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

2-Methoxyestradiol (2-ME) (5-100 μM) inhibits the assembly of purified tubulin in a concentration-dependent manner, with maximum inhibition (60%) at 200 μM 2-Methoxyestradiol (2ME2). 2-Methoxyestradiol strongly decreased mean microtubule growth rate, duration and length, and overall dynamics in viable interphase MCF7 cells with an IC50 (1.2 μM) of mitotic arrest. This was in line with its actions in vitro and did not appear to be related to microtubule depolymerization. 2. 2-Methoxyestradiol protects quiescent cells while inducing G2-M arrest and death in numerous cell types that are actively proliferating. 2. It has been demonstrated that large quantities of methylestradiol depolymerize microtubules in cells by binding to tubulin at or near the colchicine site and inhibiting microtubule assembly [1]. In cells cultivated under hypoxia, 2-Methoxyestradiol (2-ME) decreases HIF-1α and HIF-2α nuclear labeling. 2. Methoxyestradiol reduces the transcriptional activity and levels of HIF-1α protein. It is an anti-angiogenic, anti-proliferative, and pro-apoptotic drug. The growth rate of A549 cells treated with 10 μM 2-Methoxyestradiol was significantly reduced at 96 hours compared to DMSO-treated cells (66.2±7.2 and 101.2±2.3%, respectively; p=0.04). When cells treated with 10 μM 2-Methoxyestradiol in normoxic conditions were compared to cells under low O2 concentrations (5.8±0.2%; p=0.003), a significant increase in apoptosis was seen [2].

In order to investigate the impact of 2-Methoxyestradiol (2-ME2) on the progression of uveitis, C57BL/6 mice were split into two groups at random and given an IRBP peptide vaccination. From day 0 to day 13, the 2ME2 group got intraperitoneal injections of 15 mg/kg of 2-Methoxyestradiol, whereas the control group received a vehicle. With five mice in each group, the 2-Methoxyestradiol (2ME2) group had an illness score of 0.30±0.30, considerably lower than the 2.09±0.28 in the control group (p<0.05) [3]. The administration of 60-600 mg/kg/d of 2-methylestradiol led to a dose-dependent suppression of tumor development. In comparison to the vehicle treatment group (86.5%), the 2-Methoxyestradiol-treated group had a much lower percentage of cells with strong pimonidazole-positive staining (+++) (36.0% at 60 mg/kg/d, 0% at 200 and 600 mg/kg/d). This could be because 2-Methoxyestradiol therapy significantly and dose-dependently inhibited the growth of tumors [4].

Mass of MAP-Containing Tubulin In vitro[1]
Microtubule protein (2.75 mg/mL; ref. 16) was assembled to steady-state [in 100 mmol/L PIPES containing 1 mmol/L EGTA and 1 mmol/L MgSO4 (PEM100) and 1 mmol/L GTP, 35jC for 45 minutes] containing 2ME2 (final drug concentrations of 1 – 500 Amol/L).Final DMSO and ethanol concentrations were adjusted to 1% and 5%, respectively. Concentrations of 2ME2 V 5 Amol/L had no effect on microtubule polymer mass, and thus 20 to 500 Amol/L 2ME2 was used for most of the experiments.Incubation with 2ME2 was carried out for 30 minutes, at which time microtubule depolymerization was maximal, and microtubules were centrifuged at 35jC for 30 minutes and the supernatant was removed from the pellets.Microtubule pellets were solubilized overnight in 0.2 mol/L NaOH and the protein concentrations of supernatants and pellets were determined. We examined the effects of 10 Amol/L vinblastine on depolymerization F 1% DMSO to test whether the DMSO that was necessary in the 2ME2 experiments might influence the depolymerization level.We found no effect of the DMSO on the depolymerization level.Podophyllotoxin (20 Amol/L) was used as a positive control.

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Determination of Effects on Microtubule Polymer Mass of MAP-Free Tubulin In vitro [1]
Purified bovine brain tubulin (3.0 mg/mL) was assembled in the presence of 2ME2 (final drug concentrations of 1 – 500 Amol/L) in 100 mmol/L PIPES containing 1 mmol/L EGTA, 1 mmol/L MgSO4 (PEM100), and 1 mmol/L GTP, at 30jC.Final DMSO and ethanol concentrations were adjusted to V1% and 5%, respectively, and assembly was monitored by light scattering at 350 nm in a Beckman DU 640 spectrophotometer.Microtubules were centrifuged at 20,000 rpm for 60 minutes, at 30jC, in a Sorvall RC5B plus centrifuge with an SS-34 rotor.Supernatants were removed from pellets, and the protein concentrations of the pellets were determined.

