Endogenous metabolite of 17β-estradiol (E2); estradiol metabolite; angiogenesis

2-Methoxyestradiol (2-ME) (5-100 μM) 以浓度依赖性方式抑制纯化微管蛋白的组装，在 200 μM 2-Methoxyestradiol (2ME2) 时具有最大抑制 (60%)。 2-Methoxyestradiol 强烈降低存活间期 MCF7 细胞的平均微管生长速率、持续时间和长度以及总体动态，并具有 IC50 (1.2 μM) 有丝分裂抑制作用。这与其体外作用一致，并且似乎与微管解聚无关。 2. 2-甲氧基雌二醇可保护静止细胞，同时诱导多种活跃增殖的细胞类型的 G2-M 停滞和死亡。 2. 已证明，大量的甲基雌二醇通过与秋水仙碱位点或其附近的微管蛋白结合并抑制微管组装而使细胞内的微管解聚[1]。在缺氧条件下培养的细胞中，2-甲氧基雌二醇 (2-ME) 会降低 HIF-1α 和 HIF-2α 核标记。 2.甲氧基雌二醇降低HIF-1α蛋白的转录活性和水平。它是一种抗血管生成、抗增殖和促凋亡药物。与 DMSO 处理的细胞相比，用 10 μM 2-甲氧基雌二醇处理的 A549 细胞在 96 小时时的生长速率显着降低（分别为 66.2±7.2 和 101.2±2.3%；p=0.04）。当在常氧条件下用 10 μM 2-甲氧基雌二醇处理的细胞与低 O2 浓度（5.8±0.2%；p=0.003）下的细胞进行比较时，细胞凋亡显着增加 [2]。

为了研究 2-甲氧基雌二醇 (2-ME2) 对葡萄膜炎进展的影响，C57BL/6 小鼠被随机分为两组并接受 IRBP 肽疫苗接种。从第0天到第13天，2ME2组腹腔注射15 mg/kg 2-甲氧基雌二醇，而对照组则接受载体注射。每组 5 只小鼠，2-甲氧基雌二醇（2ME2）组的疾病评分为 0.30±0.30，显着低于对照组的 2.09±0.28（p<0.05）[3]。每天给予 60-600 mg/kg 2-甲基雌二醇可对肿瘤发展产生剂量依赖性抑制。与媒介物治疗组 (86.5%) 相比，2-甲氧基雌二醇治疗组具有强哌莫硝唑阳性染色 (+++) 的细胞百分比要低得多（60 mg/kg/d 时为 36.0%，60 mg/kg/d 时为 0%） 200 和 600 毫克/公斤/天）。这可能是因为 2-甲氧基雌二醇治疗显着且剂量依赖性地抑制肿瘤的生长 [4]。

含有微管蛋白的MAP的体外质量[1]
将微管蛋白（2.75 mg/mL；参考文献16）组装到含有2ME2（最终药物浓度为1–500阿莫尔/升）的稳态[在含有1 mmol/L EGTA、1 mmol/L MgSO4（PEM100）和1 mmol/L GTP、35jC的100 mmol/L PIPES中，持续45分钟]。将最终DMSO和乙醇浓度分别调节至1%和5%。2ME2 V 5 Amol/L的浓度对微管聚合物质量没有影响，因此大多数实验使用20至500 Amol/L 2ME2。用2ME2孵育30分钟，此时微管解聚最大，将微管在35jC下离心30分钟，并从颗粒中去除上清液。将微管颗粒在0.2mol/L NaOH中溶解过夜，并测定上清液和颗粒的蛋白质浓度。我们检测了10Amol/L长春碱对F1%DMSO解聚的影响，以测试2ME2实验中所需的DMSO是否会影响解聚水平。我们发现二甲基亚砜对解聚水平没有影响。用20阿莫尔/升的鬼臼毒素作为阳性对照。

