New use of table berberine

Abstract

The application discloses a new use of table berberine and an experimental method for inhibiting breast cancer cell-induced osteoclast differentiation. The application proves for the first time that table berberine does not inhibit the proliferation of osteoclast precursor cells at a higher concentration (25 μM), and has higher biological safety. In addition, table berberine has a significant inhibitory effect on breast cancer-induced osteoclast formation and metastatic breast cancer-induced osteolysis.

Description

Technical Field

[0001] This invention relates to the field of bone disease treatment technology, specifically to the use of epiberberine in the preparation of an inhibitor for inducing osteoclast differentiation in breast cancer cells. Background Technology

[0002] Breast cancer is the second leading cause of cancer death among women worldwide. More than three-quarters of breast cancers metastasize to bone in the later stages of the disease, leading to bone destruction.

[0003] Reports indicate that bone is the initial site of metastasis in 83% of breast cancer patients, and bone metastasis occurs in over 90% of breast cancer deaths. After metastasizing into the bone marrow, tumor cells deposit in bone tissue, interacting with osteoblasts, bone matrix cells, and osteoclasts in the bone microenvironment. This induces excessive activation of osteoclasts, disrupting the balance between bone resorption and bone regeneration, leading to bone tissue destruction and erosion. Ultimately, this results in osteolysis, causing severe pain and pathological fractures, significantly reducing patients' mobility and quality of life.

[0004] Therefore, the search for novel drug molecules with good stability and high safety to inhibit osteoclast production induced by breast cancer and to inhibit osteolysis induced by metastatic breast cancer is crucial for the advancement of treatment for breast cancer-related bone destruction diseases. Summary of the Invention

[0005] The present invention aims to at least partially solve one of the technical problems existing in the prior art. To this end, the present invention provides a new use of epiberberine and an experimental method for inhibiting the differentiation of breast cancer cells into osteoclasts.

[0006] According to one aspect of the invention, the use of epiberberine in the preparation of an inhibitor of breast cancer cell-induced osteoclast differentiation is provided.

[0007] According to another aspect of the invention, the use of epiberberine in the preparation of medicaments for the prevention, treatment or improvement of osteolysis caused by metastatic breast cancer cells is provided.

[0008] According to another aspect of the present invention, an experimental method for inhibiting osteoclast differentiation induced by breast cancer cells is provided, wherein breast cancer cells are pretreated with epiberberine; the treated breast cancer cells and osteoclast precursor cells are co-cultured in α-MEM medium.

[0009] Preferably, the breast cancer cells are pretreated with epiberberine, comprising culturing the breast cancer cells in a culture medium containing 10% fetal bovine serum until the confluence reaches 60%; treating the breast cancer cells with epiberberine at a concentration of 3.12 μM for 24 h; and washing off any residual epiberberine with 1× phosphate-buffered saline.

[0010] According to another aspect of the present invention, a medicament is provided for the prevention, treatment or improvement of osteolysis caused by metastatic breast cancer cells, characterized in that the raw material component of the medicament contains epiberberine.

[0011] Preferably, the mass percentage of epiberberine in the drug is 0.05%-99.5%.

[0012] Preferably, the drug further contains pharmaceutically acceptable excipients.

[0013] Preferably, the drug is an oral or non-oral formulation.

[0014] Preferably, the oral preparation is one or more of the following: capsules, tablets, oral liquids, granules, pills, powders, elixirs, or ointments.

[0015] Preferably, the non-oral preparation is one or more of the following: injection, cream, patch, ointment or spray.

[0016] This invention provides a novel use for epiberberine and an experimental method for inhibiting osteoclast differentiation induced by breast cancer cells. This invention demonstrates for the first time that epiberberine does not inhibit the proliferation of osteoclast precursor cells even at high concentrations, indicating its high biocompatibility. Furthermore, epiberberine significantly inhibits osteoclast formation induced by breast cancer and osteolysis induced by metastatic breast cancer. Attached Figure Description

[0017] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

[0018] Figure 1 The effects of different concentrations of berberine on the proliferation of osteoclast precursor cells were shown.

