CN107198685B - Pharmaceutical composition for treating renal cell carcinoma and application thereof - Google Patents

Pharmaceutical composition for treating renal cell carcinoma and application thereof Download PDF

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CN107198685B
CN107198685B CN201710448010.5A CN201710448010A CN107198685B CN 107198685 B CN107198685 B CN 107198685B CN 201710448010 A CN201710448010 A CN 201710448010A CN 107198685 B CN107198685 B CN 107198685B
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sunitinib
wogonin
cell carcinoma
cells
renal cell
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CN107198685A (en
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邹永新
史本康
王勇
陈守臻
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Shandong University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 

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Abstract

The invention relates to a pharmaceutical composition for treating renal cell carcinoma and application thereof. The invention discloses the application of a composition of wogonin and sunitinib serving as medicinal components in a medicament for treating renal cell carcinoma for the first time. The experimental results prove that the abnormal rapid DNA replication and the cell proliferation are one of the important characteristics of the tumor, and the inhibition of the DNA replication can induce the apoptosis of the tumor cells. Compared with the two medicines used alone, the wogonin and sunitinib composition has higher activity of inhibiting the DNA replication and proliferation of renal cell carcinoma cells and inducing apoptosis, and the wogonin and sunitinib have the mutual synergistic promotion effect. Meanwhile, the research finds that the combination of wogonin and sunitinib can effectively overcome the drug resistance of renal cell carcinoma cells to sunitinib when used together. The wogonin and sunitinib composition has wide application prospect in the treatment of renal cell carcinoma, and provides more drug choices for the treatment of renal cell carcinoma.

Description

Pharmaceutical composition for treating renal cell carcinoma and application thereof
Technical Field
The invention relates to a pharmaceutical composition for treating renal cell carcinoma and application thereof, belonging to the technical field of drug development.
Background
With the change of life style and living environment of people, the incidence rate and the mortality rate of cancer are in an increasing trend, and the disease with the second highest human mortality rate after coronary heart disease becomes a hot health problem in the world. Renal cancer is one of the most common malignant tumors of the urinary system, and the incidence rate accounts for about 3% of all malignant tumors, and the incidence rate of renal cancer is on the rise in recent years. According to the data of the cancer center in the world, the number of new renal cancer patients is predicted to be 61,560 in the United states in 2015, the number of tumor-specific death patients is also as high as 14,080, and the death rate is in the front. The number of patients with kidney cancer in China is also increasing year by year, and the new and dead cases of kidney cancer are 668000 cases and 234000 cases, which are respectively located at the 15 th and 17 th of morbidity and mortality, and become the second-place tumor of the urogenital system.
Renal Cell Carcinoma (RCC) originates in renal parenchymal tubular epithelial cells, with a high incidence rate, with approximately 90% of renal cancers being RCC. RCC is not sensitive to traditional radiotherapy, chemotherapy. With the increasing level of diagnosis and the innovation of clinical drugs, especially with the development of molecular genetics, more and more molecular signaling pathways have been found to serve important functions in RCC. On this basis, more and more targeted drugs, such as inhibitor drugs that target multiple tyrosine kinases, such as sunitinib, etc., are approved by various countries for the treatment of advanced metastatic RCC. The diagnosis and treatment of kidney cancer has achieved certain curative effect in recent years and is inseparable from the use of these molecular targeted therapeutic drugs. Because patients have extremely limited benefit from immunotherapy, targeted drug therapy has become a recommended treatment strategy for each of the chief guidelines for RCC, especially for mid-to-late metastatic RCC, as well as a first-line treatment for advanced kidney cancer. However, since the tumor is a highly heterogeneous disease, the drug resistance problem of the targeted drug is gradually exposed along with the application of the molecular targeted drug, for example, although the initial curative effect of the patient receiving the sunitinib treatment is significant, most of the patients generate drug resistance within 5-10 months, and few patients completely relieve the drug resistance problem, the curative effect of the RCC treatment is seriously affected, and the difficulty of clinically treating the renal cell carcinoma is increased. In addition, the targeted drugs also have certain side effects and are expensive, which become the bottleneck of targeted therapy. Therefore, the development of new therapies for increasing the sensitivity of targeted drugs, reducing the dosage and overcoming the drug resistance becomes the key research direction for the treatment of kidney cancer.
The traditional Chinese medicine shows a certain effect in the treatment of malignant tumors and has small toxic and side effects. The active natural compound separated from the traditional Chinese medicine with the effective anti-tumor effect has the overall regulation characteristic of multiple targets, has the advantage of potential improvement or reversal of drug resistance, and is one of important means for research and development of new anti-tumor drugs. Scutellaria baicalensis of Labiatae (Labiatase) is a common Chinese medicine in China. Wogonin is a flavonoid extracted from the root of scutellaria and the corresponding plants in the same genus, and is one of the effective components of scutellaria drugs. Research has shown that wogonin has biological activity in anti-inflammatory, antioxidant and antiviral aspects.
