阅读说明：本技术 拓扑替康与17β-雌二醇的药物组合物及其应用 (Medicinal composition of topotecan and 17 beta-estradiol and application thereof ) 是由 郭建丽 许建强 徐卫平 贺高红 包永明 闫志刚 代新英 于 2021-11-19 设计创作，主要内容包括：本发明提供一种拓扑替康与17β-雌二醇的药物组合物及其应用,所述药物组合物包括拓扑替康和17β-雌二醇,所述拓扑替康与17β-雌二醇的药物组合物通过以下方法制备而成：称取拓扑替康和17β-雌二醇,以二甲基亚砜和/或乙醇分别溶解,配成拓扑替康浓度为50-100mmol/L,17β-雌二醇浓度为50-100mmol/L的贮存液,在-20℃下保存,制备得到拓扑替康与17β-雌二醇的药物组合物。该药物组合物在治疗乳腺癌中具有显著的协同效用,相比单一采用拓扑替康,提高了药物的疗效,降低了用药剂量,减少了副作用的发生。(The invention provides a pharmaceutical composition of topotecan and 17 beta-estradiol and application thereof, wherein the pharmaceutical composition comprises the topotecan and the 17 beta-estradiol, and the pharmaceutical composition of the topotecan and the 17 beta-estradiol is prepared by the following method: weighing topotecan and 17 beta-estradiol, dissolving with dimethyl sulfoxide and/or ethanol respectively to prepare stock solution with topotecan concentration of 50-100mmol/L and 17 beta-estradiol concentration of 50-100mmol/L, and storing at-20 ℃ to prepare the pharmaceutical composition of topotecan and 17 beta-estradiol. The pharmaceutical composition has remarkable synergistic effect in treating breast cancer, and compared with the single use of topotecan, the pharmaceutical composition improves the curative effect of the drug, reduces the dosage and reduces the occurrence of side effects.)

1. A pharmaceutical composition of topotecan and 17 β -estradiol comprising topotecan and 17 β -estradiol.

2. The pharmaceutical composition of topotecan and 17 β -estradiol according to claim 1, characterized by being prepared by the following process: weighing topotecan and 17 beta-estradiol, dissolving with dimethyl sulfoxide and/or ethanol respectively to prepare stock solution with topotecan concentration of 50-100mmol/L and 17 beta-estradiol concentration of 50-100mmol/L, and storing at-20 ℃ to prepare the pharmaceutical composition of topotecan and 17 beta-estradiol.

3. The pharmaceutical composition of topotecan and 17 beta-estradiol according to claim 1, wherein the molar ratio of topotecan to 17 beta-estradiol is 0.03-2: 1.

4. The pharmaceutical composition of topotecan and 17 β -estradiol according to claim 1, wherein the concentration of topotecan and 17 β -estradiol in the pharmaceutical composition is 0.3 to 25 μ M and 5 to 30 μ M, respectively.

5. The pharmaceutical composition of topotecan and 17 beta-estradiol according to claim 1, wherein the diluent solvent for topotecan and 17 beta-estradiol is dimethyl sulfoxide or ethanol.

6. Use of a pharmaceutical composition of topotecan according to any one of claims 1 to 5 and 17 β -estradiol for the manufacture of a medicament for the treatment of breast cancer.

7. The use of claim 6, wherein the pharmaceutical composition of topotecan and 17 β -estradiol is administered sequentially and simultaneously.

8. The use of claim 6, wherein the pharmaceutical composition of topotecan and 17 β -estradiol is administered for a period of 24 to 72 hours.

Technical Field

The invention relates to a pharmaceutical composition technology, in particular to a pharmaceutical composition of topotecan and 17 beta-estradiol and application thereof.

Background

In 2020, the global cancer statistical data shows that 1930 ten thousand new cancer cases and nearly 1000 ten thousand cancer death cases occur globally in 2020, and the statistical data shows that female breast cancer surpasses lung cancer for the first time and becomes the most common cancer, so the treatment of breast cancer becomes a problem to be solved urgently. At present, the treatment methods of breast cancer mainly comprise surgery, radiotherapy and chemotherapy, wherein the most common method is chemotherapy. Topotecan is an incompetent ingredient in chemotherapeutic drugs. Topotecan (TPT) is a semi-synthetic water-soluble plant alkalide that targets Topo I, which inhibits the re-ligation of fragmented DNA by Topo I by forming a triple complex with Topo I and DNA, leading to extensive DNA single strand damage, inducing cell death. At present, the medicine is widely applied to second-line treatment of solid tumors. Topotecan has been used as a second-line drug primarily because its disadvantages limit its clinical application. Topotecan has one major drawback: the topotecan has poor selectivity on tumor cells, belongs to a cycle-specific drug, can cause cell cycle arrest in S phase, belongs to a DNA toxic drug, and has killing effect on all cells with DNA replication, including normal cells, so that great toxic and side effects can be generated. The second major drawback of topotecan is: the target of action is single, and the tumor cell is easy to generate drug resistance.

