CN109453164B - Anti-tumor combined medicine - Google Patents

Abstract

The invention provides an anti-tumor pharmaceutical composition and a combined medicament, which are prepared from daphnetin and an anti-tumor medicament according to a certain proportion. The experimental result proves that the daphnetin has obvious anti-tumor effect after being used together with other anti-tumor drugs.

Description

Anti-tumor combined medicine

Technical Field

The invention belongs to the field of medicines, and particularly relates to an anti-tumor combined medicine.

Background

Malignant tumors are currently the major killers of human health and are among the most important diseases that severely threaten human life. The comprehensive treatment of tumor mainly comprises surgery, radiotherapy and tumor chemotherapy. The medicine plays an important role in the chemotherapy of malignant tumors. In recent years, the research and development work of antitumor drugs makes great progress in tumor chemotherapy, and the application of carboplatin, paclitaxel and other drugs in the last century makes certain specific tumors have high cure rate. However, because of the defects of poor selectivity, large toxic and side effects, drug resistance and the like of the antitumor drugs, more than half of tumor patients do not respond to treatment or resist the drug, so that treatment failure is finally caused, particularly the treatment of solid tumors.

Daphnetin (daphnetin), a chemical name of 7, 8-dihydroxycoumarin (7, 8-dihydroycoumarin), and daphnetin are representative monomer components of coumarin compounds, are one of natural medicines independently developed in China, and are one of traditional Chinese medicine components used for anti-tumor research earlier.

At present, no report about the combined use of daphnetin and chemotherapeutic drugs for treating tumors is found.

Disclosure of Invention

In order to solve the technical problems, the invention firstly provides the application of daphnetin in preparing an IDO1 inhibitor.

The invention also provides an anti-tumor pharmaceutical composition, which consists of daphnetin and an anti-tumor drug.

Further, the using dosage ratio of the daphnetin to the anti-tumor medicine is 1 (0.033-5); wherein the antitumor drug is cyclophosphamide, doxorubicin, cisplatin or paclitaxel; preferably, the anti-tumor drug is paclitaxel.

The invention also provides a preparation method of the pharmaceutical composition, which is characterized by comprising the following steps: it comprises the following steps: taking daphnetin and the anti-tumor drug according to the proportion, preparing the solution and mixing.

The invention also provides an anti-tumor medicinal preparation which is prepared by taking the medicinal composition as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

The invention also provides an anti-tumor combined medicine which contains the daphnetin and the anti-tumor medicine which are of the same or different specifications and are simultaneously or respectively administered, and a pharmaceutically acceptable carrier.

Further, the using dosage ratio of the daphnetin to the anti-tumor medicine is 1 (0.033-5); wherein the antitumor drug is cyclophosphamide, doxorubicin, cisplatin or paclitaxel; preferably, the anti-tumor drug is paclitaxel.

The invention also provides the application of the daphnetin and the antitumor drug in preparation of the antitumor drug.

Further, the using dosage ratio of the daphnetin to the anti-tumor medicine is 1 (0.033-5); wherein the antitumor drug is cyclophosphamide, doxorubicin, cisplatin or paclitaxel; preferably, the anti-tumor drug is paclitaxel.

Further, the tumor is selected from breast cancer, liver cancer, lung cancer, melanoma; preferably, the tumor is selected from breast cancer.

The experimental result proves that the daphnetin has obvious anti-tumor effect after being used together with other anti-tumor drugs.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical concepts of the invention.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1 shows the IC50 of daphnetin against the IDO1 enzyme.

FIG. 2 shows the inhibition of over-expressed IDO1 by daphnetin.

Figure 3 is daphnetin downregulating IDO1 expression.

FIG. 4 shows the effect of daphnetin on the proliferation of HeLa cells.

FIG. 5 shows the effect of daphnetin at various concentrations on the proliferation of MCF-7 cells.

FIG. 6 shows the effect of daphnetin in combination with chemotherapeutic drugs on tumor cells.

Detailed Description

Example 1 Effect of daphnetin on the enzymatic Activity of IDO1

1 materials of the experiment

1.1 cells and plasmids

Human cervical cancer cells HeLa and human embryonic kidney cells HEK-293A are purchased from cell resource center of Shanghai Life sciences college of China academy of sciences and are preserved by a natural product center laboratory of the institute of biology of the Chinese academy of sciences; IDO cDNA was purchased from kyoto-warping shenzhou technologies ltd.

1.2 reagents

Daphnetin (Daphnetin) was purchased from shanghai-derived leaf biotechnology limited; epacadostat is available from Shanghai blue Wood chemical Co., Ltd; human interferon IFN-gamma was purchased from Nanjing Kinsrui Biotechnology Ltd; IDO1 rabbit polyclonal antibody and GAPDH rabbit polyclonal antibody were purchased from wuhan sanying biotechnology limited; transfection reagent LIPOFECTAMINE 2000 was purchased from SEMEL FEISHIER scientific; EasySee Western Blot Kit luminescence solution purchased from Beijing Quanyujin Biotechnology Ltd; the enhanced CCK-8 kit was purchased from Shanghai Bin Yuntian Biotechnology Co.