In addition, wogonin can exert an anti-tumor function in gastric cancer in various ways such as increasing the content of active oxygen in cells, inducing apoptosis of tumor cells, inhibiting angiogenesis around tumors, reversing drug resistance of chemotherapeutic drugs and the like. Nevertheless, there is no research on the application of wogonin in preparing drugs for treating renal cell carcinoma and the molecular mechanism, especially the effect of wogonin in increasing the sensitivity of targeted drugs for renal cell carcinoma and reversing the drug resistance of targeted drugs for renal cell carcinoma.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a pharmaceutical composition for treating renal cell carcinoma and application thereof.
The technical scheme of the invention is as follows:
application of sunitinib and wogonin composition in preparation of medicines for treating renal cell carcinoma is provided.
A pharmaceutical composition for treating renal cell carcinoma, comprising: sunitinib and wogonin.
The wogonin and sunitinib drug combination can effectively inhibit the proliferation of renal cell carcinoma cells, induce DNA replication disorder and induce the apoptosis of the renal cell carcinoma cells.
According to the invention, the molar ratio of sunitinib to wogonin is preferably 1 to (1-10).
According to the invention, the molar ratio of sunitinib to wogonin is preferably 1 (1-8).
According to the invention, the molar ratio of sunitinib to wogonin is preferably 1 (4-8).
According to the invention, the weight of sunitinib and wogonin accounts for 1-99.5% of the pharmaceutical composition.
The pharmaceutical composition also comprises pharmaceutically acceptable excipient.
According to some embodiments of the invention, the molar ratio of the temsunitinib to the baicalein in the pharmaceutical composition is 1: 1-10, such as 1: 1-9, 1: 1-8, 1: 1-7, 1: 1-6, 1: 1-5, 1: 1-4, 1: 1-3, 1: 1-2, 1: 2-10, 1: 2-9, 1: 2-8, 1: 2-7, 1: 2-6, 1: 2-5, 1: 2-4, 1: 2-3, 1: 3-10, 1: 3-9, 1: 3-8, 1: 3-7, 1: 3-6, 1: 3-5, 1: 3-4, 1: 4-10, 1: 4-9, 1: 4-8, 1: 4-7, 1: 4-6, 1: 4-5, 1: 5-10, 1: 5-9, 1: 5-5, 1: 5-6, 1: 6-6, 1: 4-9, 1: 6-6, 1: 4-9, 1: 4-6, 1: 6-8, 1: 6-7, 1: 7-10, 1: 7-9, 1: 7-8, 1: 8-10, 1: 8-9, and 1: 9-10; preferably 1:6 to 10, and more preferably 1:8 to 10.
According to one aspect of the invention, there is provided a kit comprising said wogonin and sunitinib.
According to the invention, the molar ratio of sunitinib to wogonin is preferably 1 (1-10).
According to the invention, the molar ratio of sunitinib to wogonin is preferably 1 (1-8).
According to the invention, the molar ratio of sunitinib to wogonin is preferably 1 (4-8).
According to the invention, the weight of sunitinib and wogonin accounts for 1-99.5% of the pharmaceutical composition.
According to some embodiments of the invention, the mass of sunitinib and wogonin accounts for 1-99.5% of the mass of the pharmaceutical composition or the pharmaceutical box, such as 1-99%, 1-90%, 1-80%, 1-70%, 1-60%, 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 10-99.5%, 10-99%, 10-90%, 10-80%, 10-70%, 10-60%, 10-50%, 10-40%, 10-30%, 10-20%, 20-99.5%, 20-99%, 20-90%, 20-80%, 20-70%, 20-60%, 20-50%, 20-40%, 20-30%, 30-99.5%, 30-90%, 30-80%, 30-70%, 30-60%, 30-50%, 30-40-99.5%, 40-99%, 40-90%, 40-80%, 40-70% and 40-99.5%, 40-60%, 40-50%, 50-99.5%, 50-99%, 50-90%, 50-80%, 50-70%, 50-60%, 60-99.5%, 60-99%, 60-90%, 60-80%, 60-70%, 70-99.5%, 70-99%, 70-90%, 70-80%, 80-99.5%, 80-99%, 80-90%, 90-99.5%, or 90-99%.
According to certain embodiments of the present application, the amount of sunitinib and wogonin is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, or 1%, 2%, 3%, 4%, 5%, 7%, 8%, or 10% of the weight of the pharmaceutical composition or kit, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Wogonin (wogonin), also known as 5, 7-dihydroxy-8-methoxy-2-phenyl-4H-1-benzofuran-4-one. Is yellow needle crystal at normal temperature, is insoluble in water, and is easily soluble in organic solvent such as ethanol, acetone, etc. Wogonin is a flavonoid extracted from root of Scutellaria baicalensis Georgi, and has antiinflammatory, blood lipid reducing, and antithrombotic effects.
