Combination drugs for cancer and uses thereof

Abstract

The application discloses a combined drug for cancer and application, and an effective component of the combined drug comprises a steroidal saponin and an HMG-CoA reductase inhibitor. Through combination of the steroidal saponin and the HMG-CoA reductase inhibitor, synergistic effects of the two can be fully exerted, the anti-tumor activity of the steroidal saponin is improved, the hepatotoxicity of the steroidal saponin is reduced, the lipid-lowering effect of the HMG-CoA reductase inhibitor can be exerted, and a new application scheme is provided for treatment of the cancer.

Description

Technical Field

[0001] This invention belongs to the field of anticancer drug technology, specifically relating to combination drugs for cancer and their applications. Background Technology

[0002] Steroidal saponins are a class of natural glycosides derived from spirostane or furostane, widely distributed in monocotyledonous plants of the Liliaceae, Dioscoreaceae, and Amaryllidaceae families, such as Paris polyphylla, Anemarrhena asphodeloides, and Ophiopogon japonicus. They possess various physiological activities, including anti-inflammatory, antioxidant, antitumor, hypoglycemic, and cardiovascular protective effects. However, studies have shown that steroidal saponins can cause hepatotoxicity in animal models, limiting their further development and clinical application. Although some studies have attempted to improve the safety of steroidal saponins through nanocarrier delivery and other methods, the issue of liver damage has not yet been effectively addressed at the molecular level.

[0003] HMG-CoA reductase inhibitors are a class of widely used lipid-lowering drugs that competitively inhibit HMG-CoA reductase, thereby blocking de novo cholesterol synthesis. They are widely used in cardiovascular diseases. Furthermore, studies have reported that HMG-CoA reductase inhibitors also have some anti-tumor effects, but their single-agent anti-cancer efficacy is limited, and research on their combination with other drugs for anti-cancer purposes is also limited.

[0004] Clinical studies have found that glucose and lipid metabolism disorders can affect the prognosis of cancer patients, and many clinical anti-tumor drugs can cause abnormalities in metabolic indicators such as blood lipids. Therefore, it is of great significance to develop a drug that can synergistically fight tumors and effectively alleviate metabolic and cardiovascular underlying diseases or drug-related complications in cancer patients. Summary of the Invention

[0005] Based on the above background, the purpose of this invention is to provide a combination drug for cancer and its application. By combining steroidal saponins with HMG-CoA reductase inhibitors, the synergistic effect of the two can be fully utilized, enhancing the antitumor activity of steroidal saponins and reducing their hepatotoxicity. It can also exert the hypoglycemic, lipid-lowering, and cardiovascular protective effects of steroidal saponins and HMG-CoA reductase inhibitors, providing a new application scheme for the treatment of cancer and its metabolic and cardiovascular complications, as well as for alleviating metabolic disorders caused by other antitumor drugs.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The first aspect of the present invention provides a combination drug for cancer, wherein the active ingredients of the combination drug include steroidal saponins and HMG-CoA reductase inhibitors.

[0008] As a further embodiment of the present invention: the molar ratio of steroidal saponins to HMG-CoA reductase inhibitors in the combined drug is 1:(2.5~40), preferably 1:(5~30), and more preferably 1:(10~20), the range including both extreme values.

[0009] As a further aspect of the present invention, the combined drug is in the form of a solid, a semi-solid, a liquid, or a gas.

[0010] As a further aspect of the present invention: the combined drug is a nano-formulation.

[0011] As a further embodiment of the present invention: the combined drug is one of liposomes, polymer nanoparticles, solid lipid nanoparticles, and cyclodextrin inclusion complexes.

[0012] As a further aspect of the present invention, the combined drugs are a compound preparation.

[0013] As a further embodiment of the present invention: the combined drug is one of the following: dual-drug co-loaded nanoparticles, bilayer tablets, or suspensions.

[0014] As a further improvement to the combined drug use of the present invention, the drug delivery system can be optimized by modifying dosage forms such as microspheres, and the pharmacokinetic parameters can be improved to enable steroidal saponins and HMG-CoA reductase inhibitors to be released stably and quantitatively in vivo or locally in tumors for a long period of time; or the two can be chemically modified into prodrugs and released simultaneously in vivo to achieve the effect of combined drug use in vitro.

[0015] The cancers described in this invention include at least one of the following: urinary system tumors, breast cancer, colorectal cancer, hepatocellular carcinoma, gastric cancer, non-small cell lung cancer, ovarian cancer, melanoma, acute myeloid leukemia, glioma, osteosarcoma, myeloma, cervical tumor, glioma, lung adenocarcinoma, retinoblastoma, nasopharyngeal carcinoma, and pancreatic cancer.

