Tubulin and microtubule-associated proteins (MAPs).[3]

PC3 cell proliferation is inhibited by estramustine sodium phosphate (1 µg/mL, 48 hours) [1]. By decreasing miR-31, estramustine sodium phosphate (2 µg/mL, 48 h) can cause PC3 cells to undergo more phosphatidylserine eversion and ultimately undergo apoptosis [1]. In RAW 264.7 cells, estramustine sodium phosphate (0–40 µM, 24-72 hours) suppresses tubulin cytoskeleton and cell growth [2]. By blocking Smad3 activation, estramustine sodium phosphate (10 µM, 24 hours) prevents TGF-β-induced RAW 264.7 cell migration and TGF-β-induced uPA generation [2].

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Estramustine phosphate sodium inhibits purified tubulin polymerization in vitro through direct interaction with tubulin.[3] (No quantitative data provided)

Estramustine sodium phosphate (ip, 4 or 12 mg/kg daily for 2 weeks) reduced PAC120 tumor growth by 53% on day 35 [3].
In PAC120 (androgen-dependent human prostate cancer xenograft), Estramustine phosphate sodium (4 mg/kg, daily for 2 consecutive weeks, intraperitoneal) inhibited tumor growth by 53% by day 35 (p=0.03), leading to survival of 55 days vs 35 days in control group.[3]
Increasing the dose of Estramustine phosphate sodium up to 3-fold alone or combined with docetaxel did not improve its antitumoral effect in PAC120.[3]
In HID variants (androgen-independent xenografts), Estramustine phosphate sodium as single agent had no effect on tumor growth.[3]
In HID25, Estramustine phosphate sodium (4 mg/kg for 4 days with docetaxel every 3 weeks) potentiated the antitumoral effect of docetaxel, leading to growth stabilization; increasing the dose up to 3-fold provided the same result.[3]
In HID28, Estramustine phosphate sodium did not potentiate the efficacy of docetaxel regardless of host hormonal status.[3]
In PAC120, combination of Estramustine phosphate sodium with docetaxel did not improve the therapeutic response compared to docetaxel alone.[3]

Cell viability assay [1]
Cell Types: PC3 Cell
Tested Concentrations: 1 µg/mL
Incubation Duration: 48 hrs (hours)
Experimental Results: Inhibition of PC3 cell growth.

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Immunofluorescence[2]
Cell Types: RAW 264.7 Cell
Tested Concentrations: 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Disruption of interphase microtubules.

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Cell migration assay[2]
Cell Types: RAW 264.7 cells treated with TGF-β for 18 hrs (hours)
Tested Concentrations: 10 µM
Incubation Duration: 24 hrs (hours)
Experimental Results: Inhibition of TGF-β induced cell migration.

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Proliferation assay (MTT): Cells were treated with indicated EP concentrations for 24 or 72 hours. MTT (0.5 mg/ml) was added for 2 hours, then formazan crystals dissolved in isopropanol:DMSO (3:2), absorbance read at 630 nm.[2]
Immunofluorescence for α-tubulin and pSmad3: 10^5 cells seeded on coverslips, grown to 50% confluence, treated, fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, incubated with primary antibodies, then fluorescent secondary antibodies and DAPI (1 µg/ml). Mounted with DABCO-Mowiol, examined by epi-fluorescence microscopy. pSmad3 positive cells quantified by counting at least 5 fields per condition.[2]
Wound healing migration assay: 5×10^4 cells/well in 24-well plates grown to confluence, scratch made with 200 µl pipette tip, washed, treated for 24h, fixed with ice-cold methanol, stained with 0.1% crystal violet, migration quantified by TScratch software.[2]
Boyden chamber migration assay: RAW 264.7 cells labeled with CFSE, seeded in upper chamber (10^5 cells/well in 150 µl) with 8.0 µm pore filters. Lower chamber contained 0.5 ml medium with or without TGF-β (5 ng/ml) and EP. After 18h, non-migrating cells removed, bottom cells fixed, mounted, counted under epi-fluorescence microscope (8 random fields per insert).[2]
uPA activity - radial caseinolysis assay: Protein-normalized conditioned medium (50 µl) added to wells in 1% agarose gel containing 0.5% casein, 2 µg/ml plasminogen, 10 mM CaCl2. Clear area diameters measured after 24h at 37°C, uPA activity as fold increase vs untreated. Quantified by densitometry (NIH-ImageJ).[2]
uPA zymography: Protein-normalized conditioned medium subjected to 10% SDS-PAGE under non-reducing conditions. Gels washed with 2.5% Triton X-100, placed on 1% agarose gels with 0.5% casein and 2 µg/ml plasminogen, incubated 24h at 37°C. Clear bands quantified by densitometry.[2]
Western blot: Cells lysed in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris pH 7.5, 1 mM Na3VO4, protease inhibitors). Equal proteins separated by SDS-PAGE, transferred to PVDF, blocked in 4% nonfat milk with 0.5% Tween 20, incubated with primary then HRP-secondary antibodies, visualized with ECL reagent, quantified by densitometry (NIH-ImageJ).[2]
Luciferase reporter assays: uPA promoter activity using p-4.8 murine uPA-Luc construct; Smad3 activity using p(CAGA)12-Luc construct. Firefly luciferase activity normalized to β-galactosidase activity.[2]

Animal/Disease Models: Swiss nu/nu (nude) male mice (5 weeks old) with PAC120 tumors [3]
Doses: 4 mg/kg, 12 mg/kg
Route of Administration: intraperitoneal (ip) injection; daily; for 2 weeks
Experimental Results: Inhibition of skin lesion development and resulting dissociation between DTH response and antibody production.

