Estrogen Receptor/ER (IC50 = 9.4 nM)

FuLvestrant (ICI 182780; ZD 9238; ZM 182780) is a very effective and selective oestrogen action inhibitor that shows superior growth suppression in animal models and human breast cancer cells. With an IC50 of 0.29 nM, fuLvestrant stops the development of MCF-7 human breast cancer cells. Fulvestrant has a relative binding affinity of 0.89. FuLvestrant maintains its pure estrogen antagonist activity while having a markedly increased antiestrogenic potency [1]. Fulvestrant, an ER antagonist that downregulates ER, is the first new class of endocrine control medication[3]. ERα expression in MCF-7 cells was not affected by 1 μM ICI 47699 treatment, while it was fully suppressed by 100 nM FuLvestrant [4].

When given by itself, fulvestrant (ICI 182,780) exhibits no uterotropic activity parenterally (sc) in immature female rats. Fulvestrant completely opposes the effects of estrogen at a dose of 0.5 mg/kg/day sc. Oral fulvestrant (5 mg/kg/day) treatment and subcutaneous administration are qualitatively comparable [1]. in two human breast cancer models in naked mice. After a single injection, fulvestrant (5 mg) in one of the models totally stopped the growth of MCF-7 tumor xenografts for at least 4 weeks. Fulvestrant suppressed existing tumor growth for twice as long and delayed tumor growth more than treatment with ICI 47699 in additional experiments conducted in nude mice carrying MCF-7 xenografts. Large[3]. At day 40, fulvestrant showed 88% tumor growth inhibition (TGI) [4].

Previous studies from this laboratory have described a series of 7 alpha-alkylamide analogues of estradiol with pure antiestrogenic activity, exemplified by ICI 164,384. A new compound, 7 alpha-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1,3,5(10 )- triene-3,17 beta-diol (ICI 182,780) has now been identified which has significantly increased antiestrogenic potency and retains pure estrogen antagonist activity. The antiuterotrophic potency of ICI 182,780 in the immature rat was more than 10-fold greater than that of ICI 164,384 (50% effective doses of 0.06 and 0.9 mg/kg, respectively). This order of magnitude increase of in vivo potency was also reflected, in part, by intrinsic activity at the estrogen receptor. The relative binding affinities of ICI 182,780 and ICI 164,384 were 0.89 and 0.19, respectively, compared with that of estradiol (1.0). Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded that of ICI 164,384 in MCF-7 human breast cancer cells, where 50% inhibitory concentrations of 0.29 and 1.3 nM, respectively, were recorded. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. This increased efficacy was reflected by a greater reduction of the proportion of cells engaged in DNA synthesis in ICI 182,780-treated cell cultures compared with tamoxifen-treated cells.[1]

---

Due to their favourable tolerability profiles, endocrine therapies have long been considered the treatment of choice for hormone-sensitive metastatic breast cancer. However, the oestrogen agonist effects of the available selective oestrogen receptor modulators, such as tamoxifen, and the development of cross-resistance between endocrine therapies with similar modes of action have led to the need for new treatments that act through different mechanisms. Fulvestrant ('Faslodex') is the first of a new type of endocrine treatment--an oestrogen receptor (ER) antagonist that downregulates the ER and has no agonist effects. This article provides an overview of the current understanding of ER signalling and illustrates the unique mode of action of fulvestrant. Preclinical and clinical study data are presented in support of the novel mechanism of action of this new type of ER antagonist[3].

Cell viability assay[2]
MCF7 cells seeded in 6-well plate were transfected with 100 nM negative control or miR-214 mimics and inhibitors for 24 h. Cells were trypsinized into 96-well plates at a density of 8 × 103 cells/well and then treated with 5 μΜ 4-OHT, or 1 μM fulvestrant (FUL) for 72 h.. Cell viability was estimated by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.