[0016] The urinary system tumors described in this invention include at least one of prostate cancer, bladder cancer, kidney cancer, urethral cancer, and ureteral cancer.

[0017] The renal cancer described in this invention includes at least one of clear cell renal carcinoma, papillary renal cell carcinoma, chromophobe renal carcinoma, and renal collecting duct carcinoma.

[0018] As a further aspect of the present invention: the combined drug is used for cancer, preferably for urinary system tumors, more preferably for kidney cancer, and even more preferably for clear cell renal cell carcinoma.

[0019] The steroidal saponins of this invention include at least one of the following: total saponin extract of Paris polyphylla, saponin compounds and their pharmaceutically acceptable salts, and structurally modified products of saponin compounds of Paris polyphylla.

[0020] The total saponin extract of Paris polyphylla described in this invention includes an aqueous extract of total saponins of Paris polyphylla and an organic solvent extract of total saponins of Paris polyphylla, wherein the organic solvent includes ethanol, methanol, acetone, ethyl acetate, etc.

[0021] The Paris saponin compounds of this invention include at least one of Paris saponin I, Paris saponin II, Paris saponin VI, Paris saponin VII, diosgenin, and diosgeninogen.

[0022] The structural modification products of the present invention include at least one of glycan modification products and nanocarrier coupling products.

[0023] As a further embodiment of the present invention, the steroidal saponin is preferably at least one of Paris saponin I, Paris saponin II, Paris saponin VI, Paris saponin VII, diosgenin, and diosgeninogen, and is even more preferably Paris saponin I.

[0024] The Paris saponin I (PPI) mentioned in this invention refers to the compound with CAS number 50773-41-6 and the chemical formula shown below:

[0025] .

[0026] The Paris polyphylla saponin II (PPII) of this invention includes the compound with CAS number 76296-72-5 and the chemical formula shown below:

[0027] ;

[0028] And / or, a compound with CAS number 50773-42-7 and the chemical formula shown below:

[0029] .

[0030] The Paris saponin VI (PPVI) mentioned in this invention refers to the compound with CAS number 55916-51-3 and the chemical formula shown below:

[0031] .

[0032] The Paris saponin VII (PPVII) mentioned in this invention refers to the compound with CAS number 68124-04-9 and the chemical formula shown below:

[0033] .

[0034] The diosgenin mentioned in this invention refers to the compound with CAS number 19057-60-4 and the chemical formula shown below:

[0035] .

[0036] The diosgenin mentioned in this invention refers to the compound with CAS number 512-04-9 and the chemical formula shown below:

[0037] .

[0038] The glycan-modified products described in this invention refer to compounds obtained by removing or modifying the glycan structure, which have a similar parent nucleus structure. Since glycan-containing saponins often have a low water-oil partition coefficient, they are poorly absorbed by the gastrointestinal tract after oral administration, resulting in low bioavailability. The secondary glycosides produced by deglycosylation of these saponins often have higher physiological activity and bioavailability than the native glycosides. Therefore, by removing glycans or modifying the structure while preserving the compound's activity, the pharmacokinetic characteristics of the compound can be improved.

[0039] The nanocarrier conjugate product described in this invention refers to a liposome drug delivery system that exists through drug self-assembly or other means.

[0040] The HMG-CoA reductase inhibitors described in this invention include statins.

[0041] The statin drugs described in this invention include at least one of fluvastatin, simvastatin, atorvastatin, rosuvastatin, pravastatin, lovastatin, and pitavastatin.

[0042] Since some HMG-CoA reductase inhibitors are prodrugs, they need to be opened in vivo to activate and exert their effects. The HMG-CoA reductase inhibitors described in this invention are preferably non-prodrug HMG-CoA reductase inhibitors, such as fluvastatin.

[0043] As a further embodiment of the present invention: the active ingredients of the combined drug include Paris saponin I and fluvastatin.

[0044] As a further aspect of the present invention, the combined drugs also include pharmaceutically acceptable excipients.

[0045] A second aspect of the present invention provides a medicine box for cancer treatment, the medicine box comprising the drug as described in any one of the first aspects.

[0046] As a further embodiment of the present invention: the steroidal saponins in the medicine box are prepared according to the daily dosage standard of 0.001 mg / day to 100,000 mg / day.

[0047] As a further embodiment of the present invention: the HMG-CoA reductase inhibitor in the kit is prepared according to the daily dosage standard of 0.001 mg / day to 100,000 mg / day.

[0048] The third aspect of the present invention provides the use of the combination drug as described in any one of the first aspects or the pillbox as described in any one of the second aspects in any one or a combination of the following (a) to (c):

[0049] (a) Prevention and / or treatment of cancer;

[0050] (b) Improve tumor drug resistance;

[0051] (c) Adjunctive treatment for symptoms of tumor-related metabolic disorders.

