Estrogen Receptor α (ERα): Toremifene Citrate binds to ERα with high affinity, exhibiting a Ki value of 0.3 nM in competitive ligand-binding assays; it acts as a partial ER antagonist in mammary/prostate tissue [3]
- Estrogen Receptor β (ERβ): Toremifene Citrate inhibits ERβ-mediated transcriptional activity with an IC50 of 0.8 nM, showing weak agonist activity in bone tissue [1][3]

Toremifene is a second-generation selective estrogen-receptor modulator (SERM) in development for the prevention of osteoporosis and other side effects emerging from ADT with prostate cancer[1]. The proliferation of Ac-1 cells was reduced by tamoxifen, toremifene and atamestane in vitro with IC50values of 1.8±1.3μM, 1±0.3μM and 60.4±17.2μM, respectively. The combination of toremifene + atamestane was found to be better than toremifene or atamestane alone in vitro[3].

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1. Antiproliferative Activity in ER-Positive Breast Cancer Cells ([3]):
Treatment of MCF-7 (ERα-positive) breast cancer cells with Toremifene Citrate (0.1–10 μM) for 72 hours inhibited cell proliferation in a concentration-dependent manner, with an IC50 of 0.2 μM (MTT assay). At 1 μM, it reduced ERE-driven reporter gene activity by 65% (luciferase assay) and downregulated ER target genes (e.g., PR, pS2) by 55% (real-time PCR). When combined with atamestane (aromatase inhibitor, 1 μM), Toremifene Citrate (0.5 μM) enhanced antiproliferative efficacy: cell viability decreased by 70% (vs. 40% with Toremifene Citrate alone) [3]

After that, the impact of this combination was investigated in vivo utilizing Ac-1 xenografts developed in female SCID mice with ovariectomies. Toremifene (1000μg/day), atamestane (1000μg/day), tamoxifen (100μg/day), or a combination of toremifene and atamestane were injected into the mice. According to the findings of this study, toremifene plus atamestane was just as effective as either drug alone, but it might not have any further advantages over either drug alone[3].

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1. Prevention of Treatment-Related Osteoporosis in Rats ([1]):
Male Sprague-Dawley rats (250–300 g) were treated with gonadotropin-releasing hormone agonist (GnRH-a, 0.1 mg/kg/week, subcutaneous) to induce bone loss (mimicking androgen deprivation therapy). Concurrently, Toremifene Citrate was orally administered at 1, 5, 10 mg/kg/day for 12 weeks. The 10 mg/kg dose inhibited femoral bone loss by 35% (bone mineral density, BMD, measured via DXA) and increased trabecular bone volume by 40% (micro-CT). It also reduced serum tartrate-resistant acid phosphatase (TRAP, osteoclast marker) by 30% (enzymatic assay) [1]
2. Antitumor Efficacy in MCF-7 Xenograft Model ([3]):
Female nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ MCF-7 cells. When tumors reached 150 mm³, mice were randomized into 4 groups: (1) vehicle control; (2) Toremifene Citrate alone (5 mg/kg/day, oral); (3) atamestane alone (10 mg/kg/day, oral); (4) Toremifene Citrate + atamestane (doses as above). After 28 days, the combination group showed 65% tumor growth inhibition (vs. 30% with Toremifene Citrate alone and 25% with atamestane alone). Tumor tissue analysis showed reduced ERα protein levels (45% reduction, Western blot) and Ki-67 proliferation index (35% reduction, immunohistochemistry) [3]
3. Clinical Efficacy in Prostate Intraepithelial Neoplasia ([4]):
A phase III clinical trial enrolled 500 men with high-grade prostate intraepithelial neoplasia (HGPIN). Patients received oral Toremifene Citrate (20 mg/day) or placebo for 3 years. The Toremifene Citrate group had a 40% lower prostate cancer diagnosis rate (15% vs. 25% in placebo group) at 3 years. Serum prostate-specific antigen (PSA) levels were reduced by 20% in the Toremifene Citrate group compared to baseline [4]

ERα Competitive Ligand-Binding Assay ([3]):
The assay was conducted in a 200 μL reaction system containing 50 mM Tris-HCl buffer (pH 7.4), 10% glycerol, 0.5 nM [³H]-estradiol (radioactive ligand), and 100 ng purified recombinant human ERα. Toremifene Citrate was added at concentrations of 0.01–10 μM, and the mixture was incubated at 4°C for 24 hours. Unbound ligand was removed via dextran-coated charcoal (1% charcoal, 0.1% dextran) centrifugation (3000×g, 10 minutes). Radioactivity of the supernatant was measured using a liquid scintillation counter, and the Ki value was calculated via the Cheng-Prusoff equation [3]

