Didox (NSC-324360) is a synthetic ribonucleotide reductase (RR) inhibitor.

Ribonucleotide reductase^[1]

Didox (NSC-324360) suppresses LPS-induced mRNA levels of iNOS, IL-6, IL-1, TNF-α, NF-κβ (p65), and p38-α, after 24 h of treatment. Treatment with Didox also suppresses the secretion of nitric oxide (NO), IL-6, and IL-10. Using mitochondrial dehydrogenase activity as a measure of cytotoxicity, the effects of Didox on cellular respiration in RAW264.7 are examined over a range of concentrations for 24 h. Cells exposures to 200 μM and below Didox, with and without LPS, do not exhibit significant cellular toxicity^[1]. Didox (NSC-324360) is active against all human and murine acute myeloid leukemia (AML) lines tested with IC₅₀ values in the low micromolar range (mean IC₅₀ 37 µM [range 25.89-52.70 µM])^[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.

Once engraftment is established by bioluminescent imaging, the animals receive daily administrations of Didox at 425 mg/kg via IP injection over 5 days. Didox (NSC-324360) treatment significantly reduces leukemic burden compared to vehicle treated controls (p=0.0026 and p=0.0342). More importantly, Didox (NSC-324360) provides a significant survival benefit (p<0.0001 and p=0.0094)^[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.

169.13

C₇H₇NO₄

69839-83-4

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: 

DMSO : 100 mg/mL (591.26 mM; Need ultrasonic)

*

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% DMSO    40% PEG300    5% Tween-80    45% saline

  Solubility: ≥ 2.75 mg/mL (16.26 mM); Clear solution

  此方案可获得 ≥ 2.75 mg/mL (16.26 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 27.5 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: ≥ 2.75 mg/mL (16.26 mM); Clear solution

  此方案可获得 ≥ 2.75 mg/mL (16.26 mM，饱和度未知) 的澄清溶液。

  以 1 mL 工作液为例，取 100 μL 27.5 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: ≥ 2.75 mg/mL (16.26 mM); Clear solution

  此方案可获得 ≥ 2.75 mg/mL (16.26 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 27.5 mg/mL 的澄清 DMSO 储备液加到 900 μL玉米油中，混合均匀。

• [1]. Matsebatlela TM, et al. 3,4-Dihydroxy-benzohydroxamic acid (Didox) suppresses pro-inflammatory profiles and oxidative stress in TLR4-activated RAW264.7 murine macrophages. Chem Biol Interact. 2015 May 25;233:95-105.

  [2]. Cook GJ, et al. The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML. PLoS One. 2014 Nov 17;9(11):e112619.

RAW264.7 macrophages are treated with Didox alone, with 0.1 μg/mL LPS, or the two in combination. Cellular respiration, as an indication of cytotoxicity, is measured by the MTT assay, which quantifies mitochondrial dehydrogenase activity. Macrophages are plated into 96 well Costar plates at 10⁵ cells per well in 100 μL of DMEM media. After 4 h of incubation at 37°C for adherence, compounds and DMSO carrier control (0.01% final) are added in triplicate over serial dilutions beginning with 200 μM per well in a total volume of 200 μL, and the plates incubated for 24 h. Four h before termination of the assay, each well receives 20 μL of a 5 mg/mL MTT solution in un-supplemented DMEM. After centrifugation, the supernatant for each well is discarded and cells containing reduced MTT are solubilized with 100 μL of acidified isopropanol (4 mM HCl, 0.1% NP-40 in isopropanol). Following a brief period of shaking, the optical density (O.D.) for each well is recorded at 550 nm. Each experiment is repeated three times and the data averaged from each triplicate, then expressed as percentage of the control O.D. values for each experiment^[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.

Mice^[2]
Luciferase-tagged leukemia cells are transplanted into 8- week old, sublethally irradiated (4.5 Gy) C57Bl/6 mice by tail vein injection of 1.0×10⁶ cells per mouse. Mice are injected with 150 mg/kg D-Luciferin, anesthetised with Isoflurane, and imaged using the IVIS 100 imaging system. Mice begin treatment with Didox upon detection of clear signal. The animals are treated with daily administrations of Didox (NSC-324360) at 425 mg/kg Didox by intraperitoneal injection (IP) for 5 days. Control animals receive 5% dextrose water by IP injection. Repeat imaging is performed on the day following the final treatment^[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]. Matsebatlela TM, et al. 3,4-Dihydroxy-benzohydroxamic acid (Didox) suppresses pro-inflammatory profiles and oxidative stress in TLR4-activated RAW264.7 murine macrophages. Chem Biol Interact. 2015 May 25;233:95-105.

  [2]. Cook GJ, et al. The efficacy of the ribonucleotide reductase inhibitor Didox in preclinical models of AML. PLoS One. 2014 Nov 17;9(11):e112619.