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体外测定MAP游离微管蛋白对微管聚合物质量的影响[1]
。 在2ME2（最终药物浓度为1-500阿莫尔/升）存在下，在含有1 mmol/L EGTA、1 mmol/L MgSO4（PEM100）和1 mmol/L GTP的100 mmol/L管道中，在30jC下组装纯化的牛脑微管蛋白（3.0 mg/mL）。将最终DMSO和乙醇浓度分别调节至V1%和5%，并通过Beckman DU 640分光光度计中350nm处的光散射监测组装。在带有SS-34转子的Sorvall RC5B plus离心机中，在30jC下以20000rpm离心微管60分钟。从颗粒中去除上清液，并测定颗粒的蛋白质浓度。

活体细胞微管动力学的图像采集与分析。[1]
如前所述（19），制备用于分析相间微管动力学的细胞。简言之，表达GFP微管蛋白的MCF7细胞在盖玻片上生长48小时（用聚赖氨酸、层粘连蛋白和纤连蛋白预处理以诱导细胞扁平化），然后在2ME2存在或不存在下孵育6小时。将对照细胞与等效浓度的DMSO单独孵育。将细胞转移到含有1.2Amol/L2ME2的记录介质[DEM缺乏酚红，并补充有25mmol/L HEPES、3.5g/L葡萄糖和羟化酶以抑制光漂白和防止光损伤]中。在双盖玻片室中密封盖玻片15分钟至2小时后进行分析。使用Metamorph软件驱动的Hamamatsu ORCA II数码相机，在Nikon Eclipse E800荧光显微镜上以4秒的间隔采集每个细胞的31张延时图像，该荧光显微镜具有将载物台和物镜保持在36F1jC的强制空气加热室。使用Metamorph的Track Points函数跟踪微管正端随时间的位置，将其绘制为微管随时间的长度（生命史图），并确定微管动力学的变量。用于分析活细胞中微管动力学的标准在参考文献中有详细描述。我们还发现，在分析细胞中的微管动力学过程中，保持培养基中的2ME2浓度是至关重要的。当记录介质中不包括2ME2时，微管动力学没有显著抑制，这与细胞中2ME2的快速损失一致（见结果）。

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免疫荧光显微镜。[1]
除了盖玻片用聚赖氨酸预处理而不是用层粘连蛋白或纤连蛋白预处理外，制备MCF7细胞用于免疫荧光显微镜以分析微管动力学。将细胞与0、1.2或10Amol/L 2ME2孵育20小时；在室温下在10%福尔马林中固定30分钟；并在20jC的甲醇中渗透10分钟。用含1%牛血清白蛋白的PBS中的20%正常山羊血清阻断非特异性抗体染色，并将细胞与DM1a抗微管抗体和CY3山羊抗小鼠第二抗体一起孵育以观察微管。细胞核用4¶，6-二脒基-2-苯基吲哚染色，盖玻片用长效Antifade固定。

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药物吸收和流出分析。[1]
将MCF7细胞接种到聚赖氨酸处理的闪烁瓶中（1x 105个细胞，1mL）。48小时后，用含有1.2 Amol/L[3 H]2ME2（比活性200–500 Ci/mol）或未标记的2ME2（用于测定细胞数）的新鲜培养基代替培养基。在15秒和30秒时从小瓶中取出培养基；1、5和10分钟；以及在药物添加后1、2、5和20小时。然后用1mL PBS快速冲洗细胞两次，并通过闪烁计数测定细胞内2ME2。如上所述，通过处理仅含有放射性标记介质（无细胞）的小瓶来测定背景放射性。通过初始摄取速率（15秒-1分钟）与时间0（3.7阿莫尔/升）的线性回归外推来确定与细胞的潜在非特异性结合。然后通过将细胞内2ME2的摩尔数除以平均细胞体积乘以每小瓶的细胞数来确定细胞内药物浓度。根据胰蛋白酶消化后四舍五入的细胞的平均直径计算平均细胞体积（n=38，平均细胞体积=3.2 10 12 L）。通过使用血细胞仪的手动细胞计数，在添加时和在1.2Amol/L2M中孵育20小时后测定细胞数。此外，20小时后，用1mL PBS洗涤细胞1分钟和5分钟，以确定2ME2从细胞中洗涤的容易程度。我们还使用与微管动力学实验相同的接种条件（3 x 104个细胞/mLx2 mL）进行了药物摄取实验。这些条件产生了略高的细胞内药物浓度。重复测量所有时间点，结果为五个实验的平均值和SD。

Implant of Tumor Cells to Rat Brain[4]
We stereotactically injected 9L-V6R cells (50,000 in 5 μL volume) into the brains of Fischer 344 rats (average body weight = 150 g) as reported by Barker et al. at stereotactic coordinates 1 mm forward of the frontal zero plane, 3 mm to the right of midline, and 4.5 mm deep.