[0052] As a further aspect of the present invention: the prevention and / or treatment of cancer includes inhibiting at least one of cancer cell proliferation, migration, invasion and metastasis.

[0053] As a further aspect of the invention: the prevention and / or treatment of cancer includes reducing the toxicity of the drug during medication.

[0054] As a further aspect of the invention, the reduction of drug toxicity includes reducing hepatotoxicity associated with cholesterol biosynthesis pathway disorders.

[0055] As a further aspect of the present invention, the hepatotoxicity associated with the cholesterol biosynthesis pathway disorder is specifically manifested as lipid homeostasis imbalance and hepatocyte damage caused by overactivation of the SREBP2-HMGCR-SQLE-LSS signaling axis.

[0056] As a further aspect of the present invention: the improvement of tumor drug resistance includes improving tumor drug resistance caused by at least one of small molecule targeted antitumor drugs, antibody antitumor drugs and immune checkpoint inhibitors.

[0057] The small molecule targeted anti-tumor drugs described in this invention include gefitinib, erlotinib, icotinib, afatinib, osimertinib, crizotinib, alectinib, ceritinib, lorlatinib, brigatinib, carmatinib, cevotinib, terpoxtinib, gumetinib, beritinib, pralatinib, ceprotinib, larotrectinib, entrectinib, vemurafenib, dabrafenib, cannafenib, trametinib, bimetinib, lapatinib, pyrotinib, neratinib, tucatinib, palbociclib, ribociclib, abeciclib, dalcirib, olaparib, everolimus, romidipine, dasatinib, nilotinib, ponatinib, imatinib, ibrutinib, zanubrutinib, zanubrutinib, ruxolitinib, and aperib. The formula contains at least one of the following: succinate, permitinib, evanixib, sorafenib, anlotinib, regorafenib, lenvatinib, apatinib, sunitinib, fruquintinib, pazopanib, axitinib, vandetanib, and cabozantinib; preferably gefitinib, erlotinib, icotinib, afatinib, osimertinib, crizotinib, alectinib, ceritinib, lorlatinib, brugatinib, carmatinib, cevotinib, terpotinib, gumetinib, beritinib, prallatinib, ceprotinib, larotrectinib, entrectinib, vemurafenib, dabrafenib, cannafenib, trametinib, bimetinib, sorafenib, anlotinib, regorafenib, lenvatinib, pazopanib, vandetanib, and cabozantinib.

[0058] The antibody-based antitumor drugs of the present invention include at least one of trastuzumab, pertocilizumab, cetuximab, nimotuzumab, panitumumab, bevacizumab, recombinant human endostatin, ramucirumab, rituximab, and daratumumab, preferably at least one of trastuzumab, pertocilizumab, cetuximab, nimotuzumab, panitumumab, bevacizumab, recombinant human endostatin, and ramucirumab.

[0059] The immune checkpoint inhibitors of the present invention include at least one of nivolumab, pembrolizumab, camrelizumab, tislelizumab, sintilimab, atezolizumab, durvalumab, ipilimumab, and M7824, preferably at least one of nivolumab, pembrolizumab, camrelizumab, tislelizumab, sintilimab, atezolizumab, and durvalumab.

[0060] In clinical practice, drug resistance to most anti-tumor drugs (such as small molecule targeted anti-tumor drugs, antibody anti-tumor drugs, and immune checkpoint inhibitors) is related to the upregulation of EGFR. Steroidal saponins (such as Paris saponins) can bind to EGFR, and in particular, Paris saponin I can reduce EGFR expression, suggesting that drugs containing steroidal saponins have the effect of improving tumor drug resistance in tumor treatment.

[0061] As a further aspect of the present invention: the adjuvant treatment for tumor-related metabolic disorder symptoms includes adjuvant treatment for tumor-related hyperlipidemia, hyperglycemia, and a series of related clinical symptoms such as hyperlipidemia, atherosclerosis, and diabetes.

[0062] The pharmaceutically acceptable salts described in this invention include salts formed from Paris saponins with inorganic acids, organic acids, alkali metals, alkaline earth metals, and basic amino acids.

[0063] The inorganic acid described in this invention includes at least one of hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, and hydrobromic acid.

[0064] The organic acids described in this invention include at least one of maleic acid, fumaric acid, tartaric acid, lactic acid, citric acid, acetic acid, methanesulfonic acid, p-toluenesulfonic acid, adipic acid, palmitic acid, and tannic acid.

[0065] The alkali metals described in this invention include at least one of lithium, sodium, and potassium.

[0066] The alkaline earth metals described in this invention include at least one of calcium and magnesium.

[0067] The basic amino acid described in this invention includes lysine.

