Pharmaceutical composition for reducing drug resistance of liver cancer cells

Abstract

The invention relates to the technical field of preparation of drug-resistant drugs, and discloses a pharmaceutical composition for reducing drug resistance of liver cancer cells, which is prepared from the following components in parts by mass: 10 to 20 parts of oxymatrine, 5 to 15 parts of curcumin, 8 to 15 parts of ginsenoside Rg38, 3 to 8 parts of sorafenib, 5 to 12 parts of quercetin and 3 to 8 parts of oridonin. Different components in the pharmaceutical composition have a synergistic effect and jointly play a role in reducing the drug resistance of the liver cancer cells, so that the drug resistance of the liver cancer cells can be effectively reduced, and the safety is high.

Description

Technical Field

[0001] The present invention relates to the technical field of drug resistance drug preparation, and in particular to a pharmaceutical composition for reducing the drug resistance of liver cancer cells. Background Art

[0002] Hepatocellular carcinoma (HCC) is one of the malignant tumors with the highest morbidity and mortality rates worldwide. In my country, the prevention and treatment of HCC is particularly challenging due to factors such as high rates of hepatitis B virus infection and aflatoxin exposure. Currently, clinical treatments for HCC include surgical resection, liver transplantation, interventional therapy, chemotherapy, and targeted therapy, but the development of drug resistance is a major cause of treatment failure.

[0003] Drug resistance in liver cancer cells is divided into primary resistance (insensitive at the beginning of treatment) and acquired resistance (gradually developed during treatment). Its mechanisms are complex and mainly include:

[0004] Enhanced drug efflux: Tumor cells highly express the ABC transporter family (such as P-glycoprotein and MRP1), which reduces intracellular drug concentrations by actively effluxing drugs. For example, resistance to drugs such as sorafenib and doxorubicin is often related to this.

[0005] Abnormal drug metabolism: Changes in the activity of the cytochrome P450 enzyme system accelerate drug degradation and reduce drug efficacy.

[0006] Enhanced DNA repair capacity: Tumor cells activate DNA repair pathways (such as the ATM / ATR pathway) to reduce drug-induced DNA damage, thereby resisting the effects of chemotherapy drugs.

[0007] Effects of the tumor microenvironment: Hypoxia and fibrosis in the tumor microenvironment can induce the expression of resistance-related genes, while the infiltration of immunosuppressive cells (such as Treg cells) further weakens the therapeutic effect.

[0008] Abnormal activation of signaling pathways: Continuous activation of pathways such as PI3K / Akt / mTOR and MAPK can make tumor cells insensitive to targeted drugs.

[0009] Existing strategies to reduce drug resistance include combination therapy (such as chemotherapy drugs combined with targeted drugs), dose escalation, and the development of novel drugs. However, these strategies are associated with cumulative side effects, poor patient tolerance, and high costs. Traditional Chinese medicine (TCM) offers unique advantages in reversing tumor drug resistance. Its multi-component, multi-target nature can synergistically regulate resistance-related pathways, but currently lacks clear formulations and mechanisms for addressing drug resistance in liver cancer.

[0010] Based on this, it is of great clinical significance to provide a pharmaceutical composition that is highly safe and can effectively reduce the drug resistance of liver cancer cells. Summary of the Invention

[0011] In view of this, the present invention proposes a pharmaceutical composition for reducing the drug resistance of liver cancer cells, aiming to solve at least one of the above-mentioned background technical problems.

[0012] The present invention provides a pharmaceutical composition for reducing drug resistance of liver cancer cells, comprising the following components in parts by mass:

[0013] 10-20 parts of oxymatrine, 5-15 parts of curcumin, 38-15 parts of ginsenoside Rg, 3-8 parts of sorafenib, 5-12 parts of quercetin, and 3-8 parts of oridonin.

[0014] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, wherein the excipient is selected from at least one of a filler, a disintegrant, a binder, and a lubricant.

[0015] Preferably, the filler is microcrystalline cellulose or lactose, with a mass fraction of 10-30 parts.

[0016] Preferably, the disintegrant is sodium starch glycolate or cross-linked polyvinylpyrrolidone, with a mass fraction of 1-5 parts.

[0017] Preferably, the binder is hypromellose or povidone, with a mass fraction of 2-8 parts.

[0018] Preferably, the lubricant is magnesium stearate or talc, with a mass fraction of 0.5-2 parts.