[0019] Figure 2 The effects of different concentrations of epiberberine on osteoclast differentiation were shown.

[0020] Figure 3 This demonstrates the effect of epiberberine treatment on osteoclast differentiation in breast cancer cells; and

[0021] Figure 4 The effect of epiberberine on osteolysis caused by metastatic breast cancer in bone was shown. Detailed Implementation

[0022] The following examples are provided to help those skilled in the art better understand the present invention. It should be noted that the following examples are not intended to limit the scope of protection claimed by the present invention, but are merely illustrative. Unless otherwise specified, the raw materials, reagents, or devices mentioned in the following examples are commercially available or obtained through known existing methods.

[0023] Extraction and separation from natural products is a crucial method for obtaining new drugs. Between 1981 and 2010, 34% of drugs approved for marketing by the U.S. Food and Drug Administration (FDA) were derived from natural products or their derivatives. Natural products, as an important source of drugs, possess novel and diverse structures and unique biological activities.

[0024] Coptis chinensis (Huanglian) is a well-known traditional Chinese herbal medicine with a history of use spanning thousands of years. Epiberberine, also known as dehydro-octanediol, is a natural molecule isolated and extracted from Coptis chinensis. This patent is the first to discover that epiberberine inhibits RANKL-induced osteoclast differentiation and formation. Compared to berberine, epiberberine did not inhibit the proliferation of osteoclast precursor cells at higher concentrations, demonstrating higher biocompatibility (epiberberine does not inhibit osteoclast precursor cell proliferation at 25 μM, while berberine significantly inhibits osteoclast precursor cell proliferation at concentrations close to 20 μM). Furthermore, epiberberine significantly inhibits osteoclast formation induced by breast cancer and osteolysis induced by metastatic breast cancer.

[0025] According to one embodiment of the present invention, the use of epiberberine in the preparation of an inhibitor for inducing osteoclast differentiation in breast cancer cells is provided.

[0026] According to yet another embodiment of the invention, the use of epiberberine in the preparation of medicaments for the prevention, treatment or improvement of osteolysis caused by metastatic breast cancer cells is provided.

[0027] According to another embodiment of the present invention, an experimental method for inhibiting osteoclast differentiation induced by breast cancer cells is provided, comprising: Step 1: pre-treating breast cancer cells with epiberberine; co-culturing osteoclast precursor cells and the breast cancer cells treated in Step 1 in α-MEM medium. The pre-treatment of breast cancer cells with epiberberine includes culturing the breast cancer cells in a medium containing 10% fetal bovine serum until the confluence reaches 60%; treating the breast cancer cells with epiberberine at a concentration of 3.12 μM for 24 h; and washing away residual epiberberine with 1× phosphate-buffered saline.

[0028] According to another embodiment of the present invention, a medicament is provided for the prevention, treatment, or improvement of osteolysis caused by metastatic breast cancer cells, wherein the raw material component of the medicament contains epiberberine. The mass percentage of epiberberine in the medicament is 0.05%-99.5%. The medicament also contains pharmaceutically acceptable excipients. The medicament is an oral or non-oral formulation.

[0029] The oral preparation is one or more of the following: capsules, tablets, oral liquids, granules, pills, powders, elixirs, or ointments. The non-oral preparation is one or more of the following: injections, creams, patches, ointments, or sprays.

[0030] Pharmaceutically acceptable excipients include various organic or inorganic carrier substances commonly used as pharmaceutical materials, used in solid dosage forms as excipients, lubricants, binders, disintegrants, and thickeners; and in liquid dosage forms as solvents, dispersants, solubilizers, suspending agents, tension agents, buffers, and analgesics, formulated in appropriate amounts. Additionally, preservatives, antioxidants, colorants, sweeteners, and other additives can be used as needed, following conventional methods.

