (IP), Nutlin 3 orally, or vehicles (4% DMSO for Inauhzin, EtOH: Tween: 5% Glucose=5:5:90 for Nutlin 3). Tumor volume is measured every other day, and inhibition of tumor growth (T/C) is calculated on the last day of treatment.

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]. Yu Z, et al. Design, synthesis and biological evaluation of sulfamide and triazole benzodiazepines as novel p53-MDM2 inhibitors. Int J Mol Sci. 2014 Sep 5;15(9):15741-53.

  [2]. Zhang Y, et al. Inauhzin and Nutlin3 synergistically activate p53 and suppress tumor growth.Cancer Biol Ther. Cancer Biol Ther. 2012 Aug;13(10):915-24.

  [3]. Supiot S, et al. Nutlin-3 radiosensitizes hypoxic prostate cancer cells independent of p53. Mol Cancer Ther. 2008 Apr;7(4):993-9.