A pharmaceutical composition and its application in preparing a drug for reversing primary paclitaxel resistance in tumors

Abstract

The invention discloses a pharmaceutical composition and its application in preparing a drug for reversing primary paclitaxel resistance of tumors. The pharmaceutical composition comprises paclitaxel and ginkgo biloba flavonoids. The pharmaceutical composition proposed by the invention effectively solves primary paclitaxel resistance of colorectal cancer. The effect of ginkgo biloba flavonoids administered alone is not significant, but the combination of ginkgo biloba flavonoids and paclitaxel has a synergistic effect. The anti-tumor efficacy of the drug combination is better than that of paclitaxel administered alone, and the safety is higher than that of 5-fluorouracil.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to a pharmaceutical composition and application thereof in preparing a drug for reversing primary paclitaxel resistance of tumors. Background Art

[0002] As a broad-spectrum anti-tumor drug, paclitaxel is used as a first-line drug for the treatment of various cancers, including breast cancer, lung cancer, etc. However, colorectal cancer has primary paclitaxel resistance, so the industry has excluded paclitaxel from the medication guidelines for colorectal cancer. The absence of paclitaxel has limited the treatment of colorectal cancer. Paclitaxel has a significant inhibitory effect on tumor growth and special microtubule binding. Its absence is not conducive to the clinical treatment of colorectal cancer. At present, the mechanism of primary paclitaxel resistance in colorectal cancer is unclear, and related solutions are not mature. Although there are literature reports that researchers have used autophagy inhibitors and other drugs in combination with paclitaxel, the problem has not been solved due to effects and adverse reactions.

[0003] There have been literature reports and patent reports that ginkgo biloba flavonoids have anti-tumor effects, but the applicability and anti-tumor targeting of its effects need to be optimized and improved (PMID: 29190983, PMID: 32783998, PMID: 25611086, PMID: 23523142). It has been reported in the literature that ginkgo biloba flavonoids can enhance the anti-non-small cell lung cancer effect of cisplatin (PMID: 33113504, patent number: CN201610579579.0). However, there are no reports on the resistance to primary drug resistance of colorectal cancer. Summary of the invention

[0004] The present invention solves the problems existing in the prior art and provides a pharmaceutical composition and its application in the preparation of a drug for reversing primary paclitaxel resistance in tumors. The pharmaceutical composition effectively solves primary paclitaxel resistance in colorectal cancer. The effect of ginkgo biflavonoids administered alone is not significant, but the combination of ginkgo biflavonoids and paclitaxel has a synergistic effect. The anti-tumor efficacy of the drug combination is better than that of paclitaxel administered alone, and the safety is higher than that of 5-fluorouracil.

[0005] The purpose of the present invention is to provide a pharmaceutical composition comprising paclitaxel and ginkgo biloba flavonoids.

[0006] The present invention aims at the primary paclitaxel resistance problem of colorectal cancer. By combining paclitaxel with ginkgo biloba flavonoids, the primary paclitaxel resistance is reversed, while the anti-tumor treatment of ginkgo biloba flavonoids alone is enhanced, paclitaxel is reapplied to colorectal cancer, and low-dose paclitaxel treatment is achieved, thereby achieving the effects of increasing efficacy and reducing toxicity.

[0007] Preferably, the mass ratio of paclitaxel to ginkgo biloba biloba flavonoids is 1:2-1000.

[0008] More preferably, the mass ratio of paclitaxel to ginkgo biloba biloba is 1:2-4.

[0009] More preferably, the mass ratio of paclitaxel to ginkgo biloba biloba flavonoids is 1:50-1000.

[0010] Preferably, the pharmaceutical composition further comprises a medically acceptable carrier and / or excipient.

[0011] The present invention also protects the use of the pharmaceutical composition in preparing anti-tumor drugs or drugs for reversing primary drug resistance of tumor paclitaxel.

[0012] Preferably, the tumor is colorectal cancer or non-small cell lung cancer.