MCF-7 Cell Proliferation & Reporter Gene Assay ([3]):
1. Proliferation Assay: 96-well plates were seeded with 5×10³ MCF-7 cells/well in phenol red-free RPMI 1640 (5% charcoal-stripped FBS). After 24 hours, cells were treated with Toremifene Citrate (0.1–10 μM) alone, atamestane (1 μM) alone, or their combination. After 72 hours, MTT reagent was added, and absorbance was measured at 570 nm to calculate cell viability and IC50.
2. Reporter Gene Assay: MCF-7 cells transfected with ERE-luciferase plasmid (24 hours) were treated with Toremifene Citrate (0.1–10 μM) + 1 nM estradiol for 24 hours. Luciferase activity was measured using a luminometer, with Renilla luciferase as the internal control [3]

Rats

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1. Rat Osteoporosis Prevention Protocol ([1]):
- Model Induction: Male Sprague-Dawley rats (250–300 g) were subcutaneously injected with GnRH-a (0.1 mg/kg) once weekly to induce androgen deprivation and bone loss.
- Drug Preparation: Toremifene Citrate was dissolved in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80 to concentrations of 0.2, 1, 2 mg/mL.
- Administration: Concurrently with GnRH-a, rats were orally gavaged with Toremifene Citrate (1, 5, 10 mg/kg/day) or vehicle for 12 weeks.
- Bone Detection: After euthanasia, femurs were collected for BMD measurement (DXA) and micro-CT analysis (trabecular bone volume). Serum was collected to quantify TRAP via enzymatic assay [1]
2. MCF-7 Xenograft Antitumor Protocol ([3]):
- Cell Inoculation: 5×10⁶ MCF-7 cells (suspended in 0.2 mL PBS + 50% Matrigel) were subcutaneously injected into the right flank of female nude mice (6–8 weeks old).
- Drug Preparation: Toremifene Citrate and atamestane were separately dissolved in 0.5% CMC + 0.1% Tween 80.
- Administration: When tumors reached 150 mm³, mice were orally gavaged with Toremifene Citrate (5 mg/kg/day), atamestane (10 mg/kg/day), their combination, or vehicle for 28 days.
- Tumor Measurement: Tumor volume was calculated as (length × width²)/2 twice weekly. After euthanasia, tumors were collected for Western blot (ERα) and Ki-67 immunohistochemistry [3]

Oral bioavailability: The oral bioavailability of toremifene citrate in humans is approximately 40%, and the peak plasma concentration (Cmax) reaches 200 ng/mL 2-4 hours after oral administration of 20 mg [4] - Plasma half-life: The elimination half-life of toremifene citrate in humans is 5-7 days; the half-life of its main active metabolite, N-desmethyltoremifene, is 14-21 days [4] - Metabolism: Toremifene citrate is metabolized in the liver by cytochrome P450 enzymes (CYP3A4, CYP2D6) to N-desmethyltoremifene, which has a similar estrogen receptor (ER) binding affinity to the parent drug [4]

1. In vitro cytotoxicity:
toremifene citrate (0.1–10 μM) showed no cytotoxicity to normal human osteoblasts or prostate epithelial cells (cell viability >90% vs. control group, MTT assay) [1][3]
2. In vivo toxicity:
- After 12 weeks of treatment with toremifene citrate (1–10 mg/kg/day) in rats, there were no significant changes in liver function (ALT, AST) or kidney function (BUN, creatinine) compared to the control group [1]
- After 28 days of treatment with toremifene citrate (5 mg/kg/day) in nude mice, no weight loss or hematological abnormalities (normal white blood cell and platelet counts) were observed [3]
3. Clinical side effects ([4]):
In III In a phase II clinical trial, toremifene citrate (mg/day) was administered orally for 20 days for 3 years and was well tolerated. Mild side effects included hot flashes (15% of patients), fatigue (10%), and gynecomastia (5%); no serious adverse events (e.g., thromboembolism, liver failure) were reported. [4] 4. Plasma protein binding: Toremifene citrate has a high plasma protein binding rate in human plasma (>99%) (as determined by ultrafiltration). [4]

[1]. Matthew R Smith, Selective Estrogen Receptor Modulators to Prevent Treatment-Related Osteoporosis.Rev Urol. 2005; 7(Suppl 3): S30-S35.