[0019] The present invention also provides a method for preparing the pharmaceutical composition described in the above technical solution, comprising the following steps:

[0020] Step 1: Take each component raw material, grind them separately and pass them through 80-120 mesh sieve for later use;

[0021] Step 2: Mix the pretreated raw materials in proportion, add auxiliary materials, and stir evenly to obtain a mixture;

[0022] Step 3: The uniformly stirred mixture is prepared into tablets, capsules, granules or freeze-dried powder for injection.

[0023] Preferably, when preparing tablets, step 3 further comprises: adding 50-70% ethanol solution to the mixture to prepare a soft material, granulating through an 18-24 mesh sieve, drying at 60-70° C., adding a lubricant after granulation, and tableting to obtain the tablets.

[0024] Preferably, when preparing a lyophilized powder for injection, step 3 further comprises: dissolving the mixture in water for injection, adding mannitol as a lyophilization protectant, filtering and sterilizing, and freeze-drying after packaging, wherein the freeze-drying conditions are: pre-freezing temperature -40 to -30°C, vacuum degree 10-20Pa.

[0025] The present invention also provides the use of the pharmaceutical composition described in the above technical solution in the preparation of a drug for reducing the drug resistance of liver cancer cells.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The components of the pharmaceutical composition of the present invention have different synergistic effects. Among them, oxymatrine and oridonin synergistically inhibit P-gp and MRP1, blocking drug efflux from different sites; sorafenib inhibits the RAF / MEK / ERK pathway, and ginsenoside Rg3 inhibits the PI3K / Akt / mTOR pathway, doubly blocking proliferation signals; curcumin reduces the expression of anti-apoptotic proteins through NF-κB inhibition, and quercetin enhances DNA damage through PARP-1 inhibition, and the two synergistically promote cancer cell apoptosis; sorafenib activates extrinsic apoptosis pathway, oxymatrine activates the endogenous apoptosis pathway, forming an apoptosis signal cascade; curcumin inhibits inflammatory factors (IL-6, TNF-α), and ginsenoside Rg3 enhances immune cell activity, jointly reversing the immunosuppressive microenvironment; quercetin reduces ROS generation and reduces the activation of oxidative stress-induced drug resistance-related signaling pathways; curcumin and quercetin inhibit CYP3A4 and P-gp, reducing the metabolic clearance of sorafenib, increasing its blood concentration and bioavailability, and the combined action of each component plays a role in reducing the drug resistance of liver cancer cells. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention are now described in detail. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention.

[0029] In addition, for numerical ranges in the present invention, it is understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The present invention provides a pharmaceutical composition for reducing drug resistance of liver cancer cells, comprising the following components in parts by mass:

[0034] 10-20 parts of oxymatrine, 5-15 parts of curcumin, 38-15 parts of ginsenoside Rg, 3-8 parts of sorafenib, 5-12 parts of quercetin, and 3-8 parts of oridonin.

[0035] The components in parts by mass are preferably: 10-15 parts of oxymatrine, 5-10 parts of curcumin, 38-12 parts of ginsenoside Rg, 3-5 parts of sorafenib, 5-10 parts of quercetin, and 3-5 parts of oridonin.

[0036] The present invention obtains a composition for reducing drug resistance of liver cancer cells by mixing various components, wherein oxymatrine has the following effects: downregulating the expression of P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1), reducing the active efflux of chemotherapy drugs by cancer cells, increasing intracellular drug concentration (IC50 value reduced by about 40%), activating caspase-3 and caspase-9, upregulating the pro-apoptotic protein Bax, downregulating the anti-apoptotic protein Bcl-2, and promoting apoptosis of drug-resistant liver cancer cells; and inhibiting the TGF-β / Smad signaling pathway, reducing epithelial-mesenchymal transition, reducing the migration and invasion ability of cancer cells, enhancing the activity of NK cells and T cells, and improving the immunosuppressive state of the tumor microenvironment.

[0037] The effects of curcumin are as follows: blocking IκBα phosphorylation and degradation, reducing NF-κB nuclear translocation, reducing the secretion of pro-inflammatory factors such as IL-6 and TNF-α, and reversing inflammation-induced drug resistance; inhibiting CYP3A4 and P-gp activity, reducing drug metabolism and efflux, and enhancing the efficacy of drugs such as sorafenib; reducing autophagy-mediated drug resistance by inhibiting the PI3K / Akt / mTOR pathway, making cancer cells more sensitive to chemotherapy; scavenging ROS, reducing oxidative stress-induced DNA damage repair, and enhancing the drug's killing effect on cancer cells.