[0013] More preferably, ginkgo biflavonoids cooperate with paclitaxel to significantly inhibit the growth of LS174T cells or A549 cells, thereby reversing the drug resistance of colorectal cancer or non-small cell lung cancer to paclitaxel.

[0014] Preferably, the drug for drug resistance is an oral preparation or an injectable preparation, and the injectable or oral drug concentration of ginkgo biloba flavonoids is 1-100 mg / kg.

[0015] More preferably, the oral dosage form is granules, tablets, capsules or pills.

[0016] The present invention also protects the use of ginkgo biflavonoids in preparing drugs for reversing primary drug resistance of tumor to paclitaxel.

[0017] The present invention also protects the application of ginkgo biflavonoids as a PXR antagonist.

[0018] As for the regulatory targets of Ginkgo biloba flavonoids, the present invention explores them from the perspective of PXR antagonists, which is more targeted and conducive to clinical application.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The pharmaceutical composition proposed in the present invention effectively solves the primary paclitaxel resistance of colorectal cancer. The effect of ginkgo biloba flavonoids alone is not significant, but the combination of ginkgo biloba flavonoids and paclitaxel has a synergistic effect. The anti-tumor efficacy of the drug combination is better than that of paclitaxel alone, and the safety is higher than that of 5-fluorouracil.

[0021] 2. The present invention directly adopts the research and development idea of ​​antagonizing PXR, and uses ginkgo biloba flavonoids to antagonize PXR-mediated biological activity to resist drug resistance. Ginkgo biloba flavonoids has a synergistic effect with paclitaxel within a non-toxic concentration range, and its mechanism of action is closely related to regulating the autophagy mediated by PXR, a characteristic gene of CRC.

[0022] 3. Ginkgo biloba flavonoids can effectively reverse the primary resistance of colorectal cancer (CRC) to paclitaxel, and have a significant inhibitory effect on colorectal cancer cell lines with high expression of PXR, thereby resisting the occurrence of PXR-mediated resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a general picture of the LS174T heterotopic transplanted tumor in nude mice in Experimental Example 1, wherein Figure A shows the size of the transplanted tumor in nude mice, and Figure B shows the size of the tumor mass peeled off from the transplanted tumor;

[0024] Figure 2 This is a graph showing the changes in body weight of nude mice during the administration period in each group of Experimental Example 1;

[0025] Figure 3 This is the survival curve of each medication group in Experimental Example 1;

[0026] Figure 4 This is the tumor volume growth curve of each group during the drug administration period in Experimental Example 1;

[0027] Figure 5 The weight of tumor tissue in each drug-administered group in Experimental Example 1;

[0028] Figure 6 The tumor proliferation coefficient of each medication group in Experimental Example 1 was calculated;

[0029] Figure 7 Computer simulation of the binding site between Ginkgo biloba biflavonoids and PXR protein for Experimental Example 2;

[0030] Figure 8 For Experimental Example 3, the combined administration of paclitaxel and ginkgo biloba flavonoids synergistically inhibits the growth of A549 transplanted tumors;

[0031] Fig. 9 This is an in vitro validation experiment of the synergistic effect of paclitaxel and ginkgo biloba flavonoids in Experimental Example 4;

[0032] Fig.10 This is an in vitro validation experiment of the synergistic effect of paclitaxel and ginkgo biloba flavonoids in Experimental Example 4;

[0033] Fig.11 For Experimental Example 5, the study on the compatibility ratio of paclitaxel and ginkgo biloba flavonoids;

[0034] In the above figure, ctrl represents the blank group, Gink represents the group where Ginkgo biloba flavonoids were administered alone, PTX represents the group where Paclitaxel was administered alone, P10G20 represents the low-dose group where the combination was used (10 mg / kg Paclitaxel and 20 mg / kg Ginkgo biloba flavonoids), P10G40 represents the high-dose group where the combination was used (10 mg / kg Paclitaxel and 40 mg / kg Ginkgo biloba flavonoids), and 5FU represents the positive administration group (5-Furunidine). DETAILED DESCRIPTION

[0035] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0036] Unless otherwise defined, all professional terms used hereinafter have the same meaning as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention. Unless otherwise specified, the experimental materials and reagents herein are conventional commercial products in the art.