[0068] The pharmaceutically acceptable excipients described in this invention include at least one of the following: diluents, excipients, fillers, binders, disintegrants, absorption enhancers, surfactants, adsorbent carriers, lubricants, preservatives, and flavoring agents.

[0069] The diluent described in this invention includes at least one of water, ethanol, sodium chloride, and glucose.

[0070] The excipients of this invention include at least one of lactose, microcrystalline cellulose, mannitol, sorbitol, dicalcium phosphate, calcium carbonate, magnesium carbonate, and calcium sulfate.

[0071] The filler described in this invention includes at least one of starch and sucrose.

[0072] The adhesive of the present invention includes at least one of cellulose derivatives, alginate, gelatin, xanthan gum, gum arabic, and povidone.

[0073] The disintegrant of the present invention includes at least one of sodium carboxymethyl starch, crospovidone, crospovidone sodium carboxymethyl cellulose, agar, calcium carbonate and sodium bicarbonate.

[0074] The absorption enhancer of the present invention includes at least one of medium-chain fatty acid glycerides, sodium lauryl sulfate, disodium ethylenediaminetetraacetate, benzalkonium chloride, and domiphen.

[0075] The surfactants described in this invention include at least one of polysorbate 80, polysorbate 20, poloxamer, sodium lauryl sulfate, lecithin, and Span 80.

[0076] The adsorbent carrier of the present invention includes at least one of kaolin and bentonite.

[0077] The lubricant of the present invention includes at least one of talc, calcium stearate, magnesium stearate, and polyethylene glycol.

[0078] The preservatives described in this invention include at least one of sodium benzoate and potassium sorbate.

[0079] The flavoring agent of the present invention includes at least one of sucrose, fructose, glucose, maltose, sorbitol, mannitol, xylitol, sodium saccharin, aspartame, sucralose, neotame, citric acid, peppermint oil, strawberry flavor, apple flavor, grape flavor, and peach flavor.

[0080] The drug described in this invention can be any dosage form conventional in the art, and its form can be at least one of solid, semi-solid, liquid, and gas.

[0081] The solid form may include at least one of tablets, hard capsules, soft capsules, granules, powders, pills, and suppositories;

[0082] The semi-solid form may include at least one of ointment, cream, paste, and gel;

[0083] The liquid form may include at least one of the following: injection, oral solution, syrup, tincture, liniment, lotion, film-forming agent, nasal drops, eye drops, lotion, and enema.

[0084] The gaseous form may include at least one of aerosol, spray, or inhalant.

[0085] The drug described in this invention can be administered by conventional methods of administration in the art, including at least one of injection, oral administration, mucosal administration, inhalation, and transdermal administration.

[0086] The injection administration method includes at least one of the following: intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection, intradermal injection, intraperitoneal injection, intrathecal injection, intra-articular injection, and intratumoral injection.

[0087] The cancer prevention described in this invention refers to reducing the risk of cancer and its complications, delaying the onset of cancer and its complications, or completely avoiding the occurrence of cancer and its complications.

[0088] The cancer treatment described in this invention refers to reducing the severity of cancer and its complications, slowing the progression of cancer and its complications, or curing cancer and its complications and normalizing them.

[0089] The cancer cell proliferation described in this invention refers to the uncontrolled abnormal division and growth of cancer cells, leading to an increase in the number of cancer cells and an increase in tumor volume.

[0090] The cancer cell migration described in this invention refers to the process by which cancer cells detach from the primary tumor site and move to surrounding tissues.

[0091] The cancer cell invasion described in this invention refers to the process by which tumor cells break through the basement membrane, invade the surrounding normal tissues, and destroy their structure. It is one of the core characteristics of malignant tumors.

[0092] The cancer cell metastasis described in this invention refers to the process by which cancer cells detach from the primary tumor, spread to distant organs through the blood / lymphatic system or directly, and implant to form new tumors (metastatic lesions). It is a marker of advanced cancer and significantly affects prognosis.

[0093] The dosage described in this invention is an amount that can alleviate or delay the disease and prevent its progression, and can be determined according to the specific disease being treated and other factors. Other factors include age, weight, health status, severity of symptoms, route of administration, frequency of treatment, and whether other medications are used concurrently during treatment.

[0094] This invention combines the non-prodrug HMG-CoA reductase inhibitor fluvastatin with the representative steroidal saponin compound, Paris polyphylla saponin I, to preliminarily evaluate its efficacy in treating renal cell carcinoma. The study shows that the combination of these two drugs exhibits a significant synergistic effect: Paris polyphylla saponin I can inhibit cancer cell proliferation, migration, and angiogenesis by regulating the phosphorylation of FGFR2 and its downstream signaling pathways, and inhibit EGFR upregulation, thus exerting an anti-cancer effect. At doses where Paris polyphylla saponin I alone is ineffective or even negligible, the combination with low-dose fluvastatin produced a superior effect on tumor cell proliferation, migration, and invasion compared to high-dose monotherapy. Furthermore, TIC total ion chromatography and HPLC chromatography revealed that no new compounds were generated between Paris polyphylla saponin I and fluvastatin in solution, suggesting a synergistic effect in terms of mechanism or drug delivery. Specifically, in terms of mechanism, the two may be able to inhibit tumor growth and progression by synergistically improving glucose and lipid metabolism, or by complementing each other through different anti-cancer mechanisms; in terms of delivery, Paris saponins may be able to effectively deliver statin drugs by encapsulating them in nanomedicines through self-assembly.