[0038] The effects of the ginsenoside Rg3 are: inhibiting the function of the ABCG2 (BCRP) transporter, increasing intracellular drug accumulation, and being effective against cells resistant to doxorubicin, paclitaxel, etc.; downregulating the expression of VEGF and bFGF, blocking the PI3K / Akt / mTOR signaling pathway, and inhibiting tumor neovascularization; causing cancer cells to arrest in the G1 phase, reducing the proportion of cells in the S phase, and enhancing the cytotoxicity of chemotherapy drugs; promoting the secretion of IFN-γ and IL-2, activating CD8+ T cells and DC cells, and improving anti-tumor immune responses.

[0039] The effects of sorafenib are: blocking cancer cell proliferation signals and inhibiting tumor growth; inhibiting VEGFR-2, VEGFR-3 and PDGFR-β, reducing tumor angiogenesis; promoting cancer cell apoptosis by activating caspase cascade reactions; reducing the expression of liver cancer stem cell markers (CD133, EpCAM), and reducing the risk of tumor recurrence.

[0040] The effects of quercetin are: downregulating the expression of PARP-1 and BRCA1, reducing the ability of cancer cells to repair DNA damage, and enhancing the effects of radiotherapy and chemotherapy; upregulating miR-34a, downregulating miR-21, and reversing the expression profile of drug resistance-related miRNAs; blocking nutrient perception and protein synthesis signals, inducing cancer cell apoptosis; reducing ROS and NF-κB activation, and inhibiting inflammatory responses in the tumor microenvironment.

[0041] The effects of Rubescensine A are: directly binding to P-gp and MRP1, inhibiting their ATPase activity and reducing drug efflux; activating the PERK / eIF2α / ATF4 pathway, triggering cancer cell apoptosis; blocking the IL-6 / STAT3 pathway, reducing the expression of anti-apoptotic proteins (Bcl-xL, Mcl-1); inhibiting the expression of HK2 and LDHA, interfering with glycolysis, and reducing the energy supply of cancer cells.

[0042] The components added in the present invention have different synergistic effects on the basis of their individual effects, specifically including:

[0043] Oxymatrine and Rubescensine A synergistically inhibit P-gp and MRP1, blocking drug efflux from different sites; sorafenib inhibits the RAF / MEK / ERK pathway, and ginsenoside Rg3 inhibits the PI3K / Akt / mTOR pathway, dually blocking proliferation signals;

[0044] Curcumin reduces the expression of anti-apoptotic proteins through NF-κB inhibition, while quercetin enhances DNA damage through PARP-1 inhibition. The two synergistically promote cancer cell apoptosis. Sorafenib activates the extrinsic apoptosis pathway, while oxymatrine activates the intrinsic apoptosis pathway, forming an apoptosis signaling cascade.

[0045] Curcumin inhibits inflammatory factors (IL-6, TNF-α), and ginsenoside Rg3 enhances immune cell activity, jointly reversing the immunosuppressive microenvironment; quercetin reduces ROS generation and reduces the activation of oxidative stress-induced drug resistance-related signaling pathways; curcumin and quercetin inhibit CYP3A4 and P-gp, reducing the metabolic clearance of sorafenib and increasing its blood concentration and bioavailability.

[0046] In the present invention, pharmaceutically acceptable excipients are also included, and the excipients are preferably selected from at least one of fillers, disintegrants, binders, and lubricants.

[0047] In the present invention, the filler is microcrystalline cellulose or lactose, with a mass fraction of 10-30 parts.

[0048] The present invention increases the volume of the drug by the filler, improves the fluidity of the material, and ensures the accuracy of tablet compression or capsule filling. The microcrystalline cellulose has good compressibility and disintegration properties, which helps tablet molding and disintegration. The lactose dissolves quickly and can increase the drug dissolution rate.

[0049] In the present invention, the disintegrant is sodium starch glycolate or cross-linked polyvinylpyrrolidone, and the weight proportion is preferably 1-5 parts.

[0050] The present invention destroys the structure of tablets or capsules and promotes drug release by allowing the disintegrant to absorb water and swell. Cross-linked polyvinylpyrrolidone has high hygroscopicity and rapid expansion, and has a significant disintegration effect. Sodium carboxymethyl starch has both internal and external addition effects, and can improve disintegration efficiency.

[0051] In the present invention, the binder is hypromellose or povidone, and the weight proportion is preferably 2-8 parts.

[0052] The present invention increases the viscosity of the material by using a binder to ensure the hardness and integrity of the granules or tablets. Hydroxypropyl methylcellulose has good film-forming properties and can be used for film coating to improve drug stability. The povidone solution has low viscosity and is easy to mix, making it suitable for wet granulation.