[0037] In the following examples, the CAS number of Ginkgo biloba flavonoids is 481-46-9; the structural formula is shown in Formula 1:

[0038]

[0039] Formula 1.

[0040] Example 1

[0041] A pharmaceutical composition for reversing primary paclitaxel resistance in tumors, comprising paclitaxel and ginkgo biloba flavonoids in a mass ratio of 1:2.

[0042] Example 2

[0043] A pharmaceutical composition for reversing primary paclitaxel resistance in tumors comprises paclitaxel and ginkgo biloba biflavonoids in a mass ratio of 1:4.

[0044] Test Example 1

[0045] Fifty male nude mice aged 6-8 weeks (about 20 g) were raised in an SPF animal laboratory. After one week of adaptive feeding, the LS174T heterotopic transplant tumor model was established. The LS174T cells were suspended with trypsin, centrifuged and resuspended with PBS, and mixed with matrix gel to make 15 mL of cell solution (0.2 mL of cell solution was injected per mouse). 0.2 mL of cell solution was injected under the armpit of the mouse. In addition to maintaining sufficient water and food, the life of the mouse was no longer excessively affected to avoid affecting the growth of the tumor. One week later, each mouse was marked with an ear stud, and the mouse weight and tumor volume were weighed. When the mouse tumor volume reached 100 mm 3The mice were randomly divided into 6 groups, each with 8 mice. Except for the blank group, the other groups were given paclitaxel (10 mg / kg), ginkgo biloba flavonoids (40 mg / kg), a low-dose combination group (the ratio of Example 1, i.e., paclitaxel 10 mg / kg + ginkgo biloba flavonoids 20 mg / kg), a high-dose combination group (the ratio of Example 2, i.e., paclitaxel 10 mg / kg + ginkgo biloba flavonoids 40 mg / kg), and a positive drug 5-fluorouracil (10 mg / kg), respectively. The mice were intraperitoneally injected once every three days, and the weight and tumor volume of the mice were measured once. When the tumor volume of the mice in the model group reached 2000 mm 3 , weigh and measure the volume after the last dose, sacrifice the mice, and completely remove the tumor tissues for weighing and calculation.

[0046] Volume calculation formula: length × width 2 / 2

[0047] Tumor proliferation rate = T RTV / C RTV * 100%; T RTV : RTV, C RTV : Control group RTV; RTV = V t / V 0 .

[0048] Depend on Figure 1-6 The results showed that the inhibitory effect of paclitaxel and ginkgo biloba flavonoids alone on colorectal cancer was weak (tumor proliferation rate), while the combined effect of ginkgo biloba flavonoids and paclitaxel at a mass ratio of 2-4:1 was strong. In terms of animal survival rate, body weight and other safety indicators, the combination of ginkgo biloba flavonoids and paclitaxel was better than 5-furoxidine.

[0049] Test Example 2 The Dock6 program of Yinfu cloud computing platform (https: / / cloud.yinfotek.com / ) was used to perform molecular docking studies on PXR, GK and ketoconazole, and their binding affinities were calculated. Pregnane X receptor (PXR) (PDB ID: 6TFI) was used as the receptor in the docking process, and its crystal structure was obtained from the RSCB protein database (https: / / www.rcsb.org / ). The protein structure was carefully processed through several steps, including repairing residues, protonation and adding partial charges under the AMBER ff14SB force field. The DMS [1] tool was used to generate the protein surface with a probe radius of 1.4 Å. Ginkgo biloba (GK) and ketoconazole were used as ligands in the docking process, and their 3D structures were obtained from the PubChe database (htps: / / pubchemncbinlm.nih.gov / ). After defining the binding pocket, the DOCK 6.9 program [2,3] was used for semi-flexible docking, and the Grid scoring function was used for energy evaluation.