---

Cell autophagy analysis[2]
Cells were transfected with GFP-LC3 plasmid (Addgene) and then treated with 0.1 % v/v ethanol vehicle or 5 μM 4-OHT or 1 μM fulvestrant (FUL) for 48 h. GFP-LC3-II-positive punctate pattern was observed under confocal microscope equipped with oil immersion lens (40×) with 405- and 488-nm excitation lasers. Numbers of autophagosomes were counted by using the Image J program

Sustained antiestrogenic effects, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in nude mice. A single injection of ICI 182,780 provided antitumor efficacy equivalent to that of daily tamoxifen treatment for at least 4 weeks. The properties of ICI 182,780 identify this pure antiestrogen as a prime candidate with which to evaluate the potential therapeutic benefits of complete estrogen withdrawal in endocrine-responsive human breast cancer.[1]

---

ICI 182,780/fulvestrant induced PKCε-dependent mechanical hyperalgesia[5]
To substantiate the finding that the novel estrogen receptor GPR30 apparently mediates the recently described effect of estrogen on nociceptive neurons, we used a second agonist of GPR30, which simultaneously blocks signalling through ERα and -β, ICI 182,780 (DeFriend et al., 1994; Molinari et al., 2000; Chan et al., 2007). The behavioural experiments established a clear dose-dependence for intradermal ICI 182,780 to produce mechanical hyperalgesia (ICI 182,780 dissolved to 10 mg/mL in 100% DMSO, diluted to final concentration in 2.5 µL in PBS; final concentration of DMSO 10%) into hind paws of male rats. Neither spontaneous pain nor redness or swelling was observed. The maximum decrease in nociceptive threshold, by 35.3 ± 2.2%, was observed after injection of 100 ng ICI 182,780 (Fig. 6a; n = 6 paws; absolute value of baseline withdrawal threshold of negative controls 114 ± 1.6 g).

---

Formulated to 50 mg/mL in arachis oil; 5 mg/mouse; s.c. injection

The human breast cancer xenografts MCF-7 in nude mice

Absorption, Distribution and Excretion
Fulvestrant was rapidly cleared by the hepatobiliary route with excretion primarily via the feces (approximately 90%). Renal elimination was negligible (less than 1%).
3 to 5 L/kg
Peak plasma concentrations of fulvestrant are attained approximately 7 days after IM administration and persist for at least 1 month. Steady-state plasma fulvestrant concentrations usually are achieved within 3-6 months when the drug is administered once-monthly by IM injection.
Fulvestrant appears to be rapidly and extensively distributed, principally into the extravascular space
99% (mainly VLDL, LDL, and HDL lipoprotein fractions).
Has been shown to cross the placenta and distribute into milk in rats.
For more Absorption, Distribution and Excretion (Complete) data for FULVESTRANT (8 total), please visit the HSDB record page.

---

Metabolism / Metabolites
Metabolism of fulvestrant appears to involve combinations of a number of possible biotransformation pathways analogous to those of endogenous steroids, including oxidation, aromatic hydroxylation, conjugation with glucuronic acid and/or sulphate at the 2, 3 and 17 positions of the steroid nucleus, and oxidation of the side chain sulphoxide. Identified metabolites are either less active or exhibit similar activity to fulvestrant in antiestrogen models. Studies using human liver preparations and recombinant human enzymes indicate that cytochrome P-450 3A4 (CYP 3A4) is the only P-450 isoenzyme involved in the oxidation of fulvestrant; however, the relative contribution of P-450 and non-P-450 routes in vivo is unknown.
Biotransformation and disposition of fulvestrant in humans have been determined following intramuscular and intravenous administration of 14C-labeled fulvestrant. Metabolism of fulvestrant appears to involve combinations of a number of possible biotransformation pathways analogous to those of endogenous steroids, including oxidation, aromatic hydroxylation, conjugation with glucuronic acid and/or sulphate at the 2, 3 and 17 positions of the steroid nucleus, and oxidation of the side chain sulphoxide.
Metabolites of fulvestrant exhibit pharmacologic activity that is similar to or less than that of the parent compound.
In vitro studies indicate that CYP3A4 is the only enzyme involved in fulvestrant oxidation; however, the relative contribution of CYP and non-CYP routes in vivo currently is not known.

---

Biological Half-Life
40 days
The elimination half-life of fulvestrant is about 40 days.