Renal cancer is one of the most common malignant tumors of the urinary system, including renal cell carcinoma, nephroblastoma, renal pelvis cancer, etc.
Renal Cell Carcinoma (RCC), also known as renal adenocarcinoma, is the most common pathological type of kidney cancer, accounting for approximately 80% to 85% of kidney cancers. RCC is a malignant tumor that originates in renal parenchymal tubular epithelial cells. RCC is not sensitive to traditional radiotherapy, chemotherapy.
The targeted drug sunitinib for treating renal cell carcinoma can remarkably improve the progression-free survival period and the overall survival period of a patient, and is a standard first-line drug for advanced renal cell carcinoma. However, the clinical application shows that the medicine has higher adverse reaction incidence rate, and even part of patients influence the curative effect or stop taking the medicine due to intolerable toxicity. In addition, after the medicine is used for 5-10 months, patients are easy to have drug resistance, and the treatment effect is seriously influenced.
The traditional Chinese medicine for treating the tumor has the characteristics of improving the disease condition, prolonging the remission stage, having small side effect, good long-term curative effect, low cost and the like, and shows unique advantages.
In renal cell carcinoma research, human renal cell carcinoma cell lines 786-O and OS-RC-2 cells are often used to analyze the effect of genes or drugs on the tumor characteristics of renal cell carcinoma cells.
The term "excipient" means a pharmaceutically acceptable ingredient without any pharmacological effect, which is commonly used in pharmaceutical technology for the preparation of granular and/or solid oral dosage forms and/or liquid injectable dosage forms. Excipients may act as carriers, diluents, or dissolution modifiers, absorption promoters, stabilizers or adjuvants of manufacture, among others. Excipients useful in preparing pharmaceutical compositions are generally safe, non-toxic and acceptable for medical use as well as pharmaceutical use. As used herein, "excipient" or "pharmaceutically acceptable excipient" includes one or more such excipients.
Pharmaceutical composition and kit
The term "pharmaceutical composition" means a mixture containing a therapeutically effective amount of one or more of the compounds and pharmaceutically acceptable tautomers, solvates, hydrates or salts thereof, together with other pharmaceutically acceptable carriers. The compounds are formulated into pharmaceutical compositions for more convenient administration to a subject.
The terms "kit" or "kit" are used interchangeably in this application. Kits comprising a therapeutically effective amount of the therapeutic agent or pharmaceutical composition are disclosed. According to certain embodiments of the present application, the kit further comprises one or more additional therapeutic agents. According to certain embodiments of the present application, the kit further comprises instructions for use. According to certain embodiments of the present application, the kit further comprises a device for the respective mode of administration, such as, but not limited to, a needle.
The term "drug resistance" refers to the resistance of an organism or cell (specifically referred to herein as tumor cells and tumor patients) to the action of a drug. For example, after the tumor cells generate drug resistance to the drug, the therapeutic effect of the drug is obviously reduced, and the drug effect can not be reduced by increasing the dosage.
The wogonin can be prepared into any pharmaceutically acceptable dosage form, including but not limited to tablets, oral agents, medicinal granules, injections, liposomes, targeted drug injections, pills, capsules, granules, powders, suppositories, powders, ointments, patches, injections, solutions, suspensions, sprays, lotions, drops, liniments and the like. The pharmaceutical compositions may be prepared in dry powder form and mixed with sterile water or buffer prior to administration to prepare solutions. The pH of the buffer solution is usually 3 to 11, preferably 5 to 9, and more preferably 7 to 8.
The terms "administering", "administering" or "administering" refer to administering a dose of a compound or pharmaceutical composition to a subject by a suitable mode of administration.
The "mode of administration" includes, but is not limited to, oral administration, intravenous administration, intrarespiratory administration, sublingual administration, topical administration, intramuscular administration, intraocular administration, transdermal absorption, parenteral administration, intraperitoneal administration, vaginal administration, buccal administration, rectal administration and the like, and any mode of administration known in the art. One skilled in the art will appreciate that the mode of administration to a subject will depend on a number of factors including the location of the disease, the age of the subject, the severity of the disease, and the components of the pharmaceutical composition, among others.
When "about" is used in this application to modify a numerical value, it is meant that the numerical value may fluctuate within a range of ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1%.
The use of the terms "a" and "an" and "the" and similar referents in the context of describing the application (including the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" as used herein are to be construed as open-ended terms (i.e., "including, but not limited to") unless otherwise indicated herein or otherwise clearly contradicted by context. All methods described herein can be performed in any suitable order, as understood by those skilled in the art, unless otherwise indicated herein or otherwise clearly contradicted by context.