[0031] Preferred examples of excipients include lactose, white sugar, D-mannitol, starch, crystalline cellulose, and light anhydrous silica. Preferred examples of lubricants include magnesium stearate, calcium stearate, talc, and colloidal silica. Preferred examples of binders include crystalline cellulose, white sugar, D-mannitol, dextrin, hydroxypropyl cellulose, and polyvinylpyrrolidone. Preferred examples of disintegrants include starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, and sodium carboxymethyl starch. Preferred examples of thickeners include natural gums, cellulose derivatives, and acrylic polymers. Preferred examples of solvents include water for injection, alcohols, propylene glycol, polyethylene glycol, sesame oil, and corn oil. Preferred examples of dispersants include Tween 80, HCO60, polyethylene glycol, carboxymethyl cellulose, and sodium alginate. Preferred examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, triaminomethane, cholesterol, triethanolamine, sodium carbonate, and sodium citrate. Preferred examples of suspending agents include stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzyl chloride, and glyceryl monostearate. Preferred examples of surfactants include hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose. Preferred examples of tension agents include sodium chloride, glycerol, and D-mannitol. Preferred examples of buffers include phosphates, acetates, carbonates, and citrates. Preferred examples of analgesics include benzyl alcohol. Preferred examples of preservatives include parabens, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid. Preferred examples of antioxidants include sulfites, ascorbic acid, etc.

[0032] Example 1

[0033] The compound epiberberine did not inhibit the normal proliferation of osteoclast precursor cells.

[0034] To analyze the effect of epiberberine on the proliferation of osteoclast precursor cells (bone marrow-derived macrophages, BMMs) in vitro, BMMs in the logarithmic growth phase were seeded into 96-well plates (5000 cells / well, 100 μl / well). After overnight adherence, 100 μl / well of fresh culture medium containing the drug (α-MEM supplemented with 10% bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, and 30 ng / mL M-CSF) was added, with three parallel wells per dose, and the cells were cultured at 37°C and 5% CO2 for 48 hours. 50 mL of cold 50% (w / v) trichloroacetic acid (TCA) was added to each well, and the cells were fixed at 4°C for 1 hour. The fixative was discarded, and the cells were rinsed five times with distilled water at a low flow rate and allowed to air dry. 100 μl of 0.4% (w / v) SRB solution was added to each well, and the cells were stained at room temperature for 10 minutes. Discard the supernatant, wash five times with 1% acetic acid to remove non-specifically bound dye, and air dry. Add 200 μl of 10 mM Tris solution (pH 10.5) to each well. After shaking for 5 minutes, measure the OD value at 515 nm, plot the drug concentration versus relative cell number curve, and calculate the 50% inhibitory concentration (IC50). Results are as follows. Figure 1 The results showed that epiberberine did not inhibit the normal proliferation of osteoclast precursor cells, meaning it was not cytotoxic to BMMs.

[0035] Example 2

[0036] The compound epiberberine inhibits osteoclast differentiation and formation.

[0037] To analyze the effect of epiberberine on osteoclast differentiation in vitro, this patent extracted BMMs from C57BL / 6 mice. BMMs in the logarithmic growth phase were seeded into 96-well plates (5000 cells / well, 100 μl / well). After overnight adherent growth, 100 μl / well of fresh culture medium containing the drug (α-MEM supplemented with 10% bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, 30 ng / mL M-CSF, and 100 ng / mL RANKL) was added, with three parallel wells per dose, and cultured at 37°C and 5% CO2. The culture medium was refreshed every two days until multinucleated osteoclast differentiation occurred. Cells were washed three times with cold 1× phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde (POM) for 15 minutes, and stained with tartaric acid-resistant (TRAP). TRAP-positive cells with more than three nuclei were identified as osteoclasts. The number and area of ​​osteoclasts were counted under a microscope. The results are as follows: Figure 2 The compound epiberberine showed that it inhibited osteoclast differentiation and formation.