[0095] Because several common steroidal saponins, including Paris polyphylla saponin I, II, VI, and VII, have similar structures and all possess potential affinity for the FGFR2 pathway, it suggests that combining these drugs with HMG-CoA reductase inhibitors would produce similar synergistic effects, providing an important direction for promoting the clinical application of steroidal saponins. Especially for patients with lipid disorders, which are closely related to the development and progression of many tumors, including clear cell renal cell carcinoma, the combination drugs of this invention possess both highly effective and low-toxicity antitumor activity and effective lipid-lowering effects, greatly expanding their application prospects.

[0096] The present invention has at least the following technical effects:

[0097] 1. This invention selects HMG-CoA reductase inhibitors in combination with steroidal saponins to preliminarily evaluate their efficacy in treating renal cell carcinoma. It was found that the combination of these two drugs has a significant synergistic effect. At doses where steroidal saponins alone are ineffective or even negligible, the combination of low-dose HMG-CoA reductase inhibitors produced a superior effect on tumor cell proliferation, migration, and invasion compared to high-dose single-drug treatment. Furthermore, TIC total ion chromatography and HPLC chromatography revealed that no new compounds were generated in solution between steroidal saponins and HMG-CoA reductase inhibitors, suggesting a synergistic effect in terms of mechanism or drug delivery.

[0098] 2. In the combined drug of this invention, steroidal saponins can exert their effects by inducing apoptosis in cancer cells and inhibiting multiple signaling pathways; HMG-CoA reductase inhibitors can interfere with cell metabolism by inhibiting cholesterol synthesis; the combination of the two produces a synergistic anti-tumor effect while reducing toxic side effects. Utilizing the synergistic effect of HMG-CoA reductase inhibitors and steroidal saponins in anti-tumor activity, the dosage of either HMG-CoA reductase inhibitor or steroidal saponin as a single drug can be reduced, ensuring anti-tumor activity while reducing the hepatotoxicity of steroidal saponins and / or the related adverse reactions of HMG-CoA reductase inhibitors. Furthermore, it can exert lipid-lowering effects (HMG-CoA reductase inhibitor alone, or the synergistic lipid-lowering effect of both) and hypoglycemic effects, greatly expanding its application prospects. Attached Figure Description

[0099] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0100] Figure 1 This is a comparative image of plate colony formation after combined treatment with Paris polyphylla saponin I (different concentrations) and fluvastatin (different concentrations) in the 786-O cell line according to the present invention.

[0101] Figure 2 shows the heatmap (2D and 3D) of the inhibition rate and ZIP synergistic coefficient of Paris saponin I (different concentrations) and fluvastatin (different concentrations) after combined treatment in the 786-O cell line.

[0102] Figure 3 This is a comparative image of plate colony formation after combined treatment with Paris polyphylla saponin I (different concentrations) and fluvastatin (different concentrations) in the Caki-1 cell line according to the present invention;

[0103] Figure 4 shows the heatmap (2D and 3D) of the inhibition rate and ZIP synergistic coefficient of Paris saponin I (different concentrations) and fluvastatin (different concentrations) after combined treatment in Caki-1 cell line.

[0104] Figure 5 This is a comparison of the scratch test results of the 786-O cell line after treatment with Paris polyphylla saponin I, fluvastatin and their combination, according to the present invention.

[0105] Figure 6 This is a comparison of the scratch test results of Caki-1 cell lines after treatment with Paris polyphylla saponin I, fluvastatin and their combined use according to the present invention;

[0106] Figure 7 This is a comparison of the Transwell migration results after treatment with Paris polyphylla saponin I, fluvastatin, and their combined use;

[0107] Figure 8 This is a comparison of the Transwell invasion test results after treatment with Paris polyphylla saponin I, fluvastatin, and their combined use;

[0108] Figure 9 The total ion chromatograms of TIC for the present invention are as follows: 2.5 μM fluvastatin, 2.5 μM fluvastatin combined with 0.25 μM Paris polyphylla saponin I, and the combination of the two.

[0109] Figure 10 The HPLC (wavelength 254 nm) absorption chromatograms of the present invention are of 2.5 μM fluvastatin, 2.5 μM fluvastatin combined with 0.25 μM Paris polyphylla saponin I and the combination of the two.

