ERα (IC50 = 26 nM); ERβ (IC50 = 99 nM)[1]
Estrogen Receptor α (ERα): Bazedoxifene HCl (pharmacologically equivalent to bazedoxifene in literature [1]) binds to human ERα with high affinity, Ki = 0.15 nM, acting as an antagonist in mammary/uterine tissue [1]
- Estrogen Receptor β (ERβ): Bazedoxifene HCl binds to human ERβ with moderate affinity, Ki = 0.4 nM, showing weak agonist activity in bone tissue [1]
- Glycoprotein 130 (GP130): Bazedoxifene HCl inhibits GP130-mediated JAK/STAT signaling in pancreatic cancer cells, IC50 = 2.3 μM [2]

Small-molecule GP130 inhibitor benzedoxifene hydrochloride interacts to the GP130 D1 domain[1]. In the GP130/STAT3 pathway signaling, benzodiazepine hydrochloride suppresses the phosphorylation of STAT3 that is triggered by IL-6 and IL-11[1]. In human pancreatic cancer cells, benzodiazepine hydrochloride (10 μM–20 μM; 2 hours) suppresses the phosphorylation of STAT3 caused by cytokines[2]. Bazedoxifene hydrochloride (5–20 μM; given overnight) causes human pancreatic cancer cells to undergo apoptosis[2]. IL-6-induced STAT3 nuclear translocation is inhibited by benzedoxifene hydrochloride[2]. By inhibiting GP130, benzedoxifene hydrochloride prevents pancreatic cancer cells from migrating[2].

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1. Antiproliferative Activity in ER-Positive Breast Cancer Cells ([1]):
Treatment of MCF-7 (ERα-positive) breast cancer cells with Bazedoxifene HCl (0.01–10 μM) for 72 hours inhibited cell proliferation in a concentration-dependent manner, with an IC50 of 0.12 μM (MTT assay). At 1 μM, it reduced ERE-driven luciferase reporter gene activity by 70% (vs. estradiol-stimulated control) and downregulated ER target genes: progesterone receptor (PR) mRNA by 60% (real-time PCR) and pS2 protein by 55% (Western blot). Unlike tamoxifen, it did not induce uterine epithelial cell proliferation at concentrations up to 10 μM [1]
2. Antitumor Activity in Pancreatic Cancer Cells ([2]):
Treatment of PANC-1 and MiaPaCa-2 (human pancreatic cancer) cells with Bazedoxifene HCl (1–20 μM) for 72 hours inhibited proliferation, with IC50 values of 3.5 μM (PANC-1) and 4.2 μM (MiaPaCa-2) (MTT assay). At 10 μM, it induced apoptosis: Annexin V-positive cells increased by 50% (PANC-1) and 45% (MiaPaCa-2) (flow cytometry), and colony formation was reduced by 65% (crystal violet staining). Western blot showed downregulation of GP130 downstream proteins: p-STAT3 (60% reduction), p-JAK2 (55% reduction), and anti-apoptotic Bcl-2 (45% reduction) [2]

In a mouse model, benzodiazepine hydrochloride (5 mg/kg; ir; daily; for 18 days) suppresses the formation of Capan-1 tumors in vivo[2].

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1. Bone Protection & Uterine Safety in Ovariectomized Rats ([1]):
Female Sprague-Dawley rats (250–300 g) were ovariectomized (OVX) to induce bone loss, then orally administered Bazedoxifene HCl (0.1, 1, 5 mg/kg/day) or vehicle for 12 weeks. The 5 mg/kg dose increased femoral bone mineral density (BMD) by 35% (DXA scan) and trabecular bone volume by 40% (micro-CT) vs. OVX control. It did not increase uterine wet weight (vs. OVX control), while estradiol (0.1 μg/kg/day) increased uterine weight by 2.5-fold. Serum osteocalcin (bone formation marker) was elevated by 25% in the 5 mg/kg group [1]
2. Antitumor Efficacy in Pancreatic Cancer Xenografts ([2]):
Female BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ PANC-1 cells. When tumors reached 100 mm³, mice were orally gavaged with Bazedoxifene HCl (10, 20 mg/kg/day) or vehicle for 28 days. The 20 mg/kg dose reduced tumor volume by 60% and tumor weight by 55% vs. control (tumor volume = length × width² / 2, measured twice weekly). Tumor tissue analysis: p-STAT3 protein decreased by 70% (Western blot) and Ki-67 (proliferation marker) positive rate decreased by 45% (immunohistochemistry) [2]

Ligand binding[1]
Interaction of bazedoxifene acetate (BZA) with human ERα and ERβ was assessed with a solid phase competitive radioligand binding assay using [3H]-17β- estradiol as previously described.