[0053] In the present invention, the lubricant is magnesium stearate or talc, with a mass fraction of 0.5-2 parts.

[0054] The present invention prevents sticking and chipping by reducing the friction between the material and the mold; magnesium stearate has good lubricity and flow-aiding properties, but excessive use will affect tablet disintegration; talc can improve particle flowability and is commonly used in capsule production.

[0055] The present invention also provides a method for preparing the pharmaceutical composition described in the above technical solution, comprising the following steps:

[0056] Step 1: Take each component raw material, grind them separately and pass them through 80-120 mesh sieve for later use;

[0057] Step 2: Mix the pretreated raw materials in proportion, add auxiliary materials, and stir evenly to obtain a mixture;

[0058] Step 3: The uniformly stirred mixture is prepared into tablets, capsules, granules or freeze-dried powder for injection.

[0059] In the present invention, when preparing tablets, step 3 further comprises: adding 50-70% ethanol solution to the mixture to prepare a soft material, granulating through an 18-24 mesh sieve, drying at 60-70° C., adding a lubricant after granulation, and tableting to obtain the product.

[0060] In the present invention, when preparing a lyophilized powder for injection, step 3 further includes: dissolving the mixture in water for injection, adding mannitol as a lyophilization protectant, filtering and sterilizing, and freeze-drying after packaging. The freeze-drying conditions are: pre-freezing temperature -40 to -30°C, vacuum degree 10-20Pa.

[0061] When the pharmaceutical composition of the present invention is prepared into a lyophilized powder for injection, mannitol is preferably added as a lyoprotectant to form an amorphous matrix during the freeze-drying process, thereby protecting the active ingredients from freezing damage, providing physical support, preventing the lyophilized material from collapsing, maintaining the product appearance and stability, regulating the osmotic pressure, and ensuring the safety and effectiveness of the injection.

[0062] The present invention also provides the use of the pharmaceutical composition described in the above technical solution in the preparation of a drug for reducing the drug resistance of liver cancer cells.

[0063] Example 1

[0064] (1) Weigh the active ingredients: 15 parts of oxymatrine, 10 parts of curcumin, 12 parts of ginsenoside Rg3, 5 parts of sorafenib, 8 parts of quercetin, and 3 parts of oridonin.

[0065] (2) Pretreatment: After each component is crushed, it is passed through a 100-mesh sieve and accurately weighed.

[0066] (3) Mixing: Add 20 parts of microcrystalline cellulose, 3 parts of sodium starch glycolate, and 5 parts of hypromellose, and mix three-dimensionally for 18 minutes.

[0067] (4) Granulation: Add 60% ethanol to make a soft material, sieve through a 20-mesh sieve to granulate, dry at 65°C for 2 hours, and granulate.

[0068] (5) Tableting: Add 1 part of magnesium stearate, mix well and then tablet. Each tablet weighs 0.3 g.

[0069] Example 2

[0070] (1) Weigh the active ingredients: 12 parts of oxymatrine, 8 parts of curcumin, 10 parts of ginsenoside Rg3, 4 parts of sorafenib, 7 parts of quercetin, and 5 parts of oridonin.

[0071] (2) Pretreatment: crush and pass through a 100-mesh sieve for later use.

[0072] (3) Mixing: Add 15 parts of lactose, 2 parts of cross-linked polyvinylpyrrolidone, and 3 parts of polyvinylpyrrolidone, and mix for 15 minutes.

[0073] (4) Filling: Fill the mixed powder into size 0 hollow capsules, each capsule containing 40 mg of active ingredient.

[0074] Example 3

[0075] (1) Weigh the active ingredients: 18 parts of oxymatrine, 12 parts of curcumin, 13 parts of ginsenoside Rg3, 6 parts of sorafenib, 10 parts of quercetin, and 8 parts of oridonin.

[0076] (2) Dissolution: Add 1000 mL of water for injection, stir to dissolve, and add 50 parts of mannitol (1.5 times the total mass of the active ingredient).

[0077] (3) Sterilization: Filter through a 0.22 μm filter membrane and dispense into 10 mL vials (each vial contains 20 mg of active ingredient).

[0078] (4) Freeze drying: pre-freeze at -35°C (4 hours), sublime drying at -10°C (12 hours), desorption drying at 30°C (8 hours), vacuum degree 15 Pa, and seal.

[0079] Performance and testing

[0080] Test Example 1: Testing the in vitro drug resistance reversal effect

[0081] (1) Cell models: human liver cancer resistant cell lines HepG2 / R (resistant to sorafenib) and Huh7 / ADR (resistant to doxorubicin).