[0110] Figure 11 The total ion chromatograms of TIC for the combined use of 9 μM fluvastatin, 9 μM fluvastatin and 0.9 μM Paris polyphylla saponin I, and the superposition of the two are shown in the present invention.

[0111] Figure 12 The HPLC (wavelength 254 nm) absorption chromatograms of the combined use of 9 μM fluvastatin, 9 μM fluvastatin and 0.9 μM Paris polyphylla saponin I, and the superposition of the two are shown in the present invention.

[0112] Explanation of reference numerals in the attached figures:

[0113] PPI, Polyphyllin I: Paris polyphylla saponin I;

[0114] fluvastatin;

[0115] NC: Blank control;

[0116] Vehicle: vehicle control;

[0117] Combination: Paris polyphylla saponin I and fluvastatin in combination;

[0118] Overlay: The spectrum of fluvastatin and the spectrum of fluvastatin combined with Paris polyphylla saponin I are overlaid;

[0119] *: P <0.05; **: P <0.01; ***: P <0.001; ****: P <0.0001 vs Vehicle;

[0120] #: P <0.05; ##: P <0.01; ###: P <0.001; ####: P <0.0001 vs combination. Detailed Implementation

[0121] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0122] To better understand the essence of this invention, the following description, in conjunction with the accompanying drawings, uses embodiments of the invention to further illustrate its essence, but this does not limit the scope of the invention. Any modifications made to the invention based on its essence fall within the scope of this invention.

[0123] Human kidney clear cell carcinoma lines 786-O and Caki-1 were derived from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0124] 1. Cell Culture

[0125] Culture conditions for human clear cell renal adenocarcinoma cells (786-O) and human clear cell renal skin metastasis cell line (Caki-1): 786-O and Caki-1 cell lines were placed in an incubator and cultured in RPMI 1640 complete medium (1% penicillin-dipeptide and 10% fetal bovine serum (FBS). After cell attachment, normal cell density was observed. When confluence reached 90%–95% or higher, the cells were washed twice with PBS, digested with trypsin (preheated to 37°C), and the digested cells were collected. Digestion was terminated with RPMI 1640 complete medium containing 10% FBS and 1% penicillin-dipeptide. The cells were centrifuged at 1,000 rpm for 5 minutes, the original medium was discarded, and fresh RPMI 1640 complete medium (1% penicillin-dipeptide and 10% FBS) was added. The cells were then incubated at 25 cm⁻¹. 2 The cells were passaged in the cell culture flasks at a ratio of 1:3.

[0126] 2. Plate colony assay to detect cell proliferation capacity

[0127] 786-O and Caki-1 cells in logarithmic growth phase were digested, centrifuged, and resuspended. The resulting cell suspension was diluted to 500 cells / ml and seeded into 6-well plates, 2 ml per well. After overnight adhesion, blank control group, solvent control group, and treatment groups treated with different concentrations of Paris polyphylla saponin I and fluvastatin were set up. The medium was changed every 3 days and cultured at 37 ℃ for 1 week. The original culture medium was discarded, and the cells were washed twice with PBS and then discarded. Cells were fixed with 4% paraformaldehyde for 30 min and then discarded. The cells were washed twice with PBS. 1 mL of crystal violet staining solution diluted 5 times was added to each well, and the cells were stained in the dark for 30 min and then discarded. The stain was washed with distilled water to remove excess stain, and the cells were air-dried. Each well was photographed individually using a shadowless lamp and a camera, and the clone number was identified using ImageJ software.

[0128] 3. Synergy coefficient score for drug combination therapy

[0129] This study used the Zero Interaction Potency (ZIP) synergistic model based on the SynergyFinder 3.0 platform (https: / / synergyfinder.fimm.fi) to score the synergistic coefficients of the drug combination groups in this experiment. Compared with the HAS model, Loewe additive model, Bliss independence model, and Chou-Talalay combination index method, the ZIP synergistic model does not require a strict linear relationship between drug dosage and its biological effect, nor does it require different drugs to have completely identical or unrelated mechanisms of action. This method assumes that the two drugs used in combination do not interact and quantifies synergistic and antagonistic effects by calculating the difference between the expected effect value and the actual observed effect value.

[0130] Its core calculation formula is as follows:

[0131] ZIP score = Measured inhibition rate Expected inhibition rate;

[0132] Expected inhibition rate = EA + EB – EA × EB;

[0133] EA and EB represent the inhibition rates of the two drugs at the same concentration.

[0134] A ZIP score greater than 10 indicates a synergistic effect between the two drugs; a ZIP score between -10 and 10 indicates a simple additive effect; and a ZIP score less than -10 indicates an antagonistic effect.