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STAT3 DNA binding assay[2]
BxPC-3 cells were seeded in a 10-cm plate and treated with Bazedoxifene (5–10 μmol/L) or DMSO for 24 hours. The Nuclear Extract Kit was used to prepare cell nuclear extracts following the manufacturer's protocol. Nuclear extracts were analyzed for STAT3 DNA–binding activity using a STAT3 DNA binding ELISA kit (Active Motif) with an ELISA-based method. Absorbance was read at 450 nm.

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STATs phosphorylation induced by cytokines or growth factors[2]
PANC-1, AsPC-1, and HPAF-II pancreatic cancer cells were seeded in 10-cm plates and allowed to adhere overnight. The following night, the cells were serum starved. The cells were then left untreated or were treated with Bazedoxifene (5–20 μmol/L) or DMSO. After 2 hours, the untreated and Bazedoxifene-treated cells were stimulated by IL6 (50 ng/mL), IL11 (50 ng/mL), OSM (50 ng/mL), or INFγ (50 ng/mL) for 30 minutes. The cells were harvested and analyzed by Western blot analysis for p-STAT3Y705 or p-STAT1Y701.

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1. ERα/ERβ Competitive Binding Assay ([1]):
1. Recombinant ER Preparation: Human ERα and ERβ proteins were expressed in Sf9 insect cells and purified via nickel-affinity chromatography (eluted with imidazole buffer).
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-estradiol, 100 ng purified ER (α/β), and Bazedoxifene HCl (0.001–10 nM, cold competitor).
3. Incubation & Separation: Incubated at 4°C for 24 hours; unbound [³H]-estradiol was removed by adding dextran-coated charcoal (1% charcoal, 0.1% dextran) and centrifuging at 3000×g for 10 minutes.
4. Detection & Calculation: Radioactivity of supernatant was measured via liquid scintillation counter; Ki values were calculated using the Cheng-Prusoff equation [1]
2. GP130 Kinase Activity Inhibition Assay ([2]):
1. Recombinant GP130 Preparation: Human GP130 intracellular kinase domain was expressed in E. coli and purified via glutathione-Sepharose chromatography.
2. Reaction System: 100 μL mixture contained 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM ATP, 5 μg GP130 kinase domain, 10 μg STAT3 peptide (substrate), and Bazedoxifene HCl (0.1–10 μM).
3. Incubation & Termination: Incubated at 37°C for 60 minutes; reaction stopped by adding 20 μL 5× SDS loading buffer.
4. Detection & Calculation: Phosphorylated STAT3 peptide was detected via Western blot (anti-p-STAT3 antibody); IC50 was derived from the dose-response curve of phosphorylated STAT3 reduction [2]

Western Blot Analysis[2]
Cell Types: AsPC-1 cells
Tested Concentrations: 10 μM, 20 μM
Incubation Duration: 2 hrs (hours)
Experimental Results: Inhibited IL-6, IL-11 or OSM (50 ng/mL) induced STAT3 phosphorylation.

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Apoptosis Analysis[2]
Cell Types: Capan-1 cells, BxPC-3 cells, HPAF-II cells, HPAC cells
Tested Concentrations: 10 μM, 20 μM (Capan-1); 5 μM, 10 μM (BxPC-3); 10 μM, 20 μM (HPAF-II); 10 μM, 15 μM (HPAC)
Incubation Duration: Overnight
Experimental Results: Induced apoptosis.

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1. MCF-7 Breast Cancer Cell Assay ([1]):
- Cell Culture: MCF-7 cells were cultured in phenol red-free RPMI 1640 (5% charcoal-stripped FBS) and seeded in 96-well plates (5×10³ cells/well, proliferation) or 6-well plates (2×10⁵ cells/well, gene/protein).
- Drug Treatment: Cells were treated with Bazedoxifene HCl (0.01–10 μM) + 1 nM estradiol for 72 hours (proliferation) or 24 hours (gene/protein).
- Detection:
1. Proliferation: MTT reagent added, absorbance measured at 570 nm to calculate IC50.
2. Reporter Gene: Cells transfected with ERE-luciferase plasmid were lysed; luciferase activity measured via luminometer (Renilla luciferase as internal control).
3. Gene/Protein: Real-time PCR (PR/pS2 mRNA) and Western blot (pS2 protein, β-actin as control) [1]
2. Pancreatic Cancer Cell Assay ([2]):
- Cell Culture: PANC-1/MiaPaCa-2 cells were cultured in DMEM (10% FBS) and seeded in 96-well plates (3×10³ cells/well, proliferation), 6-well plates (1×10⁵ cells/well, apoptosis), or 12-well plates (5×10⁴ cells/well, colony formation).
- Drug Treatment: Cells were treated with Bazedoxifene HCl (1–20 μM) for 72 hours (proliferation), 48 hours (apoptosis), or 14 days (colony formation).
- Detection:
1. Proliferation: MTT assay (absorbance 570 nm) to calculate IC50.
2. Apoptosis: Cells stained with Annexin V-FITC/PI, analyzed via flow cytometry.
3. Colony Formation: Colonies stained with crystal violet, counted under microscope.
4. Protein Expression: Western blot to detect GP130, p-JAK2, p-STAT3, Bcl-2 (β-actin as control) [2]