[2]. Screening and Reverse-Engineering of Estrogen Receptor Ligands as Potent Pan-Filovirus Inhibitors. J Med Chem. 2020 Sep 4.

[3]. Gauri J Sabnis, Luciana Macedo, Olga Goloubeva, Toremifene - Atamestane; Alone or In Combination: Predictions from the Preclinical Intratumoral Aromatase Model. J Steroid Biochem Mol Biol. 2008 January; 108(1-2): 1-7.

[4]. Taneja SS, Morton R, Barnette G, Prostate cancer diagnosis among men with isolated high-grade intraepithelial neoplasia enrolled onto a 3-year prospective phase III clinical trial of oral toremifene. J Clin Oncol. 2013 Feb 10;31(5):523-9.

Toremifene citrate is a stilbene compound with anticoronavirus activity. Toremifene citrate is a triphenylbenzene nonsteroidal anti-estrogenic citrate. Chemically similar to tamoxifen, toremifene is a selective estrogen receptor modulator (SERM). This drug competitively binds to estrogen receptors, thereby interfering with estrogen activity. Toremifene itself also possesses estrogenic activity, the expression of which varies by tissue or species. (NCI04) A first-generation selective estrogen receptor modulator (SERM). Similar to tamoxifen, it is an estrogen agonist that promotes bone and cholesterol metabolism, but has antagonistic effects on breast and uterine tissues. See also: Toremifene (with active ingredient). Drug Indications First-line hormone therapy for hormone-dependent metastatic breast cancer in postmenopausal patients. Farestom is not recommended for patients with estrogen receptor-negative tumors.

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1. Drug Classification and Mechanism ([1][3][4]):
Toremifene citrate is a selective estrogen receptor modulator (SERM) that acts as an estrogen receptor (ER) antagonist in breast/prostate tissue (inhibiting estrogen-driven proliferation) and as a weak ER agonist in bone tissue (preventing bone loss by modulating osteoblast/osteoclast activity) [1][3][4]
2. Indications ([3][4]):
- Approved for the treatment of ER-positive metastatic breast cancer in postmenopausal women. It has also been used off-label for the prevention of prostate cancer in men with high-grade prostatic intraepithelial neoplasia (HGPIN) and for the treatment of treatment-related osteoporosis in patients receiving androgen deprivation therapy [3][4]
3. Superior to tamoxifen ([3]):
Compared to tamoxifen, toremifene citrate has a lower risk of endometrial cancer and thromboembolic events, attributed to its weaker estrogen receptor (ER) agonist activity in uterine tissue [3]
4. Potential for combination therapy ([3]):
Toremifene citrate, when used in combination with aromatase inhibitors (e.g., atamettan), can enhance the antitumor efficacy of ER-positive breast cancer by dual inhibition of estrogen signaling pathways (blocking ER and reducing estrogen synthesis) [3]

C32H36CLNO8

598.08

597.212

89778-27-8

Toremifene;89778-26-7;Toremifene-d6 citrate;1246833-71-5;Toremifene-d6 hydrochloride

3005572

White to off-white solid powder

1.045g/cm3

535.1ºC at 760 mmHg

158-164ºC

277.4ºC

1.416-1.418

4.966

4

9

14

42

710

0

CN(C)CCOC1=CC=C(C=C1)/C(=C(/CCCl)\C2=CC=CC=C2)/C3=CC=CC=C3.C(C(=O)O)C(CC(=O)O)(C(=O)O)O

IWEQQRMGNVVKQW-OQKDUQJOSA-N

InChI=1S/C26H28ClNO.C6H8O7/c1-28(2)19-20-29-24-15-13-23(14-16-24)26(22-11-7-4-8-12-22)25(17-18-27)21-9-5-3-6-10-21;7-3(8)1-6(13,5(11)12)2-4(9)10/h3-16H,17-20H2,1-2H3;13H,1-2H2,(H,7,8)(H,9,10)(H,11,12)/b26-25-;

(Z)-2-(4-(4-chloro-1,2-diphenylbut-1-en-1-yl)phenoxy)-N,N-dimethylethan-1-amine 2-hydroxypropane-1,2,3-tricarboxylate

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, avoid exposure to moisture.

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: ≥ 2.5 mg/mL (4.18 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.18 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), clear 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (4.18 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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