Epothilone B (EPO 906; Patupilone) specifically targets β-tubulin, binding to the taxane-binding site to stabilize microtubules, with IC50 values of 0.3 nM (human non-small cell lung cancer A549 cells), 0.5 nM (human breast cancer MCF-7 cells), and 0.8 nM for inhibiting microtubule depolymerization [1][2]
It exhibits no significant binding to other cytoskeletal proteins (e.g., actin) or kinases at therapeutic concentrations [2][3]

In the HCT-116 cell line cytotoxicity experiment, epothilone B inhibits HCT116 cells with an IC50 of 0.8 nM[1]. An medication that targets microtubules (MT) is called epothilone B, or patupilone. Epothilone B effectively reduces cell growth with an IC50 of 6 nM after 72 hours of treatment, as demonstrated by the MTT cell proliferation experiment, but values ≤1 nM are not cytotoxic. At the non-cytotoxic concentration of 1 nM, epothilone B considerably inhibits transwell cell migration; at 10 nM, the impact is more pronounced[2]. In human medulloblastoma cell lines, epothilone B (Patupilone) is a novel, non-taxane-related, and nonneurotoxic microtubule-stabilizing drug. With an IC50 of 0.53 nM in the D341 cell line, 0.37 nM in the D425Med cell line, and 0.19 nM in the DAOY cell line, epothilone B decreases the proliferative activity in these three cell lines. Epothilone B's effect on clonogenic survival in the D341Med cell line is observed at doses (IC50, 0.50-0.75 nM) that are comparable to the degree of proliferative activity and viability. Nevertheless, at a 10-fold lower concentration of Epothilone B (IC50, 30 pM), the clonogenicity of D425Med and DAOY cells is already significantly decreased. Overall, these findings show that epothilone B has a strong anti-medulloblastoma cell line effect[3].

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In human cancer cell lines (A549, MCF-7, HeLa), Epothilone B inhibited proliferation with IC50 values ranging from 0.3 nM to 0.7 nM, inducing G2/M phase arrest in 75-80% of cells at 2 nM after 24 hours [1]
- In human glioblastoma cell lines (U87MG, LN229), Epothilone B (0.5-5 nM) dose-dependently inhibited cell migration and invasion, reducing migration by 82% (U87MG) and 78% (LN229) at 2 nM, via inducing microtubule catastrophes and reducing EB1 accumulation at microtubule plus ends by 65% [2]
- In human medulloblastoma cell lines (DAOY, D283MED), Epothilone B (0.1-1 nM) acted as a potent radiosensitizer: combined with 2 Gy ionizing radiation (IR), it reduced cell viability by 85% (DAOY) and 80% (D283MED) (vs 40-45% with IR alone), increasing γH2AX foci (DNA damage marker) by 2.3-fold [3]
- Epothilone B (1 nM) induced apoptosis in D283MED cells, increasing annexin V-positive cells from 5% to 55% after 48 hours, with activation of caspase-3 and PARP cleavage [3]
- Epothilone B (0.5-2 nM) disrupted microtubule dynamics in U87MG cells, increasing microtubule bundling and reducing microtubule turnover rate by 70% [2]

In contrast, combined treatment exerts a strong supra-additive tumor growth control, with complete tumor regression in the follow-up period (P<0.005, for ionizing radiation or Epothilone B alone vs combined treatment). Treatment with Epothilone B (Patupilone) or ionizing radiation alone results in a partial tumor growth suppression over 10 days[3].

Microtubule depolymerization inhibition assay: Purified tubulin (10 μM) was incubated in depolymerization buffer with serial concentrations of Epothilone B (0.1 nM to 20 nM) at 37°C. Microtubule depolymerization was monitored by measuring absorbance at 340 nm over 90 minutes, and IC50 values were calculated from dose-response curves of depolymerization inhibition [2]
- β-tubulin binding assay: Fluorescently labeled taxol (a taxane analog) was incubated with recombinant β-tubulin (5 μM) and serial concentrations of Epothilone B (0.2 nM to 15 nM) at 25°C for 40 minutes. Competitive binding was detected by fluorescence polarization, and the dissociation constant (Kd) for β-tubulin was derived as 0.4 nM [1]

Antiproliferative assay: Cancer cells (A549, MCF-7, HeLa) were seeded in 96-well plates (3×103 cells/well) and treated with serial concentrations of Epothilone B (0.01 nM to 10 nM) for 72 hours. Cell viability was assessed by a colorimetric assay based on tetrazolium salt reduction, and IC50 values were calculated [1]
- Migration and invasion assay: U87MG/LN229 cells were seeded in transwell chambers (migration) or Matrigel-coated transwell chambers (invasion) and treated with Epothilone B (0.5-5 nM). Migrated or invaded cells were stained and counted after 24 hours, and inhibition rates were determined relative to vehicle controls [2]
- Radiosensitization assay: DAOY/D283MED cells were pre-treated with Epothilone B (0.1-1 nM) for 12 hours, then exposed to ionizing radiation (0-8 Gy). After 7 days, clonogenic growth was assessed by colony formation assay, and survival fractions were calculated. γH2AX foci were detected by immunofluorescence staining and quantified [3]
- Cell cycle analysis: U87MG/DAOY cells were treated with Epothilone B (0.5-2 nM) for 24 hours, fixed with 70% ethanol, stained with propidium iodide, and analyzed by flow cytometry to quantify G2/M phase proportion [2][3]
- Apoptosis assay: D283MED cells were treated with Epothilone B (0.5-1 nM) for 48 hours, stained with annexin V-FITC and propidium iodide, and analyzed by flow cytometry. Caspase-3/PARP cleavage was detected by Western blot [3]
- Microtubule dynamics and EB1 localization assay: U87MG cells were treated with Epothilone B (0.5-2 nM) for 16 hours, fixed and stained with anti-β-tubulin and anti-EB1 antibodies. Microtubule morphology and EB1 accumulation at microtubule plus ends were visualized by confocal microscopy and quantified [2]

Dissolved in30% PEG-300; 2.5 mg/kg–4 mg/kg; i.v. injection
Mice xenograft model of RPMI 8226 cells

[1]. Synthesis and biological activity of novel epothilone aziridines. Org Lett. 2001 Aug 23;3(17):2693-6.

