Aromatase; ERα

Endoxifen, a hydroxylated Tamoxifen metabolite, is approximately 100-fold more effective than tamoxifen as an ER antagonist. It also implies that endoxifen, but not 4-hydroxytamoxifen, causes ER-alpha degradation in addition to its transcription-related effects on the ER[1]. Endoxifen is a strong antiestrogen that causes breast cancer cells' estrogen receptor α to degrade. Furthermore, research demonstrates that even in the presence of tamoxifen, N-desmethyl-tamoxifen, and 4-hydroxytamoxifen, endoxifen inhibits estrogen-induced breast cancer cell proliferation and blocks ERA transcriptional activity[2]. At 10 μM, doxifen significantly inhibits the growth of all breast cancer cell lines, with the exception of MDAMB-468, which experiences only moderate inhibition.At 10 μM concentration, cytotoxic effects on MCF7, HS 578T, and BT-549 cells are quite significant. While the inhibitory effects of Endoxifen at lower concentrations (0.01-1 μM) are not as strong as those of 10 μM, all tested cells die at concentrations of 100 μM[2].

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Treatment with Endoxifen (100 nmol/L for 24 h) reduces ERα protein levels in MCF7, T47D, Hs578T-ERα, and U2OS-ERα cells, similar to ICI, while tamoxifen and 4-hydroxy-tamoxifen stabilize ERα. No significant change is observed with N-desmethyl-tamoxifen. In Ishikawa endometrial carcinoma cells, endoxifen does not reduce ERα protein levels. [2]
- Concentration-dependent study in MCF7 cells shows that Endoxifen concentrations between 10 and 1000 nmol/L enhance ERα protein degradation; concentrations below 1 nmol/L do not affect ERα levels. [2]
- Time-course study with 100 nmol/L Endoxifen shows significant decreases in ERα protein levels within 6 hours of treatment. [2]
- Proteasome inhibition with MG132 (25 μmol/L pretreatment for 1 h, then 8 h co-treatment) blocks Endoxifen-induced ERα degradation, indicating that degradation is proteasome-dependent. [2]
- In MCF7 cells pretreated with a combination of tamoxifen (300 nmol/L), 4-hydroxy-tamoxifen (7 nmol/L), and N-desmethyl-tamoxifen (700 nmol/L) for 4 h, followed by Endoxifen (100–1000 nmol/L) for 20 h, significant decreases in ERα protein levels are observed. Low endoxifen concentrations (20–40 nmol/L) do not induce ERα degradation. [2]
- Transient transfection assay in Hs578T cells with ERα expression vector and ERE-luciferase reporter shows that Endoxifen represses estrogen-induced ERE activity. A combination of tamoxifen, 4HT, and NDT slightly reduces ERE activity, but addition of high concentrations of endoxifen (100–1000 nmol/L) completely blocks ERE activation, while low concentrations (20–40 nmol/L) are less effective. [2]
- Real-time RT-PCR in MCF7 cells shows that Endoxifen (high concentrations) completely blocks or significantly reduces estrogen-induced expression of amphiregulin and c-Myc. The combination of tamoxifen, 4HT, and NDT does not block estrogen activation of these genes, but addition of high concentrations of endoxifen (100–1000 nmol/L) does so, whereas low concentrations (20–40 nmol/L) do not. [2]
- Cell proliferation assay in MCF7 cells (8-day treatment) shows that estrogen induces a significant increase in proliferation. The combination of tamoxifen, 4HT, and NDT modestly reduces the proliferation rate. Addition of Endoxifen shows a dose response: low concentrations (20–40 nmol/L) significantly repress estrogen-induced growth, while high concentrations (100–1000 nmol/L) completely block or drastically repress this response. In Ishikawa cells, 1 nmol/L estrogen does not significantly increase proliferation; 1000 nmol/L endoxifen causes a slight but significant decrease in proliferation in the presence of estrogen and the combination of tamoxifen, 4HT, and NDT, but lower concentrations (20–100 nmol/L) have no effect. [2]

Endoxifen taken orally is quickly absorbed and available throughout the body when tested in female rats. Rats treated with Endoxifen exhibit 1,500% higher Cmax and 787% higher exposure (AUC0–∞) levels of Endoxifen than Tamoxifen-treated rats. Oral Endoxifen at doses of 2, 4, and 8 mg/kg administered once daily for 28 days in a row is safe and causes the growth of human mammary tumor xenografts in female mice to be gradually inhibited[2].