All patents, patent applications, and references cited in this application are incorporated by reference into this application in their entirety to the same extent as if each individual reference were individually incorporated by reference. In the event of a conflict between the present application and the references provided herein, the present application shall control.
Advantageous effects
The invention discloses the application of a composition of wogonin and sunitinib serving as medicinal components in a medicament for treating renal cell carcinoma for the first time. The experimental results prove that the abnormal rapid DNA replication and the cell proliferation are one of the important characteristics of the tumor, and the inhibition of the DNA replication can induce the apoptosis of the tumor cells. Compared with the two medicines used alone, the wogonin and sunitinib composition has higher activity of inhibiting the DNA replication and proliferation of renal cell carcinoma cells and inducing apoptosis, and the wogonin and sunitinib have the mutual synergistic promotion effect. Meanwhile, the research finds that the combination of wogonin and sunitinib can effectively overcome the drug resistance of renal cell carcinoma cells to sunitinib when used together. The wogonin and sunitinib composition has wide application prospect in the treatment of renal cell carcinoma, and provides more drug choices for the treatment of renal cell carcinoma.
Drawings
FIG. 1 is a bar graph of the growth inhibitory activity of a combination of wogonin and sunitinib on renal cell carcinoma in MTT experiments;
the data in the figure are the results of 3 parallel experiments, each experiment setting 5 replicate wells, using T-test; denotes p ≦ 0.05; denotes p ≦ 0.01;
FIG. 2 is a bar graph of the apoptosis activity of wogonin and sunitinib combination in the Tunel experiment in inducing renal cell carcinoma;
the data in the figure are the results of 3 parallel experiments, each experiment setting 5 random fields, each field of cells total no less than 200, using T test; denotes p ≦ 0.05 compared to placebo; denotes p ≦ 0.01 compared to the blank control;
FIG. 3 is a bar graph of the DNA replication efficiency of cells of renal cell carcinoma inhibited by the combination of wogonin and sunitinib in the EdU experiment;
the data in the figure are the results of 3 parallel experiments, each experiment setting 5 random fields, each field of cells total no less than 200, using T test; denotes p ≦ 0.05 compared to placebo; denotes p ≦ 0.01 compared to the blank control;
FIG. 4 is a bar graph of the efficacy of a combination of wogonin and sunitinib in inhibiting the proliferation of sunitinib-resistant renal cell carcinoma cells in an MTT assay;
the data in the figure are the results of 3 parallel experiments, each experiment setting 5 replicate wells, using T-test; denotes p ≦ 0.05 compared to placebo; denotes p ≦ 0.01 compared to the blank control;
FIG. 5 is a bar graph of the significant induction of sunitinib-resistant renal cell carcinoma cell apoptosis by a combination of wogonin and sunitinib in Tunel experiments;
the data in the figure are the results of 3 parallel experiments, each experiment setting 5 random fields, each field of cells total no less than 200, using T test; denotes p ≦ 0.05 compared to placebo; denotes p ≦ 0.01 compared to the blank control; and
FIG. 6 is a bar graph of the DNA replication efficiency of sunitinib-resistant renal cell carcinoma cells effectively inhibited by a combination of wogonin and sunitinib in an EdU experiment;
the data in the figure are the results of 3 parallel experiments, each experiment setting 5 random fields, each field of cells total no less than 200, using T test; denotes p ≦ 0.05 compared to placebo; denotes p ≦ 0.01 compared to the blank control.
Detailed Description
The present invention will be further illustrated with reference to specific examples, but the scope of the present invention is not limited to these examples.
Example 1: the composition of wogonin and sunitinib has higher growth inhibitory activity on renal cell carcinoma
This example shows that the growth inhibitory activity of the combination of wogonin and sunitinib on renal cell carcinoma is higher. In this example, the renal cell carcinoma cells are human renal clear cell adenocarcinoma 786-O and OS-RC-2 cells.
The test groups and final concentrations of the drug were as follows:
blank control group: equal volume of excipient dimethyl sulfoxide (DMSO)
Wogonin low dose group: 20 mu mol/L wogonin
Wogonin high dose group: 40 mu mol/L wogonin
Sunitinib low dose group: 5 mu mol/L sunitinib
Sunitinib high dose group: 10 mu mol/L sunitinib
Composition A group, 10 mu mol/L wogonin +5 mu mol/L sunitinib
Composition B group: 20 mu mol/L wogonin +5 mu mol/L sunitinib
Composition group C: 20 mu mol/L wogonin +10 mu mol/L sunitinib
Wogonin was purchased from Shanghai-derived leaf Biotechnology Ltd (product No. ZCX-00023510-005), dissolved in DMSO to prepare a 0.1M (mol/L) mother liquor and stored at-20 ℃. Sunitinib was purchased from seleck biosciences, inc (product number S7781, usa), and was dissolved in DMSO to prepare a 0.05M (mol/L) stock solution and stored at-20 ℃. MTT cell proliferation and cytotoxicity detection kits were purchased from Biyuntian biotechnology Limited (product number C0009). Human renal clear cell adenocarcinoma 786-O (product number TCTU 186) and OS-RC-2 (product number TCTU 40) cells were purchased from Chinese scienceCell bank of the institute's type culture Collection, cell culture was performed using RPMI-1640 medium (Gibco, USA, product No. 11875127) supplemented with 10% (V/V) fetal bovine serum (Gibco, USA, product No. 1099141), and with 100U/ml penicillin and 100mg/ml streptomycin, all cells were cultured at 37 ℃ under CO2The partial pressure was 5% (volume percentage) of the culture conditions.