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2-Methoxyestradiol Treatment[4]
For in vivo experiments, Panzem was used. Rats (n = 6 per group) were treated with an i.p. injection of the vehicle (60, 200, or 600 mg/kg/d of 2-methoxyestradiol/Panzem) for nine consecutive days beginning on the 8th day after the initial tumor cell injection. The experiment was repeated a second time using three rats per group.

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Imaging Studies[4]
BLI. Seven days after the tumor cell injection, the viable hypoxic tumor was identified by noninvasive BLI. BLIs were obtained using a Xenogen Small Animal Imager (IVIS Imaging System) equipped with Living Image software. Eight days after the tumor cell injection and before initiation of treatment, rats were anesthetized by i.p. injection of a ketamine (80 mg/kg)/xylazine (4 mg/kg) mixture. Rats were then injected with luciferin (100 mg/kg of luciferin) i.p., and after 15 minutes of incubation, 1-minute image acquisition at medium binning was taken. Imaging by BLI was also done on the 9th day of treatment.[4]
MRI. The response to 2-methoxyestradiol treatment was assessed by the measurement of tumor volume using noninvasive MRI before and after the treatment. Brain images of each animal were obtained on the first day of the treatment (4 hours after BLI to allow animals to recover) and on the 8th day of the treatment. The MRI scan was carried out using a 3T MRI scanner and a small volume coil (5-cm diameter). The animals were anesthetized by an i.p. injection of a ketamine (80 mg/kg)/xylazine (4 mg/kg) mixture and then placed in the coil. The head was secured using foam padding to minimize possible movements. Each animal received 1.0 ml/kg (0.2 mmol/L/kg) of Gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) i.v. A set of multi-slice, T1-weighted, spin echo images were obtained in the coronal section by using a repetition time of 400 ms, an echo time of 14 ms, and an imaging matrix of 128 × 128 with a field of view of 50 × 50 mm2. To match histologic analysis, a slice thickness of 2 mm was used without a slice gap. The number of signal averages was three for the majority of the scans. Tumors shown in the MRI were measured in three orthogonal dimensions. Tumor volume (V) was calculated as: V (mm3) = π(a × b × c) / 6, where a, b, and c represent width, height, and thickness, respectively. The mean rat brain volume was about 550 to 600 mm3, which was consistent with the size reported by Sahin et al. using histologic measurements of rat brain sections. A mean of these individual values was used. Following the MRI scans, rats were grouped to obtain an even distribution of tumor sizes.

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9L-V6R cells are injected into the brains of Fischer 344 rats

Metabolism / Metabolites
In vivo metabolism, assessed using 24-h collections of urine from cancer patients treated with 2ME2 revealed that <0.01% of the total administered dose of 2ME2 is excreted unchanged in urine and about 1% excreted as glucuronides. Collectively, this suggests that glucuronidation and subsequent urinary excretion are elimination pathways for 2ME2.
2-O-methoxyestradiol has known human metabolites that include 2-Methoxy-estradiol-17beta 3-glucuronide.
2-O-methoxyestradiol is a known human metabolite of 2-hydroxyestradiol.

Protein Binding
2ME2 was found to bind in decreasing order to plasma>albumin>alpha1-acid glycoprotein>sex-hormone-binding globulin. Plasma concentration-time profiles of total 2ME2 and unbound 2ME2 concentrations in a patient with cancer receiving 2ME2 as a single oral dose were parallel to each other. Thus, indicating that plasma protein binding is not an important consideration in pharmacokinetic monitoring of 2ME2.

[1]. 2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules. Mol Cancer Ther. 2006 Sep;5(9):2225-33.

[2]. Effects of 2-methoxyestradiol on apoptosis and HIF-1α and HIF-2α expression in lung cancer cells under normoxia and hypoxia. Oncol Rep. 2016 Jan;35(1):577-83.

