生物活性

Fulvestrant is a selective oestrogenreceptor down-regulator (SERD), which by blocking proliferation of breastcancer cells, is an effective endocrine treatment for women with hormone-sensitiveadvanced breast cancer. Fulvestrant is a high affinity estrogen receptor antagonist (IC50 = 0.29 nM), devoid of any partial agonism in vitro and Fulvestrant is also high affinity agonist at the membrane estrogen receptor GPER.

Fulvestrantinhibits the soluble epoxide hydrolase (sEH) with an IC50 of 6±1nM (Ki of 26±3 nM).[1]

Estrogen receptor activities

Anti-proliferation of fulvestrant

体外研究

体内研究

5% DMSO+95% Corn oil

激酶实验

ALP activity assays[6]

The ALP assay for osteoblastdifferentiation was performed at Day 14. Cells were lysed with a buffercontaining 10mM Tris-HCl pH 7.4 and 0.2% Triton X-100 and were then sonicatedfor 20 sec at 4˚C. Samples were incubated with 10mM p-nitrophenylphosphate as asubstrate in 100mM glycine buffer (pH 10.5) containing 1mM MgCl₂ at37˚C in a water bath. Total protein content was determined in comparison with aseries of BSA as the internal standards. The absorbance at 405nm was measuredusing a microplate reader, and ALP activities were normalized with respect tototal protein content.

细胞实验

Cell lines and culture conditions[7]

MCF-7 is a cell line that was firstisolated in 1970 from the breast tissue of a 69-year-old Caucasian woman. MCF7Lcells (derived from MCF-7 cell line by stable transfection of a luciferase geneunder constitutive promoter) were routinely maintained in DMEM-F12 medium supplementedwith 5 % FCS (FCS medium). During long term treatments, MCF7L cells were growneither in: DMEM-F12 (phenol red-free) supplemented with 3 % dextran-coatedcharcoal-treated FCS (DCC) to obtain MCF7L-DCCLT, or with DCC medium with 10nMof 4-hydroxytamoxifen to obtain MCF7L-OHTLT, or with DCC medium with 10nMICI182780 to obtain MCF7LICILT cell line (vehicle was ethanol). Before mRNA analysis,cells were hormone-deprived in DCC medium for 4 days.

Cellgrowth analysis

Cell growth analysis was tested by usingthe MTT assay. After 4 days of hormone deprivation, cells were seeded at a densityof about 1000 cells/well in 96-well tissue culture plates in DCC medium. Thenext day (day 0), 10 nM E2 or 10nM OHTam or 10 nM ICI182,780 or vehicle alone (ethanol)were added to the cultures (n = 6 wells/each condition). Medium was changedevery 2 days. At day 8, 0.5 mg/mL of MTT was added to selected wells at 37 ℃ for 4 h. MTT solution was then removed and the plates wereair-dried. Formazan crystals were then solubilized I DMSO. Absorbance was readat 540 nm on a spectrophotometer using a reference filter of 620 nm and resultswere normalized to the cell density at day 0.

动物实验

Animals and cell culture[8]

Female Fisher 344 rats (60–70 g) werehoused under a 12 h light/12 h dark cycle at 20–23°C with free access to foodand water. Animals were ovariectomized a week before estrogen treatment toexhaust endogenous estrogen. Alzet® osmotic pumps containing 10mg 17 β-estradiolin 50% (v/v) glycerol/50% (v/v) DMSO solution were implanted subcutaneously toinduce prolactinomas. After 8 weeks, fulvestrant dissolved into a mixture ofcastor oil and ethanol and benzene methanol (7:2:1) was injected into themuscles of fulvestrant rats.

Magneticresonance imaging

MRI experiments were performed on a 7.0Tvertical bore Bruker Avance nuclear magnetic resonance (NMR) spectrometer. Theparameters used in the scans were optimized for gray–white matter contrast: aT2-weighted 3D fast spin-echo sequence, with TR = 2000ms, echo train length =6, TEeff = 42ms, field-of-view (FOV) = 25 × 28 × 14 mm and matrix size = 450 ×504 × 250, giving an image with 56μm isotropic voxels. Total imaging time was 7min.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Morisseau C, Pakhomova S, Hwang SH, Newcomer ME, Hammock BD. Inhibition of soluble epoxide hydrolase by fulvestrant and sulfoxides. Bioorg Med Chem Lett. 2013;23(13):3818-3821.
[2] Wakeling AE DM, Bowler J. A potent specific pure antiestrogen with clinical potential. Cancer Res. 1991;51(15):3867-3873.
[3] Liu J, Zheng S, Akerstrom VL, et al. Fulvestrant-3 Boronic Acid (ZB716): An Orally Bioavailable Selective Estrogen Receptor Downregulator (SERD). J Med Chem. 2016;59(17):8134-8140.
[more]

体内溶解度
约35 mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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