Thiazole blue (MTT) assay: 786-O and OS-RC-2 cells in logarithmic growth phase at 10X 104The cell is inoculated into a 96-well cell culture plate with the cell concentration of one ml, each well is 100 mu L, 5 wells are arranged, and the cell is placed at 37 ℃ and 5 percent CO2Culturing in incubator for 24 hr, and adding RPMI-1640 complete culture medium containing each group of medicine. After 48 hours of incubation, 20. mu.l of 5mg/ml MTT working solution was added per well. After shaking up, the plates were returned to 37 ℃ with 5% CO2After further incubation in the incubator for 4-6 hours, the supernatant containing MTT working solution was carefully removed, 100. mu.L of DMSO solution was added to each well, and the plates were placed at 37 ℃ in 5% CO2After the purple crystals are completely dissolved and the solution transparency is uniform (about 20 minutes), the absorbance value (OD) is measured at the wavelength of 570nm by a multifunctional microplate reader, and the cell survival rate is calculated according to the OD value.
The cell survival rate was (test cell OD value-blank cell OD value)/(control cell OD value-blank cell OD value) × 100%.
The results are shown in FIG. 1, where both wogonin and sunitinib inhibited the growth of renal cell carcinoma cells. After 786-O cells were treated with 20 μ M wogonin for 48 hours, cell viability decreased by about 15% compared to 786-O cells treated without drug; about 55% reduction in cell viability after 786-O cells were treated with 40 μ M wogonin for 48 hours; after 48 hours of 5 μ M sunitinib treatment, cell viability decreased by about 21%; after 48 hours of 10 μ M sunitinib treatment, cell viability decreased by about 51%; composition a group treated for 48 hours, cell viability decreased by about 66%, drug combination index CI of 0.576; after 48 hours of treatment in composition B group, cell viability decreased by about 89%, drug combination index CI ═ 0.431; after 48 hours of treatment in composition C group, cell viability decreased by about 97% and the drug combination index CI was 0.329. Similar to 786-O cells, treatment with wogonin and sunitinib also resulted in a significant decrease in the viability of OS-RC-2 cells. About 10% less cell viability was observed after 48 hours of 20 μ M wogonin treatment of OS-RC-2 cells compared to OS-RC-2 cells treated without drug; about 28% reduction in cell viability after 48 hours of treatment of OS-RC-2 cells with 40 μ M wogonin; after 48 hours of 5 μ M sunitinib treatment, cell viability decreased by about 17%; after 48 hours of 10 μ M sunitinib treatment, cell viability decreased by about 55%; composition a group treated for 48 hours, cell viability decreased by about 64%, drug combination index CI 0.540; after 48 hours of treatment in composition B group, cell viability decreased by about 88%, drug combination index CI of 0.348; after 48 hours of treatment with composition C, cell viability decreased by about 95% with a drug combination index CI of 0.403.
These results indicate that wogonin in combination with sunitinib can more effectively inhibit the growth of renal cancer cells. And the inhibitory effect appears concentration-dependent.
Example 2: the wogonin and sunitinib composition can effectively induce the apoptosis of renal cell carcinoma cells
This example shows that wogonin and sunitinib cooperate to induce renal cell carcinoma cell apoptosis more effectively. In this example, the renal cell carcinoma cells used were human renal clear cell adenocarcinoma 786-O cells and OS-RC-2 cells.
The test groups and final concentrations of the drug were as follows:
blank control group: equal volume of excipient dimethyl sulfoxide (DMSO)
Wogonin group: 40 mu mol/L wogonin
Sunitinib group: 10 mu mol/L sunitinib
Composition group A: 10 mu mol/L wogonin +5 mu mol/L sunitinib
Composition B group: 20 mu mol/L wogonin +5 mu mol/L sunitinib
Composition group C: 20 mu mol/L wogonin +10 mu mol/L sunitinib
The one-step TUNEL apoptosis assay kit (red fluorescence) was purchased from bi yunnan biotechnology limited (product No. C1089).