1.3 instruments

Verioska Flash multifunctional reader (Thermo Fisher); ImageQuant LAS 500 Integrated imager (GE Healthcare).

2 method of experiment

2.1 cell culture

Human cervical cancer cell HeLa and human embryonic kidney cell HEK-293A are all in DMEM-high sugar medium (10% newborn bovine serum, 100U/mL ampicillin and 100mg/mL streptomycin), 5% CO₂And culturing at 37 ℃ in a cell culture box.

2.2 IDO1 inhibitor screening

HeLa cells were treated at 1.0X 10⁵One/well was inoculated into 48-well plates and incubated overnight at 37 ℃. And adding a proper amount of a drug to be tested and Epacadostat as a positive control drug into each well, and culturing for 24 h. 50ng/mL IFN- γ per well was added for 24h to induce intracellular IDO production. And (2) putting 100 mu L of cell culture solution into a centrifuge tube, adding 25 mu L of 30% TCA, carrying out water bath at 50 ℃ for 30min, centrifuging at 10000gpm for 10min, putting 100 mu L of supernatant into a 96-well plate, adding 100 mu L of 2% p-dimethylaminobenzaldehyde (PDAB) into each well, standing at room temperature for 10min after uniformly mixing, and detecting A492 by using a multifunctional reader.

2.3 immunoblot analysis

HeLa cells were treated at 1.0X 10⁶One/well was inoculated into 6-well plates and incubated overnight at 37 ℃. Daphnetin with different concentrations and Epacadostat as a positive control drug was added to each well and incubated for 24 h. 50ng/mL IFN- γ per well was added for 24h to induce intracellular IDO production. Absorbing the culture medium, rinsing the cells for 3 times by PBS, adding a proper amount of RIPA lysate, carrying out ice bath for 30min to lyse the cells, scraping the cells by a cell scraper, centrifuging at 12000gpm, and collecting supernatant as protein extracting solution. Adding 5 Xloading buffer boiling water bath for 5min to obtain protein sample. Proteins were separated by 12% SDS-PAGE electrophoresis and transferred to NC membranes by semidry methods. After the membrane transfer, the membrane was blocked in 5% BSA in TBST for 2 h. After blocking, the membrane was incubated with primary antibody (1:1000 dilution) overnight at 4 ℃, rinsed 3 times with TBST, incubated with HRP-labeled secondary antibody (1:5000 dilution) at room temperature for 2h, rinsed 3 times with TBST, developed according to EasySee Western Blot Kit luminescence instructions, and imaged by GE Health imaging system.

2.4 HEK-293A overexpresses IDO

HEK-293A cells at 1.0X 10⁶One/well was inoculated into 6-well plates and incubated overnight at 37 ℃. The culture medium was changed to serum-free and double-antibody-free culture medium 2h before transfection. Mu.g of IDO cDNA was diluted with 125. mu.L of Opti-MEM, and 5. mu.L of LIPOFECTAMINE 2000 was diluted with 125. mu.L of Opti-MEM, gently mixed, and allowed to stand at room temperature for 5 min. And (3) dropwise adding the plasmid diluent into the diluted transfection reagent, standing at room temperature for 20min, dropwise adding the mixed solution into a 6-well plate, culturing at 37 ℃ for 6h, replacing with a complete culture medium, and continuously culturing at 37 ℃ for 24h to over-express IDO.

2.5 HeLa cell proliferation

HeLa cells were seeded at 5000 cells/well in 96-well plates and cultured overnight at 37 ℃. Adding daphnetin with different concentrations, culturing for 24h, adding 10% enhanced CCK-8 solution into each well, and culturing at 37 deg.C for 0.5-1 h. When the color of the culture solution turns yellow, the absorbance at 450nm is detected by a multifunctional reading instrument.

2.6 data statistics and processing

All experiments were repeated three times, results are expressed as mean ± standard deviation, One way analysis of variance (One way-ANOVA) was performed using GraphPad Prism software, with significant differences at P < 0.05.

3 results of the experiment

3.1 IDO1 inhibitor screening

As can be seen from FIG. 1, the inhibition of IFN-. gamma.induced expression of IDO1 enzyme by HeLa cells was measured with different concentrations of daphnetin, and IC50 values were calculated using GraphPad Prism software. Wherein the IC50 value of daphnetin is 16.50 +/-0.33 mu M, which indicates that the daphnetin has good IDO1 enzyme inhibition effect in vitro.