[0038] Example 3

[0039] The compound epiberberine inhibits the formation and function of osteoclasts induced by breast cancer cells, such as... Figure 3 A. After extracting the breast cancer conditioned medium (CM), a series of experiments were conducted. First, breast cancer MDA-MB-231 cells were grown to 60% confluence in a medium containing 10% fetal bovine serum. Then, the cells were treated with 3.12 μM epiberberine for 24 h. Next, the cells were cultured in serum-free α-MEM medium for another 24 h. The culture medium was then extracted, filtered, and added to fresh medium (20% v / v). Specifically, step one: breast cancer cells were pre-treated with epiberberine, and the treated breast cancer cells were cultured in serum-free α-MEM medium for another 24 h; step two: the product from step one was extracted and filtered, and the obtained filtrate was added to serum-containing α-MEM medium (α-MEM containing 10% bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, 30 ng / mL M-CSF, and 50 ng / mL RANKL) at a ratio of 20% v / v, and osteoclast precursor cells were added for further culture.

[0040] The results showed that untreated MDA-MB-231 CM cells significantly promoted osteoclast formation and bone resorption. However, the osteoclast-inducing and bone resorption-inducing effects of MDA-MB-231 CM cells treated with epiberberine were significantly inhibited. These data indicate that epiberberine inhibits osteoclast differentiation and function induced by MDA-MB-231 CM cells.

[0041] Example 4

[0042] The compound epiberberine inhibits breast cancer cell-induced osteolysis.

[0043] To determine whether epiberberine could inhibit osteolysis caused by metastatic breast cancer, this patent describes the intramedullary injection of murine breast cancer 4T1 cells into the tibial bone marrow cavity of healthy female Balb / c mice aged 6-8 weeks. Specifically, after anesthesia with 3.5% chloral hydrate via peritoneal injection, 4T1 cell suspensions (1×10⁻⁶ cells) were injected... 5 50 μl of berberine was injected into the proximal tibial cavity of the right tibia via the tibial plateau. The mice were then randomly assigned to two groups: a control group (n=10) and a berberine group (n=10). The berberine group received intraperitoneal administration of berberine 2.5 mg / kg daily. The control group, however, received only intraperitoneal injection of PBS. The mice were euthanized after 16 days. Micro-CT scans were performed to further examine osteolysis induced by metastatic breast cancer cells. Figure 4As shown, significant osteolytic destruction was observed in the control group. In contrast, the epiberberine treatment group exhibited less osteolytic lesions and more intact bone cortex. These data suggest that epiberberine has a protective effect against osteolysis induced by metastatic breast cancer cells.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Use of epiberberine in the preparation of a drug for preventing, treating or improving the disease of bone resorption caused by metastatic breast cancer cells.

2. Use according to claim 1, characterized in that, The mass percentage of epiberberine in the drug is 0.05%-99.5%.

3. Use according to claim 1, characterized in that, The drug further contains pharmaceutically acceptable adjuvants.

4. Use according to claim 1, characterized in that, The drug is an oral preparation or a non-oral preparation.

5. Use according to claim 4, characterized in that, The oral preparation is one or more of capsules, tablets, oral liquids, granules, pills, powders, boluses or pastes.

6. Use according to claim 4, characterized in that, The non-oral preparation is one or more of injections, creams, patches, ointments or sprays.

7. An experimental method of inhibiting breast cancer cell-induced osteoclast differentiation, characterized by, The experimental method is for non-therapeutic purposes, Step one: breast cancer cells are pre-treated with epiberberine, and the treated breast cancer cells are further cultured in serum-free α-MEM medium; Step two: the product in step one is extracted and filtered, the obtained filtrate is added to serum-containing α-MEM medium and osteoclast precursor cells are added for further culture, the serum-containing α-MEM medium is α-MEM added with 10% bovine serum, 100 U / mL penicillin, 100 mg / mL streptomycin, 30 ng / mL M-CSF and 50 ng / mL RANKL.

8. The experimental method of claim 7, wherein, Step one: breast cancer cells are pre-treated with epiberberine, including The breast cancer cells are cultured in a medium containing 10% fetal bovine serum until the confluency reaches 60%; The breast cancer cells are treated with epiberberine at a concentration of 3.12 μM for 24 h; Residual epiberberine is washed with 1× phosphate buffered saline.

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