TUNEL method to detect apoptosis: the cell culture was the same as in example 1. 786-O and OS-RC-2 cells in the logarithmic growth phase are selected, the medicines are respectively added into the cells for treatment for 48 hours, the cells are washed once by PBS, and immunostaining fixing liquid is added into the cells for fixing at room temperature for 20 minutes. The fixative was discarded, washed 2 times with PBS, and treated with 0.5% TritonX 100 in PBS for 30 min at room temperature. Discard the supernatant, wash 2 times with PBS, drop 20 μ l of freshly prepared TUNEL stain onto cell-slide, incubate in the dark at 37 ℃ for 60 min, add DAPI-containing anti-quencher, and take pictures using fluorescence microscopy.
As shown in FIG. 2, the apoptosis rate of the untreated 786-O cells was only about 2%, and the apoptosis rate of the 40. mu.M wogonin-treated cells increased to about 33% after 48 hours; the proportion of apoptotic cells rose to about 43% after 48 hours of treatment with 10 μ M sunitinib; 786-O apoptosis was about 56% after 48 hours of composition group A treatment; the apoptosis rate of the composition B after 48 hours of treatment was about 79%; composition C group had an apoptosis rate of about 91% after 48 hours of treatment.
Similar to 786-O cells, the apoptosis rate of untreated OS-RC-2 cells was only about 3%, and the rate of OS-RC-2 cells apoptotic increased to 18% after 48 hours of 40. mu.M wogonin treatment; the proportion of apoptotic cells increased to 46% after 48 hours of treatment with 10 μ M sunitinib; composition A, 48 hours after treatment, the rate of OS-RC-2 apoptosis was about 50%; the apoptosis rate of the composition B after 48 hours of treatment was about 71%; composition C group had an apoptotic rate of about 88% after 48 hours of treatment.
These results indicate that wogonin and sunitinib cooperate to induce apoptosis of renal cell carcinoma cells more effectively, and the induction effect is concentration-dependent.
Example 3: the wogonin and sunitinib composition has higher activity of inhibiting the DNA replication of renal cell carcinoma cells
This example shows that wogonin and sunitinib cooperate to inhibit the replication of renal cell carcinoma cell DNA more effectively. In this example, the renal cell carcinoma cells are human renal clear cell adenocarcinoma 786-O and OS-RC-2 cells.
The test groups and final concentrations of the drug were as follows:
blank control group: equal volume of excipient dimethyl sulfoxide (DMSO)
Wogonin group: 40 mu mol/L wogonin
Sunitinib group: 10 mu mol/L sunitinib
Composition group A: 10 mu mol/L wogonin +5 mu mol/L sunitinib
Composition B group: 20 mu mol/L wogonin +5 mu mol/L sunitinib
Composition group C: 20 mu mol/L wogonin +10 mu mol/L sunitinib
The cell immunostaining fixing solution is purchased from Biyuntian biotechnology limited (product number P0098), and the EdU cell proliferation detection kit is purchased from Ruibo biotechnology limited, Guangzhou (product number C10310-1). Anti-quencher was purchased from Invitrogen (product No. P36931).
EdU cell proliferation assay: the cell culture was the same as in example 1. Selecting OS-RC-2 cells and 786-O cells in logarithmic growth phase, adding the above medicines, treating for 24 hr, adding EdU, and standing at 37 deg.C and 5% CO2Incubate for 20 minutes and wash once with PBS. EdU staining was performed using a cell immunostaining fixative overnight at 4 ℃. Anti-quencher with DAPI was added and photographed by fluorescence microscope.
The results are shown in fig. 3, where treatment with both wogonin and sunitinib significantly reduced the rate of incorporation of EdU into positive renal cell carcinoma cells. If the incorporation ratio of EdU in 786-O cells not treated with the drug was about 38%, the incorporation ratio of EdU in 786-O cells was reduced to about 18% after 24 hours of treatment with 40. mu.M wogonin; after 24 hours of treatment with 10 μ M sunitinib, the proportion of EdU incorporation in 786-O cells decreased to about 13%. After 24 hours of treatment of composition group a, the EdU incorporation rate decreased to about 12%; after 24 hours of treatment in composition B group, the EdU incorporation rate was reduced to about 6%; composition C group was treated for 24 hours and the EdU incorporation was reduced to about 1.6%.
Similarly, the EdU incorporation ratio of untreated OS-RC-2 cells was about 45%, and after 24 hours of 40. mu.M wogonin treatment, the EdU incorporation ratio of OS-RC-2 cells was reduced to about 31%; after 24 hours of treatment with 10 μ M sunitinib, the rate of incorporation of EdU by OS-RC-2 cells decreased to about 27%. Composition group a, 24 hours after treatment, EdU incorporation decreased to about 22%; after 24 hours of treatment in composition B group, cell viability decreased to about 7.5%; composition C group was treated for 24 hours and the EdU incorporation was reduced to about 3%.