3.2 HEK-293A overexpression of IDO1

IDO1 is highly expressed in tumor cells and is silent among most cells. IDO1 was overexpressed in HEK-293A and daphnetin was tested for its inhibitory effect on the IDO1 enzyme. After the pCMV3-IDO1 plasmid is transfected into HEK-293A cells for 24h, daphnetin with different concentrations is added for treatment for 6h, and cell supernatants are subjected to IDO1 activity detection, so that as shown in a figure 2, daphnetin can inhibit the activity of over-expressed IDO1 enzyme, and the inhibition is strengthened along with the increase of the concentration. The above results indicate that daphnetin can directly inhibit the activity of IDO 1.

3.3 immunoblotting of IDO1

HeLa cells were treated with daphnoretin at different concentrations, and Western blot was used to detect the expression of IDO1 protein, the results are shown in FIG. 3. 50ng/mL of IFN-gamma can promote the remarkable expression of IDO1 protein, while daphnetin can obviously inhibit the effect of IFN-gamma on up-regulating IDO1, and the inhibition effect is more obvious along with the increase of the concentration of the compound, so that the result shows that the daphnetin can inhibit the activity of IDO1 possibly by down-regulating the expression of IDO 1.

3.4 HeLa cell proliferation

HeLa cells were treated with daphnetin at different concentrations for 24h, and CCK-8 examined the effect of the compounds on the proliferation of HeLa cells, the results are shown in FIG. 4. At low concentrations, daphnetin had no significant cytotoxic effect on HeLa cells, indicating that at low concentrations the compound down-regulated the expression of IDO1 in HeLa cells was not due to a toxic effect on the cells.

Example 2 experiment of combined use of daphnetin and antitumor drug

1 materials of the experiment

1.1 cells and animals

Human breast cancer cell strains MCF-7 and MDA-MB-231 are presented by doctor Cowan of national cancer institute, and are stored in laboratories of the Natural products center of the institute of Onching biological research of the national academy of sciences of China; SPF grade ICR healthy female mice, weighing 18-22g, purchased from mastered laboratory animals ltd, license number: SYXK 2018-.

1.2 drugs and reagents

The enhanced CCK-8 kit is purchased from Shanghai Bin Yuntian biotechnology limited; daphnetin (Daphnetin) was purchased from shanghai-derived leaf biotechnology limited; human interferon IFN-gamma was purchased from Nanjing Kinsrui Biotechnology Ltd; cyclophosphamide (CTX) was purchased from Sigma company, usa; doxorubicin (Doxorubicin, Dox) injection was purchased from shanghai xu eastern sea medicine ltd; cisplatin (DDP) injection was purchased from jiangsu haofen pharmaceutical group ltd; paclitaxel (PTX) was purchased from beijing dilu pharmaceutical gmbh; bleomycin hydrochloride for injection (BLM) was purchased from hainzenkei pharmaceutical limited; RMPI 1640 medium was purchased from Hyclone, USA; high quality Fetal Bovine Serum (FBS) was purchased from Shanghai Biotechnology engineering, Inc.

2 method of experiment

2.1 cell culture

The human breast cancer MCF-7 and MDA-MB-231 cells are completely cultured in 10% FBS-containing RMPI 1640 at 37 deg.C in 5% CO₂Culturing in a cell culture box.

2.2 in situ Breast cancer mouse model establishment and grouping

Collecting MDA-MB-231 cell suspension in logarithmic growth phase, mixing with Matrigel gel at a ratio of 1:1, and aseptically adding into a container at a ratio of 1 × 10⁷0.2mL of the suspension was inoculated under the pad of the fourth pair of breasts on the right side of ICR healthy female mice and cultured continuously for 16-20 days. After the model building is successful, the mice are randomly divided into a blank control group, a chemotherapy drug group and a combination drug group of chemotherapy drugs and daphnetin.

The administration modes and routes of the chemotherapy drugs of the chemotherapy drug group and the combined drug group are shown in table 1, the daphnetin of the combined drug group is orally administered in a sustained release manner (10mg/kg/d), and the same amount of physiological saline is administered to a blank control group.

TABLE 1 administration modes and routes of different chemotherapeutic drugs

Note: i.v. intravenous injection; qd is the quaque die, once a day.