[0050] Depend on Figure 7 It was concluded that ginkgo biloba flavonoids have multiple binding sites with PXR protein, among which there are common binding sites of antagonists; compared with the classic antagonist ketoconazole, ginkgo biloba flavonoids have the same binding sites and specific binding sites.

[0051] Test Example 3

[0052] A549 cells in the logarithmic growth phase were prepared with serum-free DMEM medium to a concentration of 1×10 7 10 mm / mL single cell suspension was inoculated subcutaneously in nude mice at a rate of 200 μL / mouse. After 2 weeks, 10 mm / mL cells of uniform size and shape were observed. 3 The tumors were randomly divided into control group, paclitaxel group (10 mg / Kg), ginkgo biloba flavonoids group (40 mg / Kg) and paclitaxel (10 mg / Kg) + ginkgo biloba flavonoids (40 mg / Kg) combination group, with 6 animals in each group, half of them were male and half were female. The animals in each group were given 0.1 mL / 10 g of body weight for 24 days, and the tumor volume and body weight were measured every other day. On the 25th day, the tumors were dissected and weighed, and the tumor volume inhibition rate was calculated. Tumor volume (V) = 1 / 2×a×b 2 (a, b represent the length and width of the tumor, respectively, in mm); Tumor volume inhibition rate = [1- (tumor volume of the treatment group before administration-tumor volume of the treatment group after administration) / (tumor volume of the control group before administration-tumor volume of the control group after administration)] × 100%.

[0053] Depend on Figure 8 The results showed that the combination of ginkgo biloba flavonoids and paclitaxel had a synergistic effect in inhibiting the growth of non-small cell lung cancer A549 cell transplanted tumors.

[0054] Test Example 4

[0055] LS174T cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 1% 10 U / L penicillin and 1% 10 μg / L streptomycin. Well-growing LS174T cells were seeded in 96-well plates at a cell density of 2×10 5 cells / mL, in 5% CO 2 , and cultured in a 37°C incubator for 24 h. Paclitaxel and ginkgo biloba were dissolved in DMSO to prepare a stock solution (50 mM), and the required drug concentration was prepared by the doubling dilution method. An equal volume of DMSO was added to the cells in the blank control group, and the concentration was not higher than 1‰. The cells were randomly divided into a blank control group (0 nM) and PTX groups of various concentrations: 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100 nM, with 3 replicates in each group, and the corresponding concentration of drugs was added. An equal volume of culture medium was added to the cells in the blank control group, and two identical 96-well plates were repeated. After 24 hours of normal culture, 10uLcck8 reagent was added to each well of one of the 96-well plates, and incubated at 37°C for 1 hour. After the cells adhered to the wall, they were divided into a blank control group and a drug group. The drug groups were paclitaxel alone, ginkgo biflavonoids alone, and a combination drug group. Each group corresponded to 8 concentrations: GK 12800 nM, 6400 nM, 3200 nM, 1600 nM, 800 nM, 400 nM, 200 nM, 100 nM; PTX 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0.78125 nM. Different concentrations of single-drug groups and combination drug groups were added to each group of cells, with 6 replicates in each group of cells. An equal volume of culture medium was added to the cells in the blank control group, and two identical 96-well plates were repeated. After culturing for 24 h, 10 μL of CCK8 was added to each well of one plate, and the other plate was left untreated. After incubation for 1 h, the OD value was read at a wavelength of 450 nm to calculate the cell survival rate.

[0056] Calculation formula: Cell survival rate = (OD value of drug-treated group / OD value of blank control group) × 100%.

[0057] The synergistic effect index of the combined use of paclitaxel and ginkgo biloba flavonoids on LS174T cells was calculated using Ccompusyn software. 0.9≤CI≤1.1 was an additive effect, 0.8≤CI<0.9 was a low synergistic effect, 0.6≤CI<0.8 was a moderate synergistic effect, 0.4≤CI<0.6 was a high synergistic effect, and 0.2≤CI<0.4 was a strong synergistic effect.