Disclosure of Invention

The invention aims to provide a pharmaceutical composition of topotecan and 17 beta-estradiol, aiming at the problems of large toxic and side effects and easy generation of drug resistance of the prior topotecan, wherein the pharmaceutical composition has an obvious synergistic effect in treating breast cancer, and compared with the single use of the topotecan, the pharmaceutical composition improves the curative effect of the drug, reduces the dosage and reduces the occurrence of side effects.

In order to achieve the purpose, the invention adopts the technical scheme that: a pharmaceutical composition of topotecan and 17 β -estradiol comprising topotecan and 17 β -estradiol.

Wherein the structural formulas of topotecan and 17 beta-estradiol are shown as I and II.

Further, the preparation method comprises the following steps: weighing topotecan and 17 beta-estradiol, dissolving with dimethyl sulfoxide and/or ethanol respectively to prepare stock solution with topotecan concentration of 50-100mmol/L and 17 beta-estradiol concentration of 50-100mmol/L, and storing at-20 ℃ to prepare the pharmaceutical composition of topotecan and 17 beta-estradiol.

Further, the molar ratio of topotecan to 17 beta-estradiol is 0.03-2: 1, and the preferred molar ratio is 0.26-2: 1.

Further, the concentrations of topotecan and 17 beta-estradiol in the pharmaceutical composition are 0.3-25 mu M and 5-30 mu M respectively, and the preferred concentrations are 1.56-13 mu M and 6-25 mu M respectively.

Further, the dilution solvent of topotecan and 17 beta-estradiol is dimethyl sulfoxide or ethanol.

The invention also discloses application of the pharmaceutical composition of topotecan and 17 beta-estradiol in preparing a medicine for treating breast cancer.

Further, the sequence of administration of the pharmaceutical composition of topotecan and 17 β -estradiol is simultaneous.

Further, the pharmaceutical composition of topotecan and 17 β -estradiol is administered for a period of 24-72 hours.

Compared with the prior art, the pharmaceutical composition of topotecan and 17 beta-estradiol has the following advantages:

1) the invention can improve the drug effect of topotecan and reduce the toxic and side effects, and the invention combines the topotecan with other low toxic drugs, thereby achieving the effects of attenuation and synergy.

2) Estradiol is a steroidal estrogen, a natural estrogen secreted by the mature follicular follicles of the ovaries, and has a variety of pharmaceutical effects including the treatment of cancer. However, since it is administered through the endocrine system, it causes many side effects such as breast pain, weight gain, hypertension, etc. when administered in a large amount for a long time. However, the single estradiol drug has a weak anticancer effect, and the single estradiol drug can only play an anticancer role with high dosage, so that the single estradiol drug is unrealistic to be used for anticancer. Therefore, the invention fully utilizes the advantages of low toxicity, low price and anticancer effect of the chemotherapy drugs by a drug combination mode, thereby improving the drug effect of the chemotherapy drugs and reducing the toxic and side effects of the chemotherapy drugs.

In conclusion, the medicinal composition of topotecan and 17 beta-estradiol has obvious synergistic effect in treating breast cancer, reduces the dosage, improves the curative effect of the medicament and reduces the occurrence of side effects.

Drawings

FIG. 1 shows the effect of topotecan on MCF7 cell proliferation over 48 hours;

FIG. 2 is a graph showing the effect of 17 β -estradiol on MCF7 cell proliferation over 48 hours on single drug exposure;

figure 3 is a graph of the effect of different concentrations of topotecan in combination with 17 β -estradiol on MCF7 cell proliferation over 48 hours. Wherein: significance analysis was performed with topotecan drug alone per group as control, with P <0.01 being very significant and denoted as "x".

In each figure, TPT represents topotecan and Estradiol represents 17 β -Estradiol.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited thereto, and any changes made to the technical solution of the present invention should fall within the scope defined by the claims of the present invention without departing from the spirit and scope of the present invention.

The biomaterials, drugs and experimental methods used in examples 1-3 were as follows:

cell: MCF-7 (human breast cancer cell line) is commercially available.

Medicine preparation: topotecan and 17 beta-estradiol are accurately weighed and dissolved by dimethyl sulfoxide or ethanol respectively to prepare stock solution with the topotecan concentration of 50-100mmol/L and the 17 beta-estradiol concentration of 50-100mmol/L, the stock solution is stored at the temperature of-20 ℃, and when the stock solution is used, fresh culture medium is diluted to a proper concentration, and the culture medium used in the examples 1-3 is DMEM high-sugar medium.

Thiazole blue (MTT) assay for cell proliferation: the cells are put in a DMEM high-sugar medium containing 5-20% of fetal calf serum, 80-120kU/L of penicillin and 80-120mg/L of streptomycin by volume ratio at 37 ℃ and 5% of CO by volume ratio₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount of 8000-₂Adding topotecan and 17 beta-estradiol with different concentration gradients in a single medicine or a combined medicine after culturing for 12-36 hours in an incubator at 37 ℃, wherein the volume ratio is 5% CO₂And incubating in an incubator at 37 ℃ for 24-72 hours. The medium was discarded, washed once with PBS, and then 200. mu.L of 100-200. mu.L of a serum-free medium containing 0.4-1.0mg/mL of MTT was added to each well in a volume ratio of 5% CO₂And incubating for 4-6 hours in an incubator at 37 ℃. The medium in the wells of the 96-well plate was aspirated and then 200. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm.

Three different situations may arise when two drugs are used in combination: synergistic, additive or antagonistic. The criterion used in the present invention is the Combination Index (CI) proposed by Chou and Talalay. When the CI value is less than 1, the combination of the two medicaments is shown to be synergistic; when CI value is 1, it indicates that the combination of the two drugs is additive; when CI > 1, it indicates that the combination of the two drugs is antagonistic.

The invention is further illustrated by the following examples:

example 1

The embodiment provides a pharmaceutical composition for resisting breast cancer, which comprises the following specific steps:

(1) MTT measures the effect of topotecan and 17 β -estradiol on MCF7 cell proliferation: MCF-7 cells in a medium containing 10% fetal bovine serum, 100kU/L penicillin, 100mg/LIn DMEM high-sugar culture medium containing streptomycin, the volume ratio of CO is 5 percent at 37 DEG C₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount of 8000-10000/well, selected according to cell size and growth rate) at volume ratio of 5% CO₂Adding topotecan and 17 beta-estradiol with different concentration gradients after culturing for 24 hours in an incubator at 37 ℃, wherein the volume ratio is 5 percent CO₂And incubated at 37 ℃ for 48 hours in an incubator. The medium was discarded, washed once with PBS, and then 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well in a volume ratio of 5% CO₂And incubated at 37 ℃ for 4 hours in an incubator. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm. The influence of the single topotecan and the 17 beta-estradiol on the proliferation of the MCF7 cells is calculated according to the absorbance value at the wavelength, and the result shows that both the topotecan and the 17 beta-estradiol have the inhibition effect on the MCF7 cells, the half lethal concentration of the single topotecan on the MCF7 after 48 hours is 10.86 +/-2.81 mu M, and the half lethal concentration of the 17 beta-estradiol on the MCF7 after 48 hours is 29.21 +/-2.10 mu M, which is detailed in attached figures 1 and 2.

(2) The MTT method examined the effect of 6.25 μ M17 β -estradiol in combination with different concentrations of topotecan on MCF7 cell proliferation: MCF-7 cells are placed in DMEM high-sugar medium containing 10% fetal calf serum, 100kU/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO in volume ratio₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount of 8000-10000/well, selected according to cell size and growth rate) at volume ratio of 5% CO₂After 24 hours of culture in an incubator at 37 ℃, a drug combination of 17 beta-estradiol with the final concentration of 6.25 mu M and topotecan with the final concentration of 3.13-25 mu M is added, and the volume ratio is 5 percent CO₂And incubated at 37 ℃ for 48 hours in an incubator. The medium was discarded, washed once with PBS, and then 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well in a volume ratio of 5% CO₂And incubated at 37 ℃ for 4 hours in an incubator. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm. The combined effect of 6.25 μ M17 β -estradiol and topotecan was calculated based on the absorbance value at that wavelength and the calculation of the combination index. The results show that 6.25 μ M17 β -estradiol and 3.13-25 μ M topotecan act in combination on MCF7 cells for 48 hours, as shown in figure 3, with a combination index CI of 0.35-0.75, both less than 1, where the two drugs act synergistically.

Example 2

The embodiment provides an anti-breast cancer pharmaceutical composition, which comprises the following specific steps:

(1) MTT measures the effect of topotecan and 17 β -estradiol on MCF7 cell proliferation: MCF-7 cells are placed in DMEM high-sugar medium containing 10% fetal calf serum, 100kU/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO in volume ratio₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount 8000-10000/well, selected according to cell size and growth rate) with 5% CO in a volume of 100 μ L per well₂Adding topotecan and 17 beta-estradiol with different concentration gradients after culturing for 24 hours in an incubator at 37 ℃, wherein the volume ratio is 5 percent CO₂And incubated at 37 ℃ for 48 hours in an incubator. The medium was discarded, washed once with PBS, and then 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well in a volume ratio of 5% CO₂And incubated at 37 ℃ for 4 hours in an incubator. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm. The influence of the single topotecan and the 17 beta-estradiol on the proliferation of the MCF7 cells is calculated according to the absorbance value at the wavelength, and the result shows that both the topotecan and the 17 beta-estradiol have the inhibition effect on the MCF7 cells, the half lethal concentration of the single topotecan on the MCF7 after 48 hours is 10.86 +/-2.81 mu M, and the half lethal concentration of the 17 beta-estradiol on the MCF7 after 48 hours is 29.21 +/-2.10 mu M, which is detailed in attached figures 1 and 2.