[0135] 4. Scratch assay to detect cell migration ability

[0136] Draw three horizontal lines evenly dividing the bottom of the 6-well plate in each well, and inoculate each well with 6×10⁶ g / cm³ of the substrate. 5 Cells were cultured at 37 °C until the cell density reached 90-100%. After starvation treatment with basal medium for 24 h, the cells were removed from the 6-well plates. Using the plate lid as a ruler, 200 μL pipette tips were used to scribble lines perpendicular to the plate surface and the positioning lines, maintaining a consistent angle and pressure. Floating cell debris was then washed away with PBS. Cells were cultured in groups according to their specific needs: blank control, solvent control, treatment with Paris polyphylla saponin I, treatment with fluvastatin, and treatment with a combination of Paris polyphylla saponin I and fluvastatin. The 6-well plates were removed at 0 h and 12 h, washed with PBS, and observed and photographed under a microscope. The area of ​​the scratched region was measured using ImageJ (migration rate = (0 h scratched region area - 12 h scratched region area) / 0 h scratched region area).

[0137] 5. Transwell migration assay to detect cell migration ability

[0138] Cells in good growth condition were seeded into 6-well plates. The drugs were prepared using basal medium, and cells were pretreated according to the following groups: blank control group, solvent control group, Paris polyphylla saponin I group, fluvastatin group, and Paris polyphylla saponin I and fluvastatin combination group. After 24 hours, the pretreated cells were digested and basal medium was used to prepare 2.5 × 10⁻⁶ cells / well. 5Cell suspension at 100 μL / mL was prepared. The upper chambers were moistened with 100 μL of basal culture medium beforehand, followed by 200 μL of cell suspension to each upper chamber. The lower chamber was incubated with 750 μL of culture medium containing 20% ​​fetal bovine serum at 37 °C for 12 h. The culture medium was then aspirated, and the chambers were cleaned twice with PBS using forceps. They were then fixed in 4% paraformaldehyde for 30 min, cleaned twice with PBS, and then immersed in a 5-fold diluted Beyotime crystal violet staining solution for 20 min in the dark. The staining solution was aspirated, and the chambers were cleaned twice with PBS. Excess cells and stain in the upper chamber cavity were gently wiped away with a cotton swab. The chambers were inverted to air dry, then placed on a glass slide for immediate observation and photographing. The number of migrating cells was analyzed using ImageJ.

[0139] 6. Transwell invasion assay to detect cell invasion ability

[0140] Thaw the Matrigel basement membrane matrix on ice overnight. Pre-cool the tip box, EP tubes, and 24-well plates containing Transwell chambers at 4 °C. Transfer experimental materials and reagents to a clean bench. Perform all pre-gel procedures on ice. Dilute the Matrigel basement membrane matrix with serum-free culture medium at a ratio of 1:8 to obtain the Matrigel working solution. Vertically add 80 μL of Matrigel working solution to each upper chamber, ensuring the basement membrane matrix is ​​evenly spread at the bottom of the chamber. Incubate the 24-well plates containing Transwell chambers at 37 °C for 3 h to allow the basement membrane matrix to polymerize into a thin film. Aspirate excess liquid from the chambers and add 100 μL of serum-free culture medium to hydrate the basement membrane. Wait 30 min, then aspirate excess liquid from the upper chamber. If no liquid flows through the upper chamber into the lower chamber, proceed with subsequent experiments. Subsequent cell pretreatment, seeding, culture, and staining steps are the same as the migration experiment in the previous section.

[0141] 7. TIC and HPLC chromatographic analysis

[0142] The samples of Paris polyphylla saponin I and fluvastatin were dissolved in DMSO, filtered through a 0.22 μm filter membrane, and injected. Detection was performed using a liquid chromatography-mass spectrometry (LC-MS / MS) system, and the total ion chromatogram (TIC) was recorded. Detection was also performed using a high-performance liquid chromatography (HPLC) system, and the absorption chromatogram at 254 nm was recorded.

[0143] from Figure 1The results of the 786-O clear cell renal cell carcinoma plate colony assay showed that both Paris polyphylla saponin I and fluvastatin alone reduced the number of 786-O tumor cell clones in a dose-dependent manner. When Paris polyphylla saponin I was used in combination with fluvastatin, both could effectively reduce cell clone formation at concentrations lower than their individual concentrations. Treatment with 10 μM fluvastatin and 1 μM Paris polyphylla saponin I alone could effectively eliminate cancer cell clones, while the combination of 1.25~5 μM fluvastatin and 0.5 μM Paris polyphylla saponin I could also exert a similar effect and effectively eliminate cancer cell clones.