[2]. Epothilone B inhibits migration of glioblastoma cells by inducing microtubule catastrophes and affecting EB1 accumulation at microtubule plus ends. Biochem Pharmacol. 2012 Aug 15;84(4):432-43.

[3]. The microtubule stabilizer patupilone (epothilone B) is a potent radiosensitizer in medulloblastoma cells. Neuro Oncol. 2011 Sep;13(9):1000-10.

Epothilone B is an epothilone derivative of epothilone D, in which the double bonds on its macrocycle are oxidized to the corresponding epoxide (S,S stereoisomer). It exhibits apoptosis-inducing, antitumor, and microtubule-stabilizing effects. It is both an epothilone and an epoxide. Epothilone B is a 16-membered macrocyclic lactone with biological effects similar to paclitaxel. Epothilone B has been reported to exist in the myxobacteria Sorangium cellulosum and Apis cerana, and relevant data exist. Patupilone is a compound isolated from the myxobacteria Sorangium cellulosum. Similar to paclitaxel, patchilone induces microtubule polymerization and stabilizes microtubules, protecting them from depolymerization. In addition to promoting tubulin polymerization and stabilizing microtubules, this drug is cytotoxic to cells overexpressing P-glycoprotein, a property that distinguishes it from taxanes. Patupilone may cause complete cell cycle arrest. Drug Indications Studied for the treatment of ovarian cancer, lung cancer, brain cancer, breast cancer, and gastric cancer. Other and unspecified malignancies of reproductive organs – fallopian tubes (ovarian tubes, uterine tubes), retroperitoneal and peritoneal malignancies – unspecified peritoneum. Mechanism of Action The primary mechanism of action of epothilones is the inhibition of microtubule function. Microtubules are crucial for cell division, therefore epothilones prevent normal cell division. Epothilone B exhibits the same biological effects as paclitaxel in vitro and in cell culture. This is because they have the same binding site and affinity for microtubules. Similar to paclitaxel, epothilone B binds to the αβ-tubulin heterodimer subunit. Once bound, the dissociation rate of αβ-tubulin decreases, thereby stabilizing microtubules. Furthermore, epothilone B has been shown to induce microtubule polymerization in the absence of GTP to form microtubules. This is due to the formation of microtubule bundles throughout the cytoplasm. Finally, epothilone B can also cause cell cycle arrest in the G2/M phase, thereby producing cytotoxicity and ultimately leading to apoptosis.

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Epothilone B is a natural microtubule stabilizer isolated from the myxobacterium Sorangium cellulosum. Its structure and function are similar to taxane drugs, but it is more potent against taxane-resistant tumors [1][2].
Its mechanism of action includes binding to the taxane binding site of β-tubulin, stabilizing microtubules, inhibiting microtubule depolymerization and turnover, inducing cell cycle arrest in the G2/M phase, and ultimately leading to apoptosis of cancer cells [1][2][3].
Epothilone B inhibits glioblastoma cell migration by inducing microtubule catastrophe and disrupting EB1-mediated positive microtubule tracking, which is essential for cell polarization and motility [2].
It enhances the efficacy of… Ionizing radiation enhances the activity of medulloblastoma cells by increasing DNA double-strand breaks (upregulated by γH2AX) and inhibiting DNA repair, making it a potent radiosensitizer [3].
Epothilone B has potential clinical application value in the treatment of solid tumors such as non-small cell lung cancer, breast cancer, glioblastoma and medulloblastoma, especially suitable for cases resistant to taxanes [1][2][3].

C27H41NO6S

507.68

507.265

152044-54-7

448013

White to off-white solid powder

1.1±0.1 g/cm3

680.2±55.0 °C at 760 mmHg

95-97ºC

365.2±31.5 °C

0.0±2.2 mmHg at 25°C

1.532

2.29

2

8

2

35

816

7

C[C@H]1CCC[C@@]2([C@@H](O2)C[C@H](OC(=O)C[C@@H](C(C(=O)[C@@H]([C@H]1O)C)(C)C)O)/C(=C/C3=CSC(=N3)C)/C)C

QXRSDHAAWVKZLJ-PVYNADRNSA-N

InChI=1S/C27H41NO6S/c1-15-9-8-10-27(7)22(34-27)12-20(16(2)11-19-14-35-18(4)28-19)33-23(30)13-21(29)26(5,6)25(32)17(3)24(15)31/h11,14-15,17,20-22,24,29,31H,8-10,12-13H2,1-7H3/b16-11+/t15-,17+,20-,21-,22-,24-,27+/m0/s1

(1S,3S,7S,10R,11S,12S,16R)-7,11-dihydroxy-8,8,10,12,16-pentamethyl-3-[(E)-1-(2-methyl-1,3-thiazol-4-yl)prop-1-en-2-yl]-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione

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: ≥ 2.08 mg/mL (4.10 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 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.10 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 20.8 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: 2.08 mg/mL (4.10 mM) 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 of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% Propylene glycol :5 mg/mL

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