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Western blotting for ERα protein: Cells (MCF7, T47D, Hs578T-ERα, U2OS-ERα, Ishikawa) were plated in 12-well plates and treated with indicated ER ligands for 24 h. For Hs578T-ERα or U2OS-ERα cells, doxycycline (100 ng/mL) was included to induce ERα expression. For proteasome inhibition, cells were pretreated with either DMSO vehicle or 25 μmol/L MG132 for 1 h, then treated with ER ligands for an additional 8 h. After treatment, cells were washed with PBS and cell extracts prepared using Laemmli buffer. ERα protein was detected using anti-ERα antibody (HC-20) or flag-specific antibody; tubulin was used as loading control. Proteins were visualized using enhanced chemiluminescence. [2]
- Real-time RT-PCR: MCF7 cells were plated in 100-mm dishes and treated as indicated for 24 h. Total RNA was isolated using Trizol reagent. 500 ng of total RNA was reverse transcribed using iScript cDNA Synthesis kit. Real-time PCR was performed with primers specific for amphiregulin (forward: GGGAGTGAGATTTCCCCTGT, reverse: AGCCAGGTATTTGTGGTTCG) and c-Myc (forward: GCCACGTCTCCACACATCAG, reverse: TCTTGGCAGCAGGATAGTCCT). Control primers for human TATA binding protein were used. [2]
- Cell proliferation assay: MCF7 and Ishikawa cells were grown in 10% triple charcoal-stripped serum-containing medium for 3 days, then plated at 2000 cells per well in 96-well plates and treated as indicated every 48 h. Cell proliferation was assayed 8 days after the first treatment using a luminescent cell viability kit according to the manufacturer’s protocol. [2]
- Transient transfection and luciferase assay: Parental Hs578T cells were plated in 12-well plates and transfected in triplicate with 250 ng per well of ER-TK-luciferase reporter construct and 250 ng per well of an ERα expression construct (PCDNA4/TO Flag ERα) using a transfection reagent. After transfection, cells were treated as indicated for 24 h, then lysed, and equal amounts of extract were assayed for luciferase activity. [2]

Mice: A subcutaneous (s.c.) implant of a 30-to 40-mg fragment of MCF-7 human mammary tumor from an in vivo passage is made in six-week-old female athymic NCr-nu/nu mice near the right flank. Day 0 is the date of the implantation of the tumor fragments. Each animal has a 0.72-mg 17 β-estradiol 60-day release pellet s.c. implanted in the back of their neck one day before the tumor fragment is inserted, in order to support the growth of the estrogen-dependent MCF-7 tumor. Day 13 following tumor fragment implantation, the day treatment was started, saw the growth of individual tumors to sizes ranging from 75 to 196 mm3. One control group (12 mice/group) and four treatment groups (6 mice/group) are created from a total of thirty-six tumor-bearing mice through randomization. On day 13 after the tumor was implanted, the patient was given oral gavage once a day for 28 days, along with three different doses of Endoxifen (2, 4, and 8 mg/kg) or Tamoxifen (10 mg/kg) twice a day, three hours apart. For every treatment group, the dosage volume of 0.2 mL/10 g body weight remains unchanged. Beginning on the first day of treatment, the s.c. tumors are measured and the animals are weighed twice a week. On Day 58, the research comes to an end. The overall delay in the growth of the median tumor is determined by taking the median time to reach two tumor mass doublings. Furthermore, an additional assessment of the antitumor efficacy is conducted on Days 41, 1 day after the last treatment, and 58, the day the study is terminated, by comparing the median tumor weight in the treatment groups to the median tumor weight in the control group (T/C 9 100%)[2].