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Animal/Disease Models: Human Prostate Cancer Xenograft PAC120[3]
Doses: 4 or 12 mg/kg daily for 2 weeks.
Route of Administration: intraperitoneal (ip) injection.
Experimental Results: On day 35, PAC120 inhibited tumor growth by 53%.

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Tumor-bearing nude mice were randomly distributed into groups of 7-12 assigned to control or treatment.[3]
Estramustine phosphate sodium solution was administered intraperitoneally at a daily dose of 4 or 12 mg/kg, 4 days weekly starting the day before docetaxel injection and for 2 consecutive weeks.[3]
For combination with docetaxel: docetaxel (20 mg/kg) was injected intraperitoneally on day 2 of each 3-week cycle; Estramustine phosphate sodium was given daily for 4 days starting the day before docetaxel.[3]
In PAC120 experiments, Estramustine phosphate sodium (4 mg/kg) was given daily for 2 consecutive weeks (4 days per week? as per schedule).[3]
Tumor growth was assessed by measuring two perpendicular diameters with a caliper every 3 days. Tumor volume calculated as V = a^2 × b / 2. Growth delay was time required for tumors to achieve 5-fold increase in initial volume.[3]

[1]. Estramustine phosphate induces prostate cancer cell line PC3 apoptosis by down-regulating miR-31 levels. Eur Rev Med Pharmacol Sci. 2018 Jan;22(1):40-45.

[2]. Estramustine Phosphate Inhibits TGF- β-Induced Mouse Macrophage Migration and Urokinase-Type Plasminogen Activator Production. Anal Cell Pathol (Amst). 2018 Sep 2;2018:3134102.

[3]. Activity of docetaxel with or without estramustine phosphate versus mitoxantrone in androgen dependent and independent human prostate cancer xenografts. J Urol. 2003 May;169(5):1729-34.

Estromostatin sodium phosphate is an organosoil salt, the disodium salt of estradiol phosphate. It contains the estradiol phosphate ion (2-). Estromostatin sodium phosphate is the oral disodium salt monohydrate of estradiol phosphate, a synthetic molecule that links estradiol and nitrogen mustard together via a carbamate bond. Estromostatin and its main metabolite, estradiol, bind to microtubule-associated protein (MAP) and tubulin, thereby inhibiting microtubule dynamics and causing late-stage blockade in a dose-dependent manner. The drug also has anti-androgenic effects. A nitrogen mustard linked to estradiol (usually in phosphate form); used to treat prostate cancer; also has radiation protection. See also: Estromostatin (with active moiety).

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Mechanism of action: Estramustine phosphate sodium inhibits microtubule function by binding to tubulin and microtubule-associated proteins. It can remove MAPs from microtubules, resulting in increased number of target sites available for docetaxel binding, thus potentiating docetaxel activity.[3]
It has hormonal (androgen receptor antagonist) properties due to its estradiol conjugate.[3]
In the PAC120 hormone-dependent tumor, it showed efficacy as single agent, likely due to its androgen receptor antagonist property.[3]
Clinical background: In 6 randomized trials including 304 patients, only 14 (4.6%) had objective response to single agent Estramustine phosphate sodium. A phase II trial of estramustine combination with paclitaxel was initiated by ECOG.[3]

563.098

52205-73-9

Estramustine phosphate;4891-15-0

444000

White to off-white solid powder

1.253g/cm3

565.8ºC at 760mmHg

296ºC

6.175

0

6

7

35

735

5

[O-]P([O-])(O[C@H]1CC[C@@]2([H])[C@@]3([H])[C@](C(C=CC(OC(N(CCCl)CCCl)=O)=C4)=C4CC3)([H])CC[C@@]21C)=O.[Na+].[Na+]

IIUMCNJTGSMNRO-VVSKJQCTSA-L

InChI=1S/C23H32Cl2NO6P.2Na/c1-23-9-8-18-17-5-3-16(31-22(27)26(12-10-24)13-11-25)14-15(17)2-4-19(18)20(23)6-7-21(23)32-33(28,29)30;;/h3,5,14,18-21H,2,4,6-13H2,1H3,(H2,28,29,30);;/q;2*+1/p-2/t18-,19-,20+,21+,23+;;/m1../s1

disodium;[(8R,9S,13S,14S,17S)-3-[bis(2-chloroethyl)carbamoyloxy]-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl] phosphate

EmcytRo 21-8837/001 Leo299 estramustine Phosphate sodium EstracytRo 222296000 LS 299 NSC 89199

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

H2O : ~62.5 mg/mL (~110.75 mM)
DMSO : ~5 mg/mL (~8.86 mM)

Solubility in Formulation 1: 65 mg/mL (115.18 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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