CN106924243A - Application of the fraxinellone in antineoplastic sensitizer is prepared - Google Patents

Abstract

The invention belongs to the fields of cell biology and medicine, and relates to the application of arketone in the preparation of antitumor drug sensitizers. Ashenone is a natural product, which has the effect of reversing the multidrug resistance of tumor cells, and can be used as a reversal agent for tumor multidrug resistance; it also has the effect of increasing the sensitivity of tumor multidrug resistant cells to antitumor drugs, and can be used as a chemotherapeutic agent. Sensitizer use. The invention also provides a method for inhibiting tumor multidrug-resistant cell proliferation with the pharmaceutical composition combined with antineoplastic drugs and alkone. The small molecule compound alkone in the present invention is developed as a new antitumor drug or its auxiliary component, has obvious tumor suppressing effect, is environmentally friendly, and will provide a new way and means for treating and curing tumors.

Description

Application of alkone in preparation of antitumor drug sensitizer

technical field

The invention belongs to the field of cell biology and medicine, in particular, the invention relates to a new application of arketone. The invention also relates to the corresponding pharmaceutical composition and its application method.

Background technique

Tumor is an abnormal lesion formed by the body under the action of various carcinogenic factors when a certain cell in a local tissue loses its normal regulation of its growth at the gene level, resulting in its clonal abnormal proliferation. The number of cancer cases in my country is quite large, accounting for 55% of the total number of cases in the world.

Benign tumors have less impact on the body, mainly showing symptoms of local compression and obstruction. Malignant tumors have a serious impact on the body due to their immature differentiation, rapid growth, infiltration and damage to the structure and function of organs, and the possibility of metastasis. In addition to local compression and obstruction symptoms similar to those of benign tumors mentioned above, malignant tumors may also cause fever and intractable pain, and severe emaciation, fatigue, anemia, and systemic exhaustion may appear in the advanced stage.

Traditional Chinese medicine believes that the cause of cancer is the balance of yin and yang in the human body, and the tissue cells are caused by cell mutations under the long-term action of different carcinogenic factors. In fact, cancer tissue is also a part of the human body. Only when the balance of yin and yang is out of balance and the five elements change, the immune monitoring system of the human body will lose its monitoring and let it develop. Chinese herbal medicine can preserve life by regulating qi and blood, adjusting the balance of yin and yang, and maintaining normal vital signs; Therefore, Chinese herbal medicine extracts have become an important direction for the treatment of tumors.

Dictamnusdasy carpus Turcz , a traditional Chinese medicine, is the dried root bark of Rutaceae plant Dictamnusdasy carpus Turcz, which has the functions of expelling wind, dampness, heat and detoxification. Modern pharmacological studies have shown that Cortex radix has various activities such as anti-oxidation, anti-virus, anti-bacteria, anti-inflammation, and anti-tumor. Studies in recent years have shown that the main medicinal components of D. japonicus are the main medicinal components of D. japonicus, which have various strong biological activities. Among them, as a new type of drug for treating liver diseases, arketone has good activities such as protecting the liver, reducing enzymes, inhibiting liver fibrosis, etc., and has antibacterial and insecticidal activities.

Zhu Danni etc. measured the content of arketone in fresh bark by HPLC (high performance liquid chromatography was used to determine the content of arketone in fresh bark, Journal of China Pharmaceutical University, 1998, 29 (4): 319～ 320).

Studies at home and abroad have shown that phrysitine and alkone are the main active substances in the bark of white moss, which have trapping and killing effects on mites, inhibit growth on insects, and have strong poisonous and killing activities on third-instar armyworms. In 2005, Yuan Chunlan et al. further reported the insecticidal activity of alkone (the insecticidal activity of a new botanical insecticide ketone, Plant Protection, Volume 31, Issue 5). In 2009, Yuan Chunlan et al. conducted a detailed study on the extraction process of alkone from white moss bark, and obtained a simple and practical separation process. Using this process, the yield of alkone reaches 94.7%, which provides an effective method for the industrial separation of alkone (Volume 26, No. 7, July 2009, Extraction process of active substance alkone in white moss bark research, fine chemicals). Li Xiang etc. have also reported the method for extracting alkone from the fresh cortex (the method for simultaneously preparing alkone, white fresh alkali, and cortex ketone monomer from the fresh white peel, Journal of Sichuan University (Engineering Science Edition), V o.l 39 No. 4).

Ashenone is a natural product with high bioavailability and relatively stable properties, and has clinical application value. With the deepening of its chemical and biological research, its molecular mechanism of action will be gradually clarified, which will further promