CN106974934A - A kind of epirubicin based on calcium agent is combined chemotherapeutics and its application - Google Patents

Abstract

The invention discloses a compound chemotherapy drug of epirubicin based on calcium agent, the compound chemotherapy drug is a compound preparation of soluble calcium salt and epirubicin; wherein, the calcium element in the soluble calcium salt and the epirubicin The ratio of the mass of Rubistar to the two is 1320-15460:1. Compared with the prior art, the present invention effectively reduces the concentration of epirubicin with the same antitumor effect by improving the ingredients of the key additives that cooperate with epirubicin to more effectively exert the antitumor effect of the drug, and the addition ratio thereof. Dosage, or the same dosage of epirubicin can significantly improve the anti-cancer effect, can effectively reduce the toxic and side effects of using epirubicin in the chemotherapy process, and the calcium-based epirubicin compound chemotherapy drug can Selectively enhance the killing effect of epirubicin on tumor cells while reducing side effects.

Description

A kind of calcium-based epirubicin compound chemotherapy drug and its application

technical field

The invention belongs to the technical field of biomedicine, and more specifically relates to a calcium-based epirubicin compound chemotherapeutic drug and an application thereof. The calcium-based epirubicin compound chemotherapeutic drug is a compound chemotherapeutic drug.

Background technique

Cancer has become the number one killer of modern people's health. my country has entered the era of "cancer outbreak". For tumors, chemotherapy is still one of the commonly used methods for cancer treatment. In order to obtain a good therapeutic effect, the method of combining multiple chemotherapeutic drugs (ie comprehensive chemotherapy scheme) has been widely used clinically. On the one hand, due to the generally poor selectivity of chemotherapy drugs, the effective blood drug concentration at the target site and the uptake rate of tumor cells are not high, coupled with obvious toxic and side effects, these often affect the actual curative effect, making patients miserable, and even appear Severe heterogeneous response. On the other hand, insufficient doses of chemotherapeutic drugs will lead to incomplete killing of tumors and affect the curative effect. At the same time, chemotherapeutic drugs often take longer to act, making it difficult to achieve a radical cure on tumor cells, which limits the efficacy of drugs to a certain extent.

Contents of the invention

For the above deficiencies or improvement needs of the prior art, the object of the present invention is to provide a calcium-based epirubicin compound chemotherapy drug and its application, wherein the anti-tumor effect of the drug can be more effectively exerted by cooperating with epirubicin. The composition of the key additives and their addition ratio have been improved, which effectively reduces the amount of epirubicin used under the same anti-tumor effect compared with the existing technology, or significantly improves the anti-tumor effect under the same amount of epirubicin. Cancer effect, can effectively reduce the toxic and side effects of epirubicin in the chemotherapy process, and the calcium-based epirubicin compound chemotherapy drug can selectively enhance the killing effect of epirubicin on tumor cells, and at the same time Reduce side effects.

To achieve the above object, according to one aspect of the present invention, a calcium-based epirubicin compound chemotherapy drug is provided, characterized in that, the compound chemotherapy drug is a compound preparation of soluble calcium salt and epirubicin; wherein , the mass ratio of the calcium element in the soluble calcium salt to the epirubicin is 1320-15460:1.

As a further preference of the present invention, the soluble calcium salt is calcium chloride, calcium citrate, calcium lactate, calcium gluconate, calcium acetate or calcium nitrate.

As a further preference of the present invention, the concentration of calcium element in the application solution of the compound chemotherapy drug is 1.8-21 mmol/L, and the concentration of epirubicin is 0.1 μmol/L.

According to another aspect of the present invention, the present invention provides the application of the above calcium-based epirubicin compound chemotherapy drug in the preparation of antitumor drugs.

As a further preference of the present invention, the tumor is liver cancer tumor, colon cancer tumor or lymphoma tumor.

Through the above technical scheme conceived by the present invention, compared with the prior art, due to the use of soluble calcium salts in combination with epirubicin, the actual dosage of epirubicin can be greatly reduced under the premise of ensuring the efficacy of the drug, and the Improve its selectivity and reduce side effects on normal tissue cells. The composite calcium agent adopted in the invention has wide production sources, is easy to popularize in the market, expands the application of extracellular calcium ions, and has strong practical use value.

Calcium ions are important first and second messengers in the process of cell signal transduction, and participate in the regulation of many physiological and biochemical processes such as cell proliferation, apoptosis, and differentiation. Sustained increase in intracellular calcium concentration is often closely related to cell apoptosis and necrosis. The inventor’s previous in vitro pre-experiment found that tumor cells and normal tissue cells have different sensitivity to extracellular calcium ions, and there are differences in the range of tolerance to increased intracellular calcium concentration, and the tolerance of tumor cells to extracellular calcium ions is significantly smaller than normal cells. At the same time, the chemotherapy drug epirubicin can increase the concentration of intracellular calcium ions, but the selectivity is not good at the physiological calcium concentration level. The combination of extracellular calcium ions and epirubicin can enhance the chemotherapy effect of the latter and improve the selectivity of the target. There is no report on the combination of extracellular calcium ions and the chemotherapy drug epirubicin at home and abroad.

The epirubicin-calcium compound preparation used in the present invention can selectively inhibit the proliferation of human liver cancer cell line HepG2 and human liver cancer cell line SMMC7721, and the proliferation inhibition rate of human normal liver cell L-02 is significantly lower than that of liver cancer cell line. Calcium concentration measurement found that the amplitude of calcium influx induced by compound calcium agent was significantly greater than that of L-02, and the peak time of calcium influx occurred earlier.

Description of drawings

Figure 1 is a graph showing the effect of extracellular calcium ions on the proliferation of HepG2, SMMC7721 and L-02; among them, the cell proliferation rate was obtained by the SRB method, and calcium chloride solutions of different concentrations were added to DMEM high-glucose medium, and after 48 hours of culture , to calculate the proliferation rate of different cell lines under different extracellular calcium concentrations. Among them, "***" means p<0.001 for inter-group comparison, "**" means p<0.01, "*" means p<0.05; for intra-group comparison, "###" means p<0.001, "##" means p<0.001, "##" means p<0.01, "#" means p<0.05

Figure 2 is a diagram of the effect of extracellular calcium ions combined with epirubicin on the proliferation of HepG2, SMMC7721 and L-02; where "***" means p<0.001 in comparison between groups, "**" means p<0.01, "* "Indicates p<0.05; comparison within the group, "###" indicates p<0.001, "##" indicates p<0.01, "#" indicates p<0.05; the cell proliferation rate is obtained by the SRB method, which is shown in Figure 1 Calcium chloride solutions of different concentrations and 0.1 μM epirubicin were added to the DMEM high-glucose medium in a ratio, and the "0" group was diluted with calcium-free medium to dilute epirubicin. After 48 hours of culture, the different cells were calculated The proliferation rate under different drug ratios;

Figure 3 is a graph showing the time change of intracellular calcium ions in different dosage regimens of HepG2 cells; wherein, 10.8mM+0.1EPI means 10.8mM calcium chloride combined with epirubicin; 0.1EPI means using epirubicin alone; the abscissa 4h, 8h, 16h, and 24h represent the time points of 4h, 8h, 16h, and 24h after drug addition; the ordinate represents the increase in intracellular calcium ions compared with cells without intervention (that is, only adding culture medium) The size and intracellular calcium ion concentration were measured using Fluo-3/AM fluorescent probe, and the excitation wavelength was set to 488nm and the detection wavelength to 525nm in the fluorescence spectrophotometer;

Fig. 4 is the impact diagram of the minimum extracellular calcium ion concentration combined with epirubicin on the proliferation of HepG2 and L-02 in Example 1 of the present invention;

Fig. 5 is the magnitude map of the increase of the intracellular calcium ion concentration of HepG2 cells and L-02 cells under the lowest extracellular calcium concentration in Example 1 of the present invention; The intracellular calcium ion concentration value a ₁ of the rubicin group is different from the intracellular calcium ion concentration value a ₀ of the control group, then the multiple of intracellular calcium ion increase is n=a ₁ /a ₀ . Among them, the intracellular calcium ion concentration value. K is the dissociation constant of the fluorescent probe, which is 390 nm at 37°C. A is the measured fluorescence intensity value of each group, Amax is the maximum fluorescence intensity value after adding Triton-X100, and Amin is the minimum fluorescence intensity value after adding EGTA;

Figure 6 is a graph showing the effect of the highest extracellular calcium concentration combined with epirubicin on the proliferation of HepG2 and L-02 in Example 2 of the present invention;

Fig. 7 is the amplitude of intracellular calcium ion concentration promotion in HepG2 cells and L-02 cells under the highest extracellular calcium concentration in Example 2 of the present invention; Comparing the intracellular calcium ion concentration value a ₁ of the star group and the intracellular calcium ion concentration value a ₀ of the control group, the multiple of intracellular calcium ion increase is n=a ₁ /a ₀ . The calculation formula for the intracellular calcium ion concentration value is K(A-Amin)/(Amax-A), wherein K is the dissociation constant of the fluorescent probe, which is 390nM at 37°C. A is the measured fluorescence intensity value of each group, Amax is the maximum fluorescence intensity value after adding Triton-X100, and Amin is the minimum fluorescence intensity value after adding EGTA.

Figure 8 is a graph showing the influence of optimal extracellular calcium concentration combined with epirubicin on the proliferation of HepG2 and L-02 in Example 3 of the present invention;

Fig. 9 is a graph showing the increase in intracellular calcium ion concentration of HepG2 cells and L-02 cells under the optimal extracellular calcium concentration in Example 3 of the present invention, where * indicates p<0.05. The processing method is the same as that in Figure 4, and the intracellular calcium ion concentration value a ₁ of the epirubicin group obtained by the Fluo-3/AM method is compared with the intracellular calcium ion concentration value a 0 of the control group, and the intracellular calcium ion concentration value a ₀ increases. Multiple n=a ₁ /a ₀ . The formula for calculating the intracellular calcium ion concentration is K(A-Amin)/(Amax-A), where K is the dissociation constant of the fluorescent probe, and 37°C is 390nm. A is the measured fluorescence intensity value of each group, Amax is the maximum fluorescence intensity value after adding Triton-X100, and Amin is the minimum fluorescence intensity value after adding EGTA.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not constitute a conflict with each other.

The calcium-based epirubicin compound chemotherapeutic drug of the present invention, its composition comprises soluble calcium salt (as, calcium chloride) and epirubicin, can be used for but not limited to the chemotherapy of tumors such as liver cancer, colon cancer, lymphoma program.

The compound chemotherapeutic drug is a compound preparation of soluble calcium salt and epirubicin; wherein, the weight ratio of calcium element to epirubicin is 1320～15460:1; the concentration of calcium element in the application solution (ie the corresponding solution preparation) is 1.8～21mmol/L, the concentration of epirubicin is 0.1μmol/L.

Concrete experimental process of the present invention comprises the following steps:

(1) Material preparation

1. The cell lines required for the experiment are human liver cancer cell lines HepG2, SMMC7721, and human embryonic liver cells L-02, purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, using DMEM high-glucose medium, 10% fetal bovine serum, 1% streptomycin and penicillin Culture, and regularly give 0.5μg/mL MRA to remove potential mycoplasma contamination.

2. Preparation of main reagents:

(1) Calcium chloride (analytical pure) is dissolved with sterile three-distilled water, and is prepared into a stock solution of 1800mmol/L. Before dosing, it is diluted to a working concentration with DMEM culture solution containing 10% fetal bovine serum as required. , to ensure that the volume occupied by three-distilled water does not exceed 0.5%;

(2) Epirubicin: Weigh 5mg of solid epirubicin powder, accurately measure 8.6210ml of three-distilled water, dissolve, filter and sterilize with a 0.22μm filter, and make a 1mM stock solution. Before use, use 10% fetal bovine Dilute the DMEM culture solution of the serum to the working concentration, and ensure that the volume occupied by three-distilled water does not exceed 0.5%;

(3) Fluo-3/AM calcium ion probe: strictly protected from light, accurately add 44.2μL of anhydrous DMSO to 8mg powder, the working concentration is guaranteed to be 1.5μg/mL per tube, and at the same time, add PluronicF-127 (v/w% = 2.5%) for better entry of the dye into the cells.

3. Main instruments: multifunctional microplate reader (BioTek, USA), TX400Z electric steam pressure sterilizer (Shanghai Sanshen Medical Instrument Co., Ltd.), DK-8A constant temperature water bath (Shanghai Jinghong Experimental Equipment Co., Ltd.), Pipette (Eppendorf, Germany), automatic sampler (Eppendorf, Germany), ultra-clean bench (Suzhou Purification Equipment Co., Ltd.), carbon dioxide cell incubator (SANYO, Japan), electronic analytical balance (Sartorius, Germany) , inverted microscope (Olympus, Japan), 100ml, 250ml culture bottle (Corning, U.S.), 6-well plate, 96-well plate (Corning, U.S.), membrane syringe filter (Milipore, U.S.), low-speed large capacity Centrifuge DL-5 (Shanghai Anting Scientific Instrument Factory), fluorescence spectrophotometer (Perkin Elmer LS5, USA).

(2) Detection of cell proliferation by sulforhodamine B method (SRB)

1. Cell culture: L02 cells and HepG2 cells were cultured in DMEM medium (Hyclone, USA) containing 10% calf serum (Gibco, USA), and placed in a 37°C incubator containing 5% CO2. When the cells grew 80-90% of the bottom of the full culture bottle is passage. Discard the old culture medium, wash it gently with 3ml PBS buffer twice, discard it, digest the cells with 0.5ml 0.25% trypsin for 5min, add 0.5ml calf serum to stop the digestion, then add fresh DMEM medium to blow the cells , made into a cell suspension, and one bottle was passed to three bottles.

2. Cell treatment: Take the cells in the logarithmic growth phase, plant them in a 96-well plate at 5×10 ³ cells/well, and culture the cells for 24 hours with a total volume of culture solution in each well of 100 μl. Then, the cells were cultured with DMEM containing different concentrations of calcium ions (containing 10% newborn bovine serum) (Ca ²⁺ 4.5mmol/L, Ca ²⁺ 9mmol/L, Ca ²⁺ 18mmol/L, and the control group Ca ²⁺ 1.8mmol /L) for 48 hours.

3. Fix the cells: After the drug action reaches the end of the corresponding time, add 50 μL of 4°C pre-cooled TCA solution (final concentration 10%, w/v) to each well to fix the cells, let it stand for 5 minutes, move it into a 4°C refrigerator for 1 hour, take it out and use it. Rinse five times with deionized water and dry at room temperature.

4. Staining: After the 96-well plate was dried at room temperature, 70 μL of 0.4% (w/v) SRB dye solution (prepared with 1% acetic acid) was added to each well, and after 30 minutes of staining, the dye solution was poured out and washed with 1% (v/v). /v) Rinse 4 times with acetic acid to remove unbound dye and dry at room temperature.

5. Dissolving the dye: dissolve the dye bound to the cell protein with 100 μL of unbuffered Tris-base lye (10 mM, pH=10.5), shake on a horizontal shaker for 20 min, and measure the light absorption value at 550 nm with a microplate reader. Calculate the relative optical density value according to the formula to reflect the proliferation rate of the cells

Value-added rate (%)=(A experimental group—A blank group)/(A control group—A blank group)×100%).