These results indicate that wogonin and sunitinib cooperate to inhibit the DNA replication ability of renal cell carcinoma cells more effectively, and the inhibitory effect is concentration-dependent.
Example 4: the composition of wogonin and sunitinib can effectively inhibit sunitinib drug-resistant renal cell carcinoma cells Proliferation of
This example shows that wogonin and sunitinib cooperate to effectively inhibit the proliferation of sunitinib-resistant renal cell carcinoma cells. In this example, the renal cell carcinoma cells are human renal clear cell adenocarcinoma 786-O and OS-RC-2 cells.
The test groups and final concentrations of the drug were as follows:
blank control group: equal volume of excipient dimethyl sulfoxide (DMSO)
Wogonin low dose group: 20 mu mol/L wogonin
Wogonin high dose group: 40 mu mol/L wogonin
Sunitinib low dose group: 5 mu mol/L sunitinib
Sunitinib high dose group: 10 mu mol/L sunitinib
Combination drug group: 10 mu mol/L sunitinib and 40 mu mol/L wogonin
Establishment of sunitinib drug-resistant renal cancer cell strain: OS-RC-2 cells and 786-O cells were selected in the logarithmic growth phase, and 10% (V/V) fetal bovine serum (Gibco, product No. 1099141, USA) was added to the cell culture medium using RPMI-1640 medium (Gibco, USA, product No. 11875127) containing 10. mu. mol/L sunitinib, and 100U/ml penicillin and 100mg/ml streptomycin were added to the cell culture medium at 37 ℃ and CO2The cell obtained by culturing and subculturing for more than 10 times under the condition of partial pressure of 5 percent (volume percentage) is a sunitinib drug-resistant cell strain.
Thiazole blue (MTT) was tested as in example 1.
The results are shown in fig. 4, the activity of 786-O sunitinib-resistant cells in the low-dose sunitinib treatment group at 5 μmol/L was not significantly different from that in the blank control group, and the activity of 786-O sunitinib-resistant cells in the high-dose sunitinib treatment group at 10 μmol/L was only reduced by 10% compared with that in the blank control group, indicating that the 786-O sunitinib-resistant cells were not sensitive to sunitinib treatment; by contrast, 786-O cell viability decreased by about 13% after 48 hours of 20 μ M wogonin treatment; 786-O cell viability decreased by about 37% after 48 hours of 40 μ M wogonin treatment; after 48 hours of treatment with the combination drug group, cell viability decreased by about 72%, and the drug combination index CI ═ 0.671. Similar to 786-O cells, the activity of the OS-RC-2 sunitinib drug-resistant cells of the low-dose sunitinib treatment group is not significantly different from that of the blank control group, and the activity of the OS-RC-2 sunitinib drug-resistant cells of the high-dose sunitinib treatment group is only reduced by 11% compared with that of the blank control group, which indicates that the OS-RC-2 sunitinib drug-resistant cells are not sensitive to sunitinib treatment; in contrast, OS-RC-2 cell viability decreased by about 8% after 48 hours of 20 μ M wogonin treatment; after 48 hours of 40 μ M wogonin treatment, OS-RC-2 cell viability decreased by about 26%; after 48 hours of treatment with the combination drug group, cell viability decreased by about 64% with a drug combination index CI of 0.63.
These results indicate that wogonin in combination with sunitinib can effectively overcome the resistance of renal cell carcinoma cells to sunitinib.
Example 5: the composition of wogonin and sunitinib can effectively induce sunitinib drug-resistant renal cell carcinoma cells Apoptosis of
This example shows that wogonin and sunitinib cooperate to effectively induce apoptosis of sunitinib-resistant renal cell carcinoma cells. In this example, the renal cell carcinoma cells are human renal clear cell adenocarcinoma 786-O and OS-RC-2 cells.
The test groups and final concentrations of the drug were as follows:
blank control group: equal volume of excipient dimethyl sulfoxide (DMSO)
Wogonin group: 40 mu mol/L wogonin
Sunitinib group: 10 mu mol/L sunitinib
Combination drug group: 10 mu mol/L sunitinib and 40 mu mol/L wogonin
Establishment of sunitinib drug-resistant renal cancer cell strain: the same as in example 4.
TUNEL method to detect apoptosis: the same as in example 2. Selecting sunitinib drug-resistant 786-O and OS-RC-2 cells in logarithmic growth phase, adding each group of drugs respectively, treating for 48 hours, and detecting apoptosis by TUNEL method.
The results are shown in fig. 5, the ratio of apoptosis of 786-O sunitinib-resistant cells in the sunitinib-treated group was not significantly different from that in the blank control group, and the ratio of apoptotic cells increased to about 31% after 48 hours of 40 μ M wogonin treatment; after 48 hours of treatment of the combined drug group, the apoptosis rate of 786-O sunitinib-resistant cells increased to about 67%.