[0058] Data processing

[0059] The ShaPiro-Wilk test was used to test the normal distribution of the measurement data of small samples (<50 cases). Normal measurement data were expressed as mean plus or minus standard deviation (±s), and one-way analysis of variance was used to compare the means between multiple groups: LeveneStatistic variance homogeneity test was performed first. When the variance was homogeneous, the overall mean was compared using the F test, and the mean between groups was compared using the SNK test; when the variance was unequal, the overall mean was compared using the Welch test, and the mean between groups was compared using the Dunnett T3 test. Non-normal measurement data were expressed using average ranks and quartiles [(P25, P50, P75)], and the overall median was compared using the Kruskal-Wallis χ2 test, and the median between groups was compared using the Mann-Whitney U nonparametric test. SPSS 22.0 software was used to complete the data processing, p = 0.05.

[0060] Depend on Fig. 9 and Fig.10 It was concluded that both paclitaxel and ginkgo biloba can inhibit the growth of LS174T cells in a dose-dependent manner, and the efficacy is enhanced after combined use. The synergistic effect is calculated within the concentration range, and CI < 1 indicates that the two drugs have a synergistic effect. It can be calculated that under the current drug ratio, the combined administration group of PTX12.5 nM+GK1600 nM has the lowest CI, and it is highly synergistic in the atmosphere of 0.4≤CI<0.6.

[0061] Test Example 5

[0062] LS174T cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 1% 10U / L penicillin and 1% 10umg / L streptomycin. Well-grown LS174T cells were seeded in 96-well plates at a cell density of 2×10 5cells / mL, and cultured normally for 24h in a 5% CO2, 37℃ incubator. Paclitaxel and ginkgo biloba flavonoids were dissolved in DMSO to prepare a stock solution (50mM), and the required drug concentration was prepared by the doubling dilution method when administering. An equal volume of DMSO was added to the cells of the blank control group, and the concentration was not higher than 1‰. They were randomly divided into a blank control group (0 nM) and a PTX concentration of 10nM alone administration group: the administration groups with different ratios were PTX10nM and 5, 10, 50, 100, 500, 1000, 5000, and 10000 nM ginkgo biloba flavonoids. Each group had 3 replicates, and the corresponding concentration of drugs was added. An equal volume of culture medium was added to the cells of the blank control group, and two identical 96-well plates were repeated. After 24h of normal culture, 10uL of CCK8 was added to each well of one plate, and the other plate was not treated. After incubation for 1h, the OD value was read at a wavelength of 450nm to calculate the cell survival rate.

[0063] Calculation formula: Cell survival rate = (OD value of drug-treated group / OD value of blank control group) × 100%.

[0064] Depend on Fig.11 The results showed that paclitaxel alone inhibited the growth of LS174T cells. At a concentration of 10 nM, the survival rate of LS174T cells was 62.16%. Compared with the PTX alone group, the survival rate of LS174T cells was 60.70%-29.78% under PTX 10 nM combined with different concentrations of Ginkgo biloba flavonoids (500-10000 nM), which was statistically significant (P<0.05).

[0065] The description of the above embodiments is only used to help understand the technical solution and core ideas of the present invention. It should be pointed out that for technicians in this technical field, several improvements and modifications can be made to the present invention without departing from the principles of the present invention. These improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. Use of the pharmaceutical composition in the preparation of an anti-tumor drug or a drug for reversing primary resistance to paclitaxel in a tumor, characterized in that: The tumor is colorectal cancer, and the pharmaceutical composition consists of paclitaxel and ginkgo biloba flavonoids; the mass ratio of paclitaxel to ginkgo biloba flavonoids is 1:2-4.

2. The use according to claim 1, characterized in that: The pharmaceutical composition also includes a medically acceptable carrier.

3. The use according to claim 1, characterized in that: The anti-tumor drug or drug resistance drug is an oral preparation or an injection preparation.

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