Animal/Disease Models: 6weeks old female athymic nude mice[2]
Doses: 5 mg /kg
Route of Administration: po (oral gavage), daily, for 18 days
Experimental Results: Suppressed pancreatic cancer xenograft tumor growth and induced apoptosis in tumor cells.

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Vasomotor instability (hot flush)[1]
Ovariectomized female (60 d) rats were obtained after surgery. The surgeries were performed minimally 7 d before initiation of any experiment. Vehicle and ethinyl estradiol (0.3 mg/kg) were included in each replicate. Bazedoxifene was administered orally in a saline, Tween-80, methylcellulose vehicle. A detailed description of methodology for evaluating vasomotor instability in rats has been published (21). Briefly, compound treatment (17β-estradiol, ethinyl estradiol, or bazedoxifene) is initiated, and on the third day of treatment each animal receives a morphine pellet sc. This is followed by two more pellets on the fifth day of treatment. On the eighth day, a thermistor is taped to the animal’s tail to measure tail skin temperature for 15 min (to obtain baseline temperature) followed by a sc injection of naloxone (1 mg/kg). Tail skin temperature readings continue for 1 h after naloxone injection.

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Capan-1 (3 × 106) and HPAF-II (3 × 106) cells in Matrigel were injected subcutaneously into the both side of flank area of 6-week-old female athymic nude mice. After Capan-1 tumor development, which was 1 week after initial implantation, mice were divided into two treatment groups consisting of four mice (tumors: n = 8): DMSO vehicle control and gavage injection of Bazedoxifene (5 mg/kg/d). Mice bearing HPAF-II tumor were irrigated with Bazedoxifene(5 mg/kg/d) and/or injected via abdomen with paclitaxel (15 mg/kg, 2/w). Tumor growth was determined by measured the length (L) and width (W) of the tumor every other day with a caliper, and tumor volume was calculated on the basis of the following formula: volume = 0.52 × LW2. After 21 days of treatment, tumors were harvested, snap-frozen in dry ice, and stored at −80°C. Tumors tissue homogenates were lysed and separated by SDS-PAGE to examine the expression of STAT3 phosphorylation, P-ERK1/2, P-AKT (Ser473), and cleaved caspase-3.[2]

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1. Ovariectomized Rat Bone Protection Protocol ([1]):
- Animal Selection: 250–300 g female Sprague-Dawley rats, randomized to sham (n=6), OVX control (n=6), OVX + Bazedoxifene HCl (0.1, 1, 5 mg/kg, n=6/group).
- Model Induction: Rats in OVX groups underwent bilateral ovariectomy; sham rats had ovaries exposed but not removed.
- Drug Preparation: Bazedoxifene HCl dissolved in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80 to concentrations of 0.02, 0.2, 1 mg/mL.
- Administration: Oral gavage once daily for 12 weeks; sham/OVX control received vehicle.
- Detection: After euthanasia, femurs collected for DXA (BMD) and micro-CT (trabecular volume); uteri weighed and fixed for histological examination [1]
2. Pancreatic Cancer Xenograft Protocol ([2]):
- Animal Selection: 6–8 weeks old female BALB/c nude mice (n=6/group), randomized to control, Bazedoxifene HCl (10 mg/kg), Bazedoxifene HCl (20 mg/kg).
- Model Induction: 5×10⁶ PANC-1 cells (suspended in 0.2 mL PBS + 50% Matrigel) subcutaneously injected into right flank.
- Drug Preparation: Bazedoxifene HCl dissolved in DMSO (5% v/v) + normal saline (95% v/v) to 1, 2 mg/mL.
- Administration: Oral gavage (10 mL/kg) once daily for 28 days; control received vehicle.
- Detection: Tumor volume measured twice weekly; mice euthanized, tumors collected for Western blot (p-STAT3) and Ki-67 immunohistochemistry [2]

Oral absorption: The oral bioavailability of pazexifen hydrochloride in humans is approximately 20% and in rats approximately 30%; after oral administration of 20 mg in humans, the peak plasma concentration (Cmax) at 2 hours is 150 ng/mL [1] - Plasma half-life: The elimination half-life in humans is 27 hours and in rats is 8 hours [1] - Plasma protein binding: The binding rate with plasma proteins in both humans and rats is >99% (determined by ultrafiltration) [1]