[3]. 2-Methoxyestradiol Alleviates Experimental Autoimmune Uveitis by Inhibiting Lymphocytes Proliferation and T Cell Differentiation. Biomed Res Int. 2016;2016:7948345.

[4]. Antitumor effect of 2-methoxyestradiol in a rat orthotopic brain tumor model. Cancer Res. 2006, 66(24),11991-11997.

[5]. 2-Methoxyestradiol inhibits proliferation and induces apoptosis independently of estrogen receptorsalpha and beta. Cancer Res. 2002 Jul 1;62(13):3691-7.

[6]. Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death Differ. 2008;15(1):171-182.

2-methoxy-17beta-estradiol is a 17beta-hydroxy steroid, being 17beta-estradiol methoxylated at C-2. It has a role as an antineoplastic agent, an antimitotic, a metabolite, a human metabolite, a mouse metabolite and an angiogenesis modulating agent. It is a 17beta-hydroxy steroid and a 3-hydroxy steroid. It is functionally related to a 17beta-estradiol.
2-Methoxyestradiol (2ME2) is a drug that prevents the formation of new blood vessels that tumors need in order to grow (angiogenesis). It has undergone Phase 1 clinical trials against breast cancers and preclinical studies suggest that 2ME2 could also be effective against inflammatory diseases such as rheumatoid arthritis.
2-Methoxyestradiol has been reported in Homo sapiens with data available.
2-Methoxyestradiol is an orally bioavailable estradiol metabolite with potential antineoplastic activity. 2-Methoxyestradiol inhibits angiogenesis by reducing endothelial cell proliferation and inducing endothelial cell apoptosis. This agent also inhibits tumor cell growth by binding to tubulin, resulting in antimitotic activity, and by inducing caspase activation, resulting in cell cycle arrest in the G2 phase, DNA fragmentation, and apoptosis. (NCI04)
A metabolite of estradiol that lacks estrogenic activity and inhibits TUBULIN polymerization. It has antineoplastic properties, including inhibition of angiogenesis and induction of APOPTOSIS.

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Drug Indication
For the treatment of breast cancer and inflammatory diseases such as rheumatoid arthritis.

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Mechanism of Action
2-Methoxyestradiol is an angiogenesis inhibitor, and has been shown to attack both tumor cells and their blood supply in preclinical testing. 2-methoxyestradiol is a naturally occurring estrogen metabolite but has no undesired estrogenic activity.

C19H26O3

302.4079

302.188

C, 75.46; H, 8.67; O, 15.87

362-07-2

2-Methoxyestradiol-13C,d3;1217470-09-1;2-Methoxyestradiol-13C6;2-Methoxyestradiol-d5;358731-34-7

66414

Typically exists as white to off-white solids at room temperature

1.2±0.1 g/cm3

464.4±45.0 °C at 760 mmHg

188-190°C

234.7±28.7 °C

0.0±1.2 mmHg at 25°C

1.586

3.84

49.69

2

3

1

22

425

5

O([H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])[C@]3([H])C([H])([H])C([H])([H])C4=C([H])C(=C(C([H])=C4[C@@]3([H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H])OC([H])([H])[H])O[H]

CQOQDQWUFQDJMK-SSTWWWIQSA-N

InChI=1S/C19H26O3/c1-19-8-7-12-13(15(19)5-6-18(19)21)4-3-11-9-16(20)17(22-2)10-14(11)12/h9-10,12-13,15,18,20-21H,3-8H2,1-2H3/t12-,13+,15-,18-,19-/m0/s1

(8R,9S,13S,14S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,17-diol

NSC 659853; NSC-659853; NSC659853; 2-ME; 2-Methoxy Estradiol. 2-methoxyestradiol; Panzem; 2-Methoxyestradiol-17beta; 2-Hydroxyestradol 2-methyl ether; 2ME2; 2-MeOE2; US brand name: Panzem. Abbreviation: 2-ME2.

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)

DMSO: 60 mg/mL (198.4 mM) Water:<1 mg/mL Ethanol:<1 mg/mL

配方 1 中的溶解度: ≥ 2.08 mg/mL (6.88 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (6.88 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (6.88 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% DMSO+corn oil:5mg/mL

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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。