A experimental group represents the drug treatment group; A control group represents the normal control group; A blank group represents the blank control group. Results are expressed as mean±SD. A is the optical density value at 550 nm.

(3) Determination of intracellular calcium ion

Cell culture, treatment, and cell harvesting are the same as above. Wash twice with PBS solution, centrifuge at 2000rpm for 5min. The cells in each group were counted and leveled to 10 ⁵ /tube, the cells were loaded with 5 μM Fluo-3AM (Japanese colleagues), and incubated at 37°C for 60 min in the dark. After incubation, 13000rpm, 10min, discard the supernatant, add 1ml/tube of PBS buffer containing 1mM calcium chloride to resuspend, add 40% probenecid solution to the tube before testing on the machine, balance the background value, and put it on the fluorescence spectrophotometer Fluorescence intensity is detected, the excitation wavelength is 488nm, and the detection wavelength is 525nm.

Please refer to the following Examples 1-3 for the specific drug ratios used in the present invention and the experimental results thereof.

Example 1

As shown in Figures 4 and 5, in Example 1, the group without any treatment was used as the control group, 1.8mM calcium ions were added to the DMEM medium, and the proliferation rates of the two cell lines were detected by the SRB method.

Example 2

As shown in Figures 6 and 7, in Example 2, the group without any treatment was used as the control group, 10.8mM calcium ions were added to the culture system, and the proliferation rates of the two cell lines were detected by the SRB method.

Example 3

As shown in Figures 8 and 9, in Example 3, the group without any treatment was used as the control group, 6.3mM calcium ions were added to the culture system, and the proliferation rates of the two cell lines were detected by the SRB method.

By above-mentioned embodiment, analyze respectively as follows:

(4) Extracellular calcium ions enhance the inhibitory effect of epirubicin on the proliferation of HepG2

For the two liver cancer cell lines HepG2 and SMMC7721 cells, compared with the control group, the inhibition rates of each treatment group were significantly increased (p<0.05, and in a concentration-dependent relationship. For L02 cells, the inhibition rates of the middle and high dose groups were also Significantly increased (p<0.05, p<0.01). In each dose group, the inhibitory effect of extracellular calcium ions on HepG2 cell proliferation was significantly higher than that of L02 cell proliferation (p<0.05). Adding epirubicin alone , it is not difficult to find that the proliferation rate of L02 cells is significantly lower than that of HepG2 group (p<0.05), and the average proliferation rates of HepG2 and L02 are 50.67%±5.52% and 65.18%±6.67%, respectively, which suggests that the use of epirubicin alone Epirubicin, even at a low dose of 0.1 μM in the experiment, still has a large killing effect on normal cells, and the selectivity is not good. Figure 2 uses calcium-free culture medium to construct calcium-free extreme conditions. The killing effect on HepG2 and SMMC7721 under calcium conditions was less than that under physiological calcium concentration conditions (1.8mM), and the difference was statistically significant (p<0.01). With the addition of different extracellular calcium concentration gradients, the selectivity of epirubicin The killing effect solution gradually increased, but high doses of calcium (ie 21mM in Figure 1) had a significant inhibitory effect on the proliferation of both cells, suggesting that high doses of extracellular calcium ions had a certain toxic effect on the cells. Through improved Cole's method, fixed epirubicin concentration is 0.1 μ M, utilizes IC50 (see Table 1 for details) that the present invention provides, selects the calcium agent of 6.3mM for use, can effectively inhibit liver cancer cell line proliferation, under this dosage effect, Normal cell lines still have a proliferation rate of about 65%.

Table 1. The half maximal inhibitory concentration of the dosage regimen calcium used in the present invention

Note: the concentration of epirubicin used in the present invention is 0.1 μM, and the concentration range of calcium is between 1.8 and 21 mM. The calculation method of IC50 is based on the modified Cole's method, and the unit is mM.