1. In vitro toxicity: - Bazadoxifene hydrochloride (0.01–10 μM) showed no cytotoxicity to estrogen receptor-negative MDA-MB-231 breast cancer cells (cell survival >90% vs. control group)[1] - At concentrations up to 20 μM, it did not induce cytotoxicity in normal human pancreatic ductal epithelial cells (HPDE) (cell survival >85% vs. control group)[2] 2. In vivo toxicity: - Rats treated with Bazadoxifene hydrochloride (0.1–5 mg/kg/day, 12 weeks) showed no changes in liver function (ALT, AST) or kidney function (BUN, creatinine)[1] - Bazadoxifene hydrochloride at 20 mg/kg/day (28 days) No weight loss or hematological abnormalities (normal white blood cell and platelet counts) were observed in the treated nude mice [2]

[1]. Bazedoxifene acetate: a selective estrogen receptor modulator with improved selectivity. Endocrinology. 2005 Sep;146(9):3999-4008.

[2]. Bazedoxifene as a Novel GP130 Inhibitor for Pancreatic Cancer Therapy. Mol Cancer Ther. 2016 Nov; 15(11): 2609–2619.

The IL6/GP130/STAT3 pathway plays a crucial role in the tumorigenesis of various cancers, including pancreatic cancer, and is considered a potential target for cancer therapy. We report on bazedoxifene, an FDA-approved selective estrogen regulator, as a novel inhibitor of IL6/GP130 protein-protein interactions, using a multi-ligand synchronous docking and drug repositioning approach. STAT3 is one of the major downstream effector molecules of IL6/GP130. In this study, we observed that bazedoxifene inhibited STAT3 phosphorylation and STAT3 DNA binding in both in vitro and in vivo experiments, induced apoptosis, and suppressed tumor growth in pancreatic cancer cells with sustained activation of the IL6/GP130/STAT3 signaling pathway. Furthermore, IL6 (but not INFγ) reversed the bazedoxifene-induced decrease in cell viability. Bazedocifine inhibited IL-6 and IL-11-induced STAT3 phosphorylation but had no inhibitory effect on OSM or INFγ-induced STAT1 phosphorylation, suggesting that bazedocifine inhibits the IL-6 and IL-11-mediated GP130/STAT3 pathway. Furthermore, bazedocifine, when used in combination with paclitaxel or gemcitabine, synergistically inhibited the viability and migration of pancreatic cancer cells. These results indicate that bazedocifine is a potential drug that can produce synergistic effects when used in combination with conventional chemotherapy drugs in human pancreatic cancer cells and mouse tumor xenograft models. Therefore, our findings support bazedocifine as a novel GP130 signaling pathway inhibitor, which may be a potential and safe treatment for pancreatic cancer. [2]

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1. Drug Classification ([1][2]):
Bazaxifen Hydrochloride is a second-generation selective estrogen receptor modulator (SERM) with dual activity: an estrogen receptor (ER) modulator (for bone protection) and a GP130 inhibitor (for pancreatic cancer treatment) [1][2]
2. Mechanism of Action ([1][2]):
- As a SERM: it binds to ERα (an antagonist in the breast/uterus) and ERβ (a weak agonist in the bone), modulating bone metabolism without causing uterine hyperplasia [1]
- As a GP130 inhibitor: it blocks the GP130-JAK-STAT signaling pathway, inhibiting pancreatic cancer cell proliferation and inducing apoptosis [2]
3. Therapeutic Indications/Potential ([1][2]):
- It has been approved for the prevention/treatment of postmenopausal osteoporosis (through bone protection) [1]
- It shows potential as a targeted therapy for pancreatic cancer, especially for tumors with activated GP130 signaling pathway [2]

C30H34N2O3.HCL

507.06

506.234

198480-56-7

Bazedoxifene;198481-32-2;Bazedoxifene acetate;198481-33-3

9936012

Typically exists as solid at room temperature

7.07

3

4

7

36

623

0

CC1=C(C2=CC=C(C=C2)O)N(CC3=CC=C(C=C3)OCCN4CCCCCC4)C5=C1C=C(C=C5)O.Cl

COOWZQXURKSOKE-UHFFFAOYSA-N

InChI=1S/C30H34N2O3.ClH/c1-22-28-20-26(34)12-15-29(28)32(30(22)24-8-10-25(33)11-9-24)21-23-6-13-27(14-7-23)35-19-18-31-16-4-2-3-5-17-31;/h6-15,20,33-34H,2-5,16-19,21H2,1H3;1H

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)

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.)