(2) The MTT method measures the effect of 12.5 μ M17 β -estradiol in combination with different concentrations of topotecan on MCF7 cell proliferation: MCF-7 cells are placed in DMEM high-sugar medium containing 10% fetal calf serum, 100kU/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO in volume ratio₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount of 8000-10000/well, selected according to cell size and growth rate) at volume ratio of 5% CO₂After 24 hours of culture in an incubator at 37 ℃, a drug combination of 17 beta-estradiol with the final concentration of 12.5 mu M and topotecan with the final concentration of 0.78-25 mu M is added, and the volume ratio is 5 percent CO₂And incubated at 37 ℃ for 48 hours in an incubator. The medium was discarded, washed once with PBS, and then 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well in a volume ratio of 5% CO₂And incubated at 37 ℃ for 4 hours in an incubator. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm. The effect of combining 12.5 μ M17 β -estradiol and topotecan was calculated based on the absorbance at that wavelength and the calculation of the combination index. The results show that 12.5 μ M17 β -estradiol and 0.78-25 μ M topotecan combined on MCF7 cells for 48 hours are detailed in figure 3, where the combination index CI is 0.15-0.56, both less than 1, where the two drugs are synergistic.

Example 3

The embodiment provides an anti-breast cancer pharmaceutical composition, which comprises the following specific steps:

(1) MTT measures the effect of topotecan and 17 β -estradiol on MCF7 cell proliferation: MCF-7 cells are placed in DMEM high-sugar medium containing 10% fetal calf serum, 100kU/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO in volume ratio₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount of 8000-10000/well, selected according to cell size and growth rate) at volume ratio of 5% CO₂Adding topotecan and 17 beta-estradiol with different concentration gradients after culturing for 24 hours in an incubator at 37 ℃, wherein the volume ratio is 5 percent CO₂And incubated at 37 ℃ for 48 hours in an incubator. The medium was discarded, washed once with PBS, and then 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well in a volume ratio of 5% CO₂And incubated at 37 ℃ for 4 hours in an incubator. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm. The influence of the single topotecan and the 17 beta-estradiol on the proliferation of the MCF7 cells is calculated according to the absorbance value at the wavelength, and the result shows that both the topotecan and the 17 beta-estradiol have the inhibition effect on the MCF7 cells, the half lethal concentration of the single topotecan to the MCF7 after 48 hours is 10.86 +/-2.81 mu M, and the half lethal concentration of the single estradiol to the MCF7 after 48 hours is 29.21 +/-2.10 mu M, which are detailed in attached figures 1 and 2.

(2) The MTT method measures the effect of 25 μ M17 β -estradiol in combination with different concentrations of topotecan on MCF7 cell proliferation: MCF-7 cells are placed in DMEM high-sugar medium containing 10% fetal calf serum, 100kU/L penicillin and 100mg/L streptomycin at 37 ℃ and 5% CO in volume ratio₂Culturing under the conditions of (1). After digesting the cells with pancreatin, the cells were counted using a hemocytometer. Inoculating to 96-well cell culture plate (cell inoculation amount of 8000-10000/well, selected according to cell size and growth rate) at volume ratio of 5% CO₂And after 24 hours of culture in an incubator at 37 ℃, adding a pharmaceutical composition of 17 beta-estradiol with a final concentration of 25 mu M and topotecan with a final concentration of 0.78-25 mu M, wherein the volume ratio of the pharmaceutical composition to the topotecan is 5% CO₂And incubated at 37 ℃ for 48 hours in an incubator. The medium was discarded, washed once with PBS, and then 100. mu.L of serum-free medium containing 0.5mg/mL MTT was added to each well in a volume ratio of 5% CO₂And incubated at 37 ℃ for 4 hours in an incubator. The medium in the wells of the 96-well plate was aspirated and then 100. mu.L of dimethyl sulfoxide was added to each well. And detecting by using a microplate reader, wherein the detection wavelength is 570nm, and the reference wavelength is 630 nm. The combined effect of 25 μ M17 β -estradiol and topotecan was calculated based on the absorbance value at that wavelength and the calculation of the combination index. The results showed 25. mu.The results of 48 hours of combined action of M17 β -estradiol and 0.78-25 μ M topotecan on MCF7 cells are shown in figure 3, where the combination index CI is 0.12-0.62, both less than 1, where the two drugs act synergistically.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.