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Metabolism / Metabolites
Known metabolites of nedocoxine in the human body include nedocoxine O-glucuronide and nedocoxine O-sulfate. Nedocoxine is a known human metabolite of 4-hydroxytamoxifen and N-desmethyltamoxifen.

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Endoxifen is formed in the liver by CYP2D6-mediated oxidation of N-desmethyl-tamoxifen. [2]
- In women taking tamoxifen at 20 mg/day, average steady-state plasma concentrations are: tamoxifen 335 nmol/L, 4-hydroxy-tamoxifen 7.4 nmol/L, N-desmethyl-tamoxifen 695 nmol/L. [2]
- Plasma concentrations of Endoxifen vary widely depending on CYP2D6 activity: extensive metabolizers typically have 90 nmol/L (±40 nmol/L), intermediate metabolizers 40–60 nmol/L, and poor metabolizers <30 nmol/L. [2]
- CYP2D6 polymorphisms and drug-induced inhibition of CYP2D6 significantly reduce Endoxifen concentrations in humans. [2]

[1]. Tamoxifen, endoxifen, and CYP2D6: the rules for evaluating a predictive factor. Oncology (Williston Park). 2009 Dec;23(14):1233-4, 1236.

[2]. The tamoxifen metabolite, endoxifen, is a potent antiestrogen that targets estrogen receptor alpha for degradation in breast cancer cells. Cancer Res. 2009 Mar 1;69(5):1722-7.

4-Hydroxy-N-demethyltamoxifen is a stilbene compound.

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Endoxifen is a tamoxifen metabolite that functions as a potent antiestrogen by targeting estrogen receptor α for degradation by the proteasome, blocking ERα transcriptional activity, and inhibiting estrogen-induced breast cancer cell proliferation. Unlike tamoxifen and 4-hydroxy-tamoxifen, which stabilize ERα, endoxifen promotes ERα degradation. [2]
- The effects of Endoxifen are concentration-dependent and do not occur at concentrations observed in human CYP2D6 poor metabolizers. High concentrations (100–1000 nmol/L) mimicking CYP2D6 extensive metabolizers are more potent than low concentrations (20–40 nmol/L) mimicking poor metabolizers. [2]
- Clinical observations show that women with impaired CYP2D6 metabolism have a significantly higher risk of breast cancer recurrence while taking tamoxifen, supporting the theory that Endoxifen is the primary metabolite responsible for tamoxifen effectiveness in ER-positive breast cancer. [2]
- The study suggests that CYP2D6 genotyping may be used to individualize endocrine therapy, and that Endoxifen or related compounds could be developed as an alternative drug treatment for women with ER-positive breast tumors. [2]

C₂₅H₂₇NO₂

373.48738

373.204

110025-28-0

Endoxifen (Z-isomer);112093-28-4;Endoxifen Z-isomer hydrochloride;1032008-74-4;Endoxifen hydrochloride;1197194-41-4;Endoxifen (E-isomer);114828-90-9;Endoxifen E-isomer hydrochloride;1197194-61-8;Endoxifen-d5;1185244-45-4

10090750

White to light yellow solid

1.099g/cm3

519.3ºC at 760mmHg

267.9ºC

2.1E-11mmHg at 25°C

1.598

5.75

2

3

8

28

467

0

CC/C(C1=CC=CC=C1)=C(C2=CC=C(O)C=C2)/C3=CC=C(OCCNC)C=C3

MHJBZVSGOZTKRH-OCOZRVBESA-N

InChI=1S/C25H27NO2/c1-3-24(19-7-5-4-6-8-19)25(20-9-13-22(27)14-10-20)21-11-15-23(16-12-21)28-18-17-26-2/h4-16,26-27H,3,17-18H2,1-2H3/b25-24+

4-[(E)-1-[4-[2-(methylamino)ethoxy]phenyl]-2-phenylbut-1-enyl]phenol

Endoxifen

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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