Hepatotoxicity
Fulvestrant therapy is said to be associated with serum enzyme elevations in up to 15% of patients, but the elevations are generally asymptomatic, transient and mild, rarely requiring dose adjustment or discontinuation. ALT elevations above 5 times the upper limit of normal occurred in only 1% to 2% of patients. However, specifics on the timing and course of serum enzyme elevations during fulvestrant therapy have not been described. In addition, no cases of clinically apparent liver injury with jaundice were reported in the prelicensure controlled trials of fulvestrant and none have been published since its approval in the United States and more wide-scale use. Nevertheless, the product label for fulvestrant mentions that "hepatitis and liver failure have been reported infrequently (
Likelihood score: E* (unproven but suspected cause of clinically apprent liver injury).

---

Protein Binding
99% (mainly VLDL, LDL, and HDL)

[1]. A potent specific pure antiestrogen with clinical potential. Cancer Res. 1991 Aug 1;51(15):3867-73.

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

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

[4]. 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.

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

Therapeutic Uses
Antineoplastic Agents; Hormonal Estrogen Antagonists
Fulvestrant is indicated for the treatment of hormone receptor positive metastatic breast cancer in postmenopausal women with disease progression following antiestrogen therapy. /Included in US product label/

---

Drug Warnings
/Fulvestrant is contraindicated in/ pregnancy, known hypersensitivity to fulvestrant, benzyl alcohol, or any ingredient in the formulation.
Because fulvestrant is administered by IM injection, the drug should not be used in patients with bleeding diatheses or thrombocytopenia or in those receiving anticoagulant therapy.
The most common adverse effects of fulvestrant are adverse GI effects (e.g., nausea, vomiting, constipation, diarrhea, abdominal pain), headache, back pain, vasodilation (hot flushes), and pharyngitis, which occurred in approximately 52, 15, 14, 18, and 16% of patients, respectively, who received the drug in clinical studies.
Other adverse effects occurring in 5-23% of patients receiving fulvestrant (in order of descending frequency) include asthenia, pain, nutritional disorders, bone pain, dyspnea, injection site pain, increased cough, pelvic pain, anorexia, peripheral edema, rash, chest pain, flu syndrome, dizziness, insomnia, fever, paresthesia, urinary tract infection, depression, anxiety, and sweating. Injection site reactions with mild transient pain and inflammation were reported in 7% of patients receiving a single 5-mL injection of fulvestrant in one study and in 27% of those who received two 2.5-mL injections of the drug in another study.
For more Drug Warnings (Complete) data for FULVESTRANT (7 total), please visit the HSDB record page.

---

Pharmacodynamics
Fulvestrant for intramuscular administration is an estrogen receptor antagonist without known agonist effects.

C32H47F5O3S

606.77

606.316

C, 63.34; H, 7.81; F, 15.66; O, 7.91; S, 5.28

129453-61-8

Fulvestrant (Standard);129453-61-8;Fulvestrant (S enantiomer);1316849-17-8;Fulvestrant (R enantiomer);1807900-80-6;Fulvestrant-d3

104741

White to off-white solid powder

1.2±0.1 g/cm3

674.8±55.0 °C at 760 mmHg

104-106°C

361.9±31.5 °C

0.0±2.2 mmHg at 25°C

1.522

7.92

2

9

14

41

854

6

C[C@]12CC[C@H]3[C@H]([C@@H]1CC[C@@H]2O)[C@@H](CC4=C3C=CC(=C4)O)CCCCCCCCCS(=O)CCCC(C(F)(F)F)(F)F

VWUXBMIQPBEWFH-WCCTWKNTSA-N

InChI=1S/C32H47F5O3S/c1-30-17-15-26-25-12-11-24(38)21-23(25)20-22(29(26)27(30)13-14-28(30)39)10-7-5-3-2-4-6-8-18-41(40)19-9-16-31(33,34)32(35,36)37/h11-12,21-22,26-29,38-39H,2-10,13-20H2,1H3/t22-,26-,27+,28+,29-,30+,41?/m1/s1

(7R,8R,9S,13S,14S,17S)-13-methyl-7-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,17-diol

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)

Solubility in Formulation 1: 2.75 mg/mL (4.53 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (4.12 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), suspension solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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.

---

View More

Solubility in Formulation 3: 2.08 mg/mL (3.43 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (3.43 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 corn oil 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.

---

Solubility in Formulation 5: 5% DMSO +95%Corn oil : 30mg/mL

---

Solubility in Formulation 6: 2.5 mg/mL (4.12 mM) in 15% Solutol HS 15 10% Cremophor EL 35% PEG 400 40% water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

---

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