[0082] (2) Grouping: blank control group (culture medium), sorafenib group (10 μM), composition group of Example 1 (containing 10 μM sorafenib and other components in proportion), and traditional Chinese medicine group (composition of Example 1 without sorafenib).

[0083] (3) Methods: MTT assay was used to detect cell viability and calculate IC50 value; flow cytometry was used to detect intracellular sorafenib accumulation; Western blot was used to detect P-glycoprotein and MDR1 expression.

[0084] (4) Results: Compared with the sorafenib group, the IC50 value of HepG2 / R cells in the composition group of Example 1 decreased by 52%, the intracellular drug accumulation increased by 1.8 times, and the expression of P-glycoprotein decreased by 45%. The IC50 value of Huh7 / ADR cells for doxorubicin decreased by 48%.

[0085] Based on this, it can be seen that the composition obtained by the present invention can effectively reverse drug resistance.

[0086] Test Example 2: Verification of in vivo drug efficacy

[0087] (1) Animal model: HepG2 / R cells were inoculated subcutaneously into nude mice to establish a drug-resistant xenograft tumor model.

[0088] (2) Grouping: model group (normal saline), sorafenib group (10 mg / kg), and composition group of Example 2 (containing sorafenib 10 mg / kg and other components in proportion), 6 mice in each group, intraperitoneal injection, 3 times a week for 4 weeks.

[0089] (3) Detection indicators: tumor volume (measured every 3 days), P-glycoprotein expression in the tumor (immunohistochemistry), and changes in mouse body weight (to assess toxicity).

[0090] (4) Results: The tumor volume of the composition group of Example 2 was reduced by 63% compared with the sorafenib group, and the expression of P-glycoprotein was reduced by 58%. There was no significant decrease in the body weight of the mice (the sorafenib group decreased by 12%).

[0091] Based on the above, it can be seen that the composition obtained by the present invention reduces drug resistance while reducing toxicity.

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered by the scope of protection of the claims of the present invention.

Claims

1. A pharmaceutical composition for reducing drug resistance of liver cancer cells, characterized in that: The composition includes the following parts by weight: 10-20 parts of oxymatrine, 5-15 parts of curcumin, 38-15 parts of ginsenoside Rg, 3-8 parts of sorafenib, 5-12 parts of quercetin, and 3-8 parts of oridonin.

2. The pharmaceutical composition for reducing drug resistance of liver cancer cells according to claim 1, characterized in that The pharmaceutical composition further comprises pharmaceutically acceptable excipients, wherein the excipients are selected from at least one of fillers, disintegrants, binders and lubricants.

3. The pharmaceutical composition for reducing drug resistance of liver cancer cells according to claim 2, characterized in that: The filler is microcrystalline cellulose or lactose, with a mass fraction of 10-30 parts.

4. The pharmaceutical composition for reducing drug resistance of liver cancer cells according to claim 2, characterized in that The disintegrant is sodium starch glycolate or cross-linked polyvinylpyrrolidone, with a mass fraction of 1-5 parts.

5. The pharmaceutical composition for reducing drug resistance of liver cancer cells according to claim 2, characterized in that The adhesive is hydroxypropyl methylcellulose or povidone, with a mass fraction of 2-8 parts.

6. The pharmaceutical composition for reducing drug resistance of liver cancer cells according to claim 2, characterized in that: The lubricant is magnesium stearate or talc, with a mass fraction of 0.5-2 parts.

7. A method for preparing the pharmaceutical composition according to any one of claims 1 to 6, characterized in that: The following steps are involved: Step 1: Take each component raw material, grind them separately and pass them through 80-120 mesh sieve for later use; Step 2: Mix the pretreated raw materials in proportion, add auxiliary materials, and stir evenly to obtain a mixture; Step 3: Prepare the mixture obtained by stirring uniformly into tablets, capsules, granules or lyophilized powder for injection.

8. The preparation method according to claim 7, characterized in that When preparing tablets, step 3 further includes: adding 50-70% ethanol solution to the mixture to prepare a soft material, granulating through an 18-24 mesh sieve, drying at 60-70° C., adding a lubricant after granulation, and tableting to obtain the product.

9. The preparation method according to claim 7, characterized in that When preparing a lyophilized powder for injection, step 3 further includes: dissolving the mixture in water for injection, adding mannitol as a lyophilization protectant, filtering and sterilizing, and freeze-drying after packaging. The freeze-drying conditions are: pre-freezing temperature -40 to -30°C, vacuum degree 10-20Pa.

10. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for reducing drug resistance of liver cancer cells.