URMC-099 is an orally bioavailable and potent mixed lineage kinase type 3 (MLK3) (IC₅₀=14 nM) inhibitor with with excellent blood-brain barrier penetration properties.

The effect of URMC-099 (URMC099) on the in vitro growth of the “brain homing” MDA-MB-231 BR cells expressing eGFP (eGFP8.4) and their parental cell line, MDA-MB-231 is tested. The cells are treated with either 200 nM URMC-099 or vehicle alone. Cells treated with URMC-099 grow at a similar rate to those treated with vehicle. Cell viability is >99% in all cases^[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.

URMC-099 has moderate terminal elimination half-life (t_1/2=1.92 h, 2.14 h and 2.72 h for C57 BL/6 mice (10 mg/kg, oral dosing), C57 BL/6 mice (2.5 mg/kg, iv), C57 BL/6 mice (10 mg/kg, iv))^[1]. The effect of URMC-099 (URMC099) on tumor formation in vivo is analyzed using a well characterized mouse xenograft model of breast cancer brain metastasis. For these experiments, eGFP8.4 cells are inoculated into the left ventricle of immunodeficient nu/nu mice; animals are then treated with either URMC-099 (10 mg/kg) or vehicle alone, every 12 hours for 20 days. This dose of URMC-099 is chosen because it has been shown to be sufficient to effectively inhibit MLK3 in mice, with good penetration of the blood-brain barrier and potent inhibition of the phosphorylation of Jun N-terminal kinase (JNK) in brain tissue. On day 21 the mice are sacrificed and number of BM is assessed. Fifteen mice are used for each treatment group. BM are detected in 60% of mice, which is consistent with previous studies using this xenograft model by other investigators. URMC-099 treatment significantly (p<0.05, two-tailed t-test) increases the total number of brain metastasis (BM) in mice. For micrometastases, the pattern is similar to that observed for total BM. The number of macrometastases is statistically indistinguishable between mice treated with URMC-099 or vehicle^[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.

421.54

C₂₇H₂₇N₅

1229582-33-5

Room temperature in continental US; may vary elsewhere.

DMSO : ≥ 33 mg/mL (78.28 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。

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： 5% DMSO    40% PEG300    5% Tween-80    50% saline

  Solubility: ≥ 2.87 mg/mL (6.81 mM); Clear solution
• 2.

  请依序添加每种溶剂： 5% DMSO    95% (20% SBE-β-CD in saline)

  Solubility: ≥ 2.87 mg/mL (6.81 mM); Clear solution
• 3.

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

  Solubility: ≥ 2.08 mg/mL (4.93 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.93 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

• 4.

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

  Solubility: ≥ 2.08 mg/mL (4.93 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.93 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
• 5.

  请依序添加每种溶剂： 10% DMSO    90% corn oil

  Solubility: ≥ 2.08 mg/mL (4.93 mM); Clear solution

  此方案可获得 ≥ 2.08 mg/mL (4.93 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]. Goodfellow VS, et al. Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3. J Med Chem. 2013 Oct 24;56(20):8032-48.

  [2]. Rhoo KH, et al. Pharmacologic inhibition of MLK3 kinase activity blocks the in vitro migratory capacity of breast cancer cells but has no effect on breast cancer brain metastasis in a mouse xenograft model. PLoS One. 2014 Sep 29;9(9):e108487.

MDA-MB-231, MCF10A, HS578t and MDA-MB-231 EGFP8.4 cells are seeded in a 24 well plate at an initial density of 5.0×10⁴ cells/mL in 0.5 mL of media. The cells are treated with either 200 µM of URMC-099 or vehicle (0.002% DMSO). Cell number in each well is measured by trypsinizing the cells and counting them with a hematocytometer. The viability is tested by trypan blue dye exclusion. Each condition is tested in triplicate^[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]
6 to 8 week old female nu/nu mice are injected intraperitoneally with URMC-099 at a dose of 10 mg/kg, or vehicle, twice daily for 20 days. On day 21 mice are sacrificed by CO₂ suffocation. Brains are removed and fixed with 4% formaldehyde in PBS overnight, then transferred to 30% sucrose in PBS. The brains are then quickly frozen by immersing into isopentane cooled on dry ice. The frozen brains are sectioned coronally every 30 micrometers. Eight sections starting at bregma 2.0 and separated by 360 µm are mounted on glass slides for tumor evaluation under the microscope. The number of brain metastasis (BM) is counted by examining eGFP signals under a fluorescence 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]. Goodfellow VS, et al. Discovery, synthesis, and characterization of an orally bioavailable, brain penetrant inhibitor of mixed lineage kinase 3. J Med Chem. 2013 Oct 24;56(20):8032-48.

  [2]. Rhoo KH, et al. Pharmacologic inhibition of MLK3 kinase activity blocks the in vitro migratory capacity of breast cancer cells but has no effect on breast cancer brain metastasis in a mouse xenograft model. PLoS One. 2014 Sep 29;9(9):e108487.