Similar to 786-O cells, the activity of OS-RC-2 sunitinib-resistant cells in the sunitinib-treated group is not significantly different from that of a blank control group, and the apoptosis ratio of OS-RC-2 sunitinib-resistant cells is increased to 26% after 40 mu M wogonin is treated for 48 hours; after 48 hours of treatment with the combination group, the rate of apoptosis in OS-RC-2 sunitinib-resistant cells increased to about 53%.
These results indicate that wogonin and sunitinib in synergy can effectively reverse the anti-apoptotic activity of sunitinib-resistant renal cell carcinoma cells on sunitinib.
Example 6: the composition of wogonin and sunitinib can effectively inhibit sunitinib drug-resistant renal cell carcinoma cells The DNA of (1) is replicated.
This example shows that the combination of wogonin and sunitinib can effectively inhibit DNA replication of sunitinib-resistant renal cell carcinoma cells. In this example, the renal cell carcinoma cells are human renal clear cell adenocarcinoma 786-O and OS-RC-2 cells.
The test groups and final concentrations of the drug were as follows:
blank control group: equal volume of excipient dimethyl sulfoxide (DMSO)
Wogonin group: 40 mu mol/L wogonin
Sunitinib group: 10 mu mol/L sunitinib
Combination drug group: 10 mu mol/L sunitinib and 40 mu mol/L wogonin
Establishment of sunitinib drug-resistant renal cancer cell strain: the same as in example 4.
EdU detection of cell proliferation: the same as in example 3.
786-O and OS-RC-2 sunitinib drug-resistant cells in the logarithmic growth phase are selected, each group of drugs are added respectively, and EdU detection is carried out after 24-hour treatment.
The results are shown in fig. 6, the sunitinib-treated 786-O sunitinib-resistant cell EdU incorporation positive rate was not significantly different from that of the blank control group, and after 24 hours of 40 μ M wogonin treatment, the EdU incorporation rate of the 786-O sunitinib-resistant cell was reduced to about 22%; after 24 hours of treatment of the combination drug group, the EdU incorporation rate decreased to about 13%;
similarly, there was no significant difference in the positive rate of incorporation of EdU in the sunitinib-treated OS-RC-2 sunitinib-resistant cells compared to the blank control, and the rate of incorporation of EdU in OS-RC-2 cells was reduced to about 33% after 24 hours of 40 μ M wogonin treatment; the EdU incorporation rate decreased to about 21% after 24 hours of combination drug treatment.
These results indicate that wogonin and sunitinib cooperate to effectively inhibit the DNA replication capacity of sunitinib-resistant renal cell carcinoma cells.
The embodiments of the present application are exemplarily described above with reference to the drawings. Those skilled in the art can easily conceive of the disclosure of the present specification that various embodiments can be appropriately modified and recombined according to actual needs without departing from the spirit of the present application. The protection scope of this application is subject to the claims of this application.

Claims (10)

1. The application of sunitinib and wogonin in preparing a pharmaceutical composition for treating renal cell carcinoma is characterized in that the molar ratio of sunitinib to wogonin is 1 (1-10);
the renal cell carcinoma cells are resistant to sunitinib.
2. The use of claim 1, wherein the molar ratio of sunitinib to wogonin is 1 (1-8).
3. The use of claim 1, wherein the molar ratio of sunitinib to wogonin is 1 (4-8).
4. The use of claim 2, wherein the sunitinib and wogonin comprise 1-99.5% by weight of the pharmaceutical composition.
5. The use of claim 2, further comprising a pharmaceutically acceptable excipient.
6. The use of claim 2, wherein the molar ratio of the Hansutinib to the baicalein is 1: 2-5.
7. The use of claim 2, wherein the molar ratio of the Hansutinib to the baicalein is 1: 2-7.
8. The use of claim 2, wherein the molar ratio of the Hansutinib to the baicalein is 1: 3-6.
9. The use of claim 1, wherein the molar ratio of sunitinib to wogonin is 1:6 to 10.
10. The use of claim 1, wherein the sunitinib and wogonin are present in a molar ratio of 1:8 to 10.
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* Cited by examiner, † Cited by third party
Title
Combination of wogonin and sorafenib effectively kills human hepatocellular carcinoma cells through apoptosis potentiation and autophagy inhibition;LI WEN RONG等;《ONCOLOGY LETTERS》;20170420;第13卷(第6期);第5028-5034页，特别是摘要，第5029页左栏第31-46行，第5030页图1 *
肾癌的靶向治疗现状与进展;寿建忠等;《中国新药杂志》;20101231;第19卷(第17期);第1539-1546页，特别是第1540页右栏第20-26行，表1，第1545页右栏第5-10行 *

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