Fulvestrant  (Synonyms: ICI 182780; ZD 9238; ZM 182780)

MCF-7 or T47D cells are cultured in 10 cm dishes to ~75% confluence in EMEM growth medium. Twenty-four hours before treatment, the growth medium is replaced with phenol red-free RPMI-1640 growth medium. A stock solution of 10 mM RAD1901 is prepared in DMSO. Dilutions of RAD1901 are prepared in RPMI growth medium (doses ranging from 10 to 0.5 nM). Controls include 0.1% DMSO alone (vehicle), 100 nM Fulvestrant, and 1 µM ICI 47699. Plated cells are treated with RAD1901 or controls for 48 h, and then incubated for 15 min with ice-cold lysis buffer [1 mM EDTA, 0.5% Triton X-100, 5 mM NaF, 6 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1× HALT protease inhibitor cocktail]. Lysates are centrifuged at 2000g for 5 min, and the supernatant is diluted 1 : 1 in lysis buffer. Ninety-six-well plates are coated overnight with capture antibody (1 µg/mL), washed three times in the manufacturer’s wash buffer, blocked with blocking buffer for 2 h, and washed again. The prepared plates are incubated with 100 µL of the prepared cell lysate for 2 h, washed, incubated with biotinylated detection antibody for 2 h, and washed again. After a 20 min incubation with streptavidin-horseradish peroxidase, the plates are washed and incubated with substrate solution for 20 min. The reaction is stopped with stop solution, and the plates are analyzed on a microplate reader (OD₄₅₀)^[3].

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Rats^[1]
In studies with OVX rats, surgical preparation is performed at least 2 weeks before treatment began. To measure the duration of action of a single large dose of Fulvestrant, OVX rats are treated with a daily s.c. dose of 0.5 μg of estradici benzoate beginning on the day of Fulvestrant administration and continued until vaginal smears showed evidence of cornification. At that point the experiment is terminated and uterine weight is recorded. The arachis oil formulation used in these single dose duration of action studies contained 50 mg Fulvestrant/mL.
Mice^[3]
Female athymic nude mice [Crl:NU(NCr)-Foxn1nu] are used for tumor xenograft studies. Fourteen days after tumor cell implantation (designated as day 1 of the study), mice are 9 weeks of age, with body weights ranging from 21.4 to 32.5 g, individual tumor volumes ranging from 75 to 144 mm³, and a group mean tumor volume (MTV) of 108 mm³. The mice are randomized into nine groups of 15 animals each and treated with vehicle, ICI 47699 (1 mg/animal every other day), Fulvestrant (0.5 mg/animal daily), or RAD1901 (0.3, 1, 3, 10, 30, 60, 90, and 120 mg/kg daily). Tumor volumes are evaluated twice per week. The tumor endpoint is defined as an MTV of 1500 mm³ in the control group. Animals are also monitored for partial regression (PR) and complete regression responses.

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• [1]. Wakeling AE, et al. A potent specific pure antiestrogen with clinical potential. Cancer Res. 1991 Aug 1;51(15):3867-73.  [Content Brief]

  [2]. Yu X, et al.MiR-214 increases the sensitivity of breast cancer cells to tamoxifen and fulvestrant through inhibition of autophagy.Mol Cancer. 2015 Dec 15;14:208.  [Content Brief]

  [3]. Osborne CK, et al. Fulvestrant: an oestrogen receptor antagonist with a novel mechanism of action. Br J Cancer. 2004 Mar;90 Suppl 1:S2-6.  [Content Brief]

  [4]. Garner F, et al. RAD1901: a novel, orally bioavailable selective estrogen receptor degrader that demonstrates antitumor activity in breast cancer xenograft models. Anticancer Drugs. 2015 Oct;26(9):948-56  [Content Brief]

  [5]. Julia Kuhn, et al. GPR30 estrogen receptor agonists induce mechanical hyperalgesia in the rat. Eur J Neurosci. 2008 Apr;27(7):1700-9.  [Content Brief]