MELK (IC50 = 0.41 nM)

OTSSP167 阻断 MELK 过表达的癌细胞 A549、T47D、DU4475 和 22Rv1，IC50 值分别为 6.7、4.3、2.3 和 6.0 nM。 OTSSP167 阻止两种新型 MELK 底物 PSMA1（蛋白酶体 α 亚基 1 型）和 DBNL（类 Drebrin）的磷酸化，这两种底物对于干细胞特性和侵袭性至关重要。通过抑制 PSMA1 磷酸化，OTSSP167 可以防止乳腺癌细胞形成乳腺球。 [1]

在小鼠体内使用乳腺癌、肺癌、前列腺癌和胰腺癌细胞系进行的异种移植研究中，通过静脉内和口服给药，OTSSP167 表现出显着的肿瘤生长抑制作用。 OTSSP167以20mg/kg的剂量每两天一次静脉注射至MDA-MB-231模型，TGI为73%。每日一次口服剂量 10 mg/kg 时，TGI 为 72%。 OTSSP167 以剂量和 MELK 依赖性方式治疗各种癌症类型，且体重几乎没有减轻。 [1]

将 MELK 重组蛋白 (0.4 μg) 与 5 μg 每种底物在 20 μL 激酶缓冲液中混合，该缓冲液含有 30 mM Tris-HCl (pH)、10 mM DTT、40 mM NaF、10 mM MgCl2、0.1 mM EGTA，50 μM 冷溶液-ATP 和 10 Ci 的 [γ-32P]ATP 在 30 °C 下持续 30 分钟。在 SDS-PAGE 之前，通过添加 SDS 样品缓冲液并煮沸 5 分钟来终止反应。在室温下，将凝胶干燥并用增感屏进行放射自显影。孵育前，将 DMSO 溶解的 OTSSP167（终浓度 10 nM）添加到激酶缓冲液中。

使用 Cell Counting Kit-8 通过比色测定来测量体外细胞活力。将细胞以 100 μL 的密度接种在 96 孔板中，以产生持续线性生长（A549，1×103 个细胞；T47D，3×103 个细胞；DU4475，4×103 个细胞；22Rv1，6×103 个细胞；和 HT1197） ，2×103 个细胞，每孔 100 μL）。让细胞粘附过夜，然后在 37°C 下暴露于 OTSSP167 72 小时。在 450 nm 波长下，分光光度计读取板的读数。每个测定进行三个副本。

Injections of MDA-MB-231 cells are made into NOD's mammary fat pads.Mice CB17-Prkdcscid/J. Female BALB/cSLC-nu/nu mice are subcutaneously injected with 1 105 A549), MIAPaCa-2, and PC-14 cells. Male BALB/cSLC-nu/nu mice receive a subcutaneous injection of DU145 cells in the left flank. Animals are randomized into groups of 6 mice (apart from PC-14, for which groups of 3 mice are used) when MDA-MB-231, A549, DU145, MIAPaCa-2, and PC-14 xenografts have reached average volumes of 100, 210, 110, 250, and 250 mm3, respectively. OTSSP167 and other substances are prepared for oral administration in a vehicle containing 0.5% methylcellulose and administered orally according to the prescribed dose and schedule. Compounds are prepared in 5% glucose for intravenous administration and injected into the tail vein. For both administration routes, a volume of 10 mL per kg of body weight is used. Every other day, tumor volumes are measured using a caliper.

[1]. ncotarget . 2012 Dec;3(12):1629-40.

C25H28CL2N4O2

487.42

486.1589

C, 61.60; H, 5.79; Cl, 14.55; N, 11.49; O, 6.56

1431697-89-0

OTSSP167 hydrochloride;1431698-10-0

Solid powder

CC(=O)C1=CN=C2C=CC(=NC2=C1NC3CCC(CC3)CN(C)C)C4=CC(=C(C(=C4)Cl)O)Cl

DKZYXHCYPUVGAF-UHFFFAOYSA-N

InChI=1S/C25H28Cl2N4O2/c1-14(32)18-12-28-22-9-8-21(16-10-19(26)25(33)20(27)11-16)30-24(22)23(18)29-17-6-4-15(5-7-17)13-31(2)3/h8-12,15,17,33H,4-7,13H2,1-3H3,(H,28,29)

1-[6-(3,5-dichloro-4-hydroxyphenyl)-4-[[4-[(dimethylamino)methyl]cyclohexyl]amino]-1,5-naphthyridin-3-yl]ethanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。