(5) Extracellular calcium ions combined with epirubicin induced a significant increase in intracellular calcium ions in HepG2 cells and L02 cells

Since intracellular calcium ions are important second messengers of cells, they participate in the regulation of many physiological and biochemical processes such as cell proliferation, apoptosis, and differentiation. The increase of intracellular calcium ions can activate many calmodulins with EF domains, which can activate the activity of calmodulin kinase (CaMPK), and through the biological cascade amplification effect, mediate and regulate the activities of many transcription factors, thus directly Participate in life activities. Therefore, the detection of changes in the concentration of intracellular calcium ions is of great benefit to the study of cell-related life processes.

As shown in Figure 3, after 48 hours of drug-dosing intervention, with the gradual increase of the extracellular calcium ion concentration, the intracellular calcium ion concentration of the two cells also gradually increased under the effect of adding epirubicin, and the magnitude of the increase in HepG2 cells greater (p<0.01), at 10.8mM calcium ion concentration, the intracellular calcium ion of the two cells reached the peak; at the same time, in the absence of epirubicin, at 10.8mM calcium ion concentration, the intracellular The increase of calcium ion was less than that of 6.3mM concentration, and there was no statistical difference in the change of intracellular calcium ion concentration between the two kinds of cells.

Figure 6 reflects the dynamic changes of intracellular calcium ions at different time points. In the case of using epirubicin alone, the intracellular calcium ion concentration of HepG2 increased rapidly after 8 hours, and then gradually stabilized; while in the case of combined with extracellular calcium ions, the increase was slow in the first 8 hours, but after 16 hours The increase was rapid and surpassed that of the epirubicin group. After 18 hours, the changes of intracellular calcium ion induced by the two administration methods gradually slowed down and tended to be stable.

Except the calcium chloride in the above-mentioned embodiment, the present invention can also adopt other soluble calcium salts, as calcium citrate, calcium lactate, calcium gluconate, calcium acetate, calcium nitrate etc.

In the present invention, the calcium-based epirubicin compound chemotherapeutic drug can also include other auxiliary components harmless to cells, such as NaCl etc. (NaCl in the compound chemotherapeutic drug The concentration in can be consistent with the concentration of normal saline). The epirubicin compound chemotherapeutic drug based on calcium agent among the present invention, except being directly used as the medicine of antitumor (such as tumors such as liver cancer tumor, colon cancer tumor and lymphoma tumor), can also according to actual conditions, and other Drugs are used in combination (for example, the active ingredients of other drugs are continuously added to the compound chemotherapy drug, etc.).

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention, All should be included within the protection scope of the present invention.

Claims (5)

1. a kind of epirubicin based on calcium agent is combined chemotherapeutics, it is characterised in that the compound chemotherapeutics is solubility calcium The compound formulation of salt and epirubicin；Wherein, the quality of the calcium constituent in the soluble calcium salt and both epirubicins The ratio between be 1320~15460:1.

2. the epirubicin as claimed in claim 1 based on calcium agent is combined chemotherapeutics, it is characterised in that the soluble calcium salt For calcium chloride, calcium citrate, calcium lactate, calcium gluconae, calcium acetate or calcium nitrate.

3. the epirubicin as claimed in claim 1 based on calcium agent is combined chemotherapeutics, it is characterised in that the compound chemotherapeutic The concentration of calcium constituent is preferably 1.8~21mmol/L in the application liquid of thing, and the concentration of epirubicin is preferably 0.1 μm of ol/L.

4. the epirubicin based on calcium agent is combined chemotherapeutics and is preparing antineoplastic as described in claim 1-3 any one Application in thing.

5. the epirubicin as claimed in claim 4 based on calcium agent is combined the application of chemotherapeutics, it is characterised in that the tumour For hepatic carcinoma, colon cancer tumours or lymphom tumor.