2.3 cell viability assay

MCF-7 cells in logarithmic growth phase were seeded in 96-well plates (1.0X 10)³And/well), adding 25ng/mL IFN-gamma for incubation for 10-12 h after the cells adhere to the wall, discarding the supernatant, adding physiological saline or medicines with different concentrations for continuous culture, and arranging 6 multiple wells in each group. After 12h, 24h, 48h and 72h of culture, 20 μ l of CCK-8 solution (5mg/ml) was added to each well, the wells were incubated for 4h in the dark, the Optical Density (OD) was measured at 450nm with a microplate reader, the experiment was repeated 3 times, and the cell Inhibition Rate (IR) was calculated. IR ═ OD_{Control group}-OD_{Medicine adding device})/OD_{Control group}]×100％。

2.4 in situ tumor volume determination

Three weeks after dosing, the tumor nodules in each group of animals were measured for length (L) and width (W) with a vernier caliper according to the ellipsoidal volume formula (V ═ LW)²And/2) calculating the tumor volume, averaging the groups, and comparing the volume tumor inhibition rate of each treatment group. Tumor inhibition (%) = (transplant tumor volume in placebo-transplant tumor volume in treated group)/transplant tumor volume in placebo × 100%.

2.5 statistical treatment

SPSS 15.0 software is used for statistical treatment, the measured data is expressed by mean + -SD, more than two groups of data are compared by adopting One-way ANOVA, the comparison between the groups is carried out by adopting t test, and P <0.05 represents that the difference has statistical significance.

3 results of the experiment

3.1 determination of the combined concentration of daphnetin

MCF-7 cells were treated with daphnetin at various concentrations for 72h, and CCK-8 examined the effect of compounds on HeLa cell proliferation. FIG. 5 shows that: ruiFragrance (2.5-640 mu mol. L)^-1) Can inhibit the proliferation of MCF-7 cells in a concentration-dependent manner. 2.5. mu. mol. L^-1The IR of daphnetin to MCF cells was (8.73 + -1.45)%, less than 20%, and was selected as the combined concentration for subsequent experiments.

3.2 daphnetin can enhance the reactivity of various chemotherapeutic drugs

TABLE 2 inhibition of breast cancer in situ tumors by the combination of different chemotherapeutics and daphnetin

^**：P<0.05vs–Daphnetin groups.

After preparing the breast cancer in-situ model, the volume of in-situ tumor is determined by adopting the combination of a single chemotherapeutic drug, daphnetin and a chemotherapeutic drug. Table 2 shows that compared with the treatment group of the single chemotherapeutic drug, the combined treatment group of daphnetin and the chemotherapeutic drug has reduced tumor volume in situ and obviously increased tumor inhibition rate (P <0.05), which indicates that daphnetin can improve the sensitivity of tumor tissues to the antitumor drug.

3.3 daphnetin can enhance the sensitivity of tumor cells to paclitaxel

After IFN-gamma is used for inducing IDO1 protein expression in advance, MCF-7 cells are treated by physiological saline, daphnetin, paclitaxel and combination drugs, and the cell viability is detected by CCK-8. FIG. 6 shows that daphnetin produced only a slight antitumor effect; and 2.5. mu. mol. L^-1The combined application of daphnetin and paclitaxel can enhance the cytotoxicity (P) of paclitaxel on MCF-7<0.05), suggesting that IDO inhibitors in combination with chemotherapy may be an effective way to improve clinical treatment of breast cancer.

In conclusion, the daphnetin has obvious anti-tumor effect after being used together with other anti-tumor drugs, can improve the sensitivity of tumor tissues to the anti-tumor drugs, and has good application prospect.

Claims (8)

1. An anti-tumor pharmaceutical composition, which is characterized in that: it is composed of daphnetin and an anti-tumor drug;

the using dosage ratio of the daphnetin to the anti-tumor medicine is 1 (0.033-5); wherein the antitumor drug is cyclophosphamide, doxorubicin, cisplatin or paclitaxel;

the tumor is breast cancer.

2. The pharmaceutical composition of claim 1, wherein: the anti-tumor drug is paclitaxel.

3. A process for preparing a pharmaceutical composition according to claim 1 or 2, characterized in that: it comprises the following steps: taking daphnetin and the anti-tumor drug according to the proportion, preparing the solution and mixing.

4. An antitumor pharmaceutical preparation characterized by: the preparation is prepared by taking the pharmaceutical composition of claim 1 or 2 as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

5. An anti-tumor drug combination, which is characterized in that: it contains daphnetin and antitumor drugs with the same or different specifications which are simultaneously or respectively administered, and a pharmaceutically acceptable carrier;

the using dosage ratio of the daphnetin to the anti-tumor medicine is 1 (0.033-5); wherein the antitumor drug is cyclophosphamide, doxorubicin, cisplatin or paclitaxel;

the tumor is breast cancer.

6. The combination as defined in claim 5, wherein: the anti-tumor drug is paclitaxel.

7. The application of the daphnetin and the antitumor drug combination in preparing the antitumor drug;

the using dosage ratio of the daphnetin to the anti-tumor medicine is 1 (0.033-5); wherein the antitumor drug is cyclophosphamide, doxorubicin, cisplatin or paclitaxel;

the tumor is breast cancer.

8. Use according to claim 7, characterized in that: the anti-tumor drug is paclitaxel.

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