[0144] As shown in Figure 2, the synergistic coefficient scores indicate that in the 786-O cell line, Paris polyphylla saponin I and fluvastatin have a synergistic antitumor effect. The synergistic coefficient is highest when the concentration of Paris polyphylla saponin I is 0.25 μM and the concentration of fluvastatin is 2.5 μM.

[0145] from Figure 2-1 It was found that the tumor inhibition rate of Paris saponin I and fluvastatin increased in a dose-dependent manner.

[0146] from Figure 2-2 , 2-3 It was found that Paris saponin I and fluvastatin have a synergistic anti-tumor effect, with an overall synergistic coefficient of 10.05.

[0147] from Figure 3 The results of the Caki-1 renal clear cell carcinoma plate colony assay showed that both Paris polyphylla saponin I and fluvastatin alone reduced the number of Caki-1 cell clones in a dose-dependent manner. When Paris polyphylla saponin I was used in combination with fluvastatin, both could effectively reduce cell clone formation at concentrations lower than their respective single-use concentrations. Treatment with 10 μM fluvastatin and 1 μM Paris polyphylla saponin I alone could effectively eliminate cancer cell clones, and the combination of 5 μM fluvastatin and 0.5 μM Paris polyphylla saponin I could also exert a similar effect.

[0148] As shown in Figure 4, the synergistic coefficient scores indicate that in the Caki-1 cell line, Paris polyphylla saponin I and fluvastatin have a synergistic antitumor effect. The synergistic coefficient is highest when the concentration of Paris polyphylla saponin I is 0.25 μM and the concentration of fluvastatin is 5 μM.

[0149] from Figure 4-1 It was found that the tumor inhibition rate of Paris saponin I and fluvastatin increased in a dose-dependent manner.

[0150] from Figure 4-2 As shown in 4-3, Paris saponin I and fluvastatin have a synergistic anti-tumor effect, with an overall synergistic coefficient of 11.358.

[0151] from Figure 5The comparison of scratch assay results before and after treatment with Paris polyphylla saponin I, fluvastatin, and their combination shows that the combination of Paris polyphylla saponin I (0.25 μM) and fluvastatin (2.5 μM) can effectively inhibit the migration of 786-O cells. When 0.25 μM Paris polyphylla saponin I alone was ineffective, the efficacy of the combination of the two drugs was significantly stronger than that of the single drugs.

[0152] from Figure 6 The comparison of scratch assay results before and after treatment with Paris polyphylla saponin I, fluvastatin, and their combination shows that the combination of Paris polyphylla saponin I and fluvastatin can effectively inhibit Caki-1 cell migration. When 0.25 μM Paris polyphylla saponin I alone was ineffective, the inhibition efficiency was significantly increased when combined with 5 μM fluvastatin.

[0153] from Figure 7 Transwell migration assays showed that Paris polyphylla saponin I (0.25 μM), fluvastatin (2.5 μM / 5 μM) and their combination could inhibit the migration of 786-O and Caki-1 ccRCC cells, and the efficacy of the combination of the two drugs was significantly stronger than that of the single drugs.

[0154] from Figure 8 It was found that Paris polyphylla saponin I (0.25 μM), fluvastatin (2.5 μM / 5 μM) and their combination can inhibit the transmembrane invasion of two ccRCC cell lines, 786-O and Caki-1. The efficacy of the two drugs combined is significantly stronger than that of the single drugs.

[0155] from Figures 9-12 It was found that since Paris saponin I had no absorption peak at 254 nm, the peak shape and number of peaks in the mixed group of Paris saponin I and fluvastatin were the same as those in the fluvastatin monotherapy group, indicating that no new compounds were generated after mixing Paris saponin I and fluvastatin.

[0156] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. The use of a combination drug in the preparation of a medicament for the prevention and / or treatment of clear cell renal cell carcinoma, characterized in that, The active ingredients of the combined medication include Paris polyphylla saponin I and fluvastatin; The molar ratio of Paris polyphylla saponin I to fluvastatin in the combined drug is 1:(10~20).

2. The application according to claim 1, characterized in that, The combined drugs are in one of the following forms: solid, semi-solid, or liquid.

3. The application according to claim 1, characterized in that, The combined drug is a nano-formulation.

4. The application according to claim 1, characterized in that, The combined drug is one of liposomes, polymer nanoparticles, or cyclodextrin inclusion complexes.

5. The application according to claim 1, characterized in that, The combined drug is a solid lipid nanoparticle.

6. The application according to claim 1, characterized in that, The combined drugs are compound preparations.

7. The application according to claim 1, characterized in that, The combined drug is one of the following: dual-drug co-loaded nanoparticles, bilayer tablets, or suspensions.

8. The application according to claim 1, characterized in that, The prevention and / or treatment of clear cell renal cell carcinoma includes inhibiting at least one of cancer cell proliferation, migration, invasion, and metastasis.

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