Image Acquisition and Analysis of Microtubule Dynamics In Living Cells. [1]
Cells were prepared for analysis of interphase microtubule dynamics as described previously (19).Briefly, MCF7 cells expressing GFP-tubulin were grown for 48 hours on coverslips (pretreated with polylysine, laminin, and fibronectin to induce cell flattening) and then incubated in the presence or absence of 2ME2 for 6 hours.Control cells were incubated with an equivalent concentration of DMSO alone.Cells were transferred to recording medium [DMEM lacking phenol red and supplemented with 25 mmol/L HEPES, 3.5 g/L glucose, and Oxyrase to inhibit photobleaching and prevent photodamage] containing 1.2 Amol/L 2ME2.Analysis was carried out 15 minutes to 2 hours after sealing coverslips in a double coverslip chamber.Thirty-one time-lapse images of each cell were acquired at 4-second intervals using a Hamamatsu ORCA II digital camera driven by Metamorph software on a Nikon Eclipse E800 fluorescence microscope with a forced air heating chamber maintaining the stage and objective at 36 F 1jC.The positions of the plus ends of microtubules over time were tracked using the Track Points function of Metamorph, graphed as microtubule length over time (life history plots) and the variables of microtubule dynamics were determined.The criteria used to analyze microtubule dynamics in living cells are described in detail in ref. We also found that it was critical to maintain the 2ME2 concentration in the medium during analysis of microtubule dynamics in cells.When 2ME2 was not included in the recording medium, there was no significant suppression of microtubule dynamics, consistent with rapid loss of 2ME2 from cells (see Results).

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Immunofluorescence Microscopy. [1]
MCF7 cells were prepared for immunofluorescence microscopy as for analysis of microtubule dynamics except that coverslips were pretreated with poly-lysine but not laminin or fibronectin.Cells were incubated with 0, 1.2, or 10 Amol/L 2ME2 for 20 hours; fixed in 10% formalin for 30 minutes at room temperature; and permeabilized in methanol at
20jC for 10 minutes.Nonspecific antibody staining was blocked with 20% normal goat serum in PBS containing 1% bovine serum albumin and cells were incubated with DM1a anti-a-tubulin antibody followed by CY3 goat antimouse secondary antibody to visualize microtubules.Nuclei were stained with 4¶,6-diamidino-2-phenylindole and coverslips were mounted with Prolong Antifade.

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Analysis of Drug Uptake and Efflux.[1]
MCF7 cells were seeded into poly-lysine-treated scintillation vials (1x 105 cells, 1 mL).After 48 hours, medium was replaced with fresh medium containing 1.2 Amol/L [3 H]2ME2 (specific activity 200 – 500 Ci/mol) or unlabeled 2ME2 (for determination of cell number).Medium was removed from vials at 15 and 30 seconds; 1, 5, and 10 minutes; and 1, 2, 5, and 20 hours after drug addition.Cells were then rapidly rinsed twice with 1 mL PBS and intracellular 2ME2 was determined by scintillation counting.Background radioactivity was determined by treating vials containing only radiolabeled medium (no cells) as above.Potential nonspecific binding to cells was determined by extrapolation of the linear regression of the initial rate of uptake (15 seconds – 1 minute) to time 0 (3.7 Amol/L).The intracellular drug concentration was then determined by dividing the moles of intracellular 2ME2 by the average cell volume times the number of cells per vial.The mean cell volume was calculated from the mean diameter of cells rounded up after trypsinization (n = 38, mean cell volume = 3.2 10
12 L).Cell number was determined at the time of addition and 20 hours after incubation in 1.2 Amol/L 2M by manual cell counting using a hemacytometer.Additionally, after 20 hours, cells were washed with 1 mL PBS for 1 minute and 5 minutes to determine how readily 2ME2 is washed out of cells.We also did drug uptake experiments using the same seeding conditions as we used in the microtubule dynamics experiments (3 x 104 cells/mLx2 mL).These conditions yielded a slightly higher intracellular drug concentration.All time points were measured in duplicate, and results are the mean and SD of five experiments.

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.

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Pharmacodynamics
2-Methoxyestradiol belongs to the family of drugs called angiogenesis inhibitors. It also acts as a vasodilator.

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

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

Solubility in Formulation 1: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (6.88 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2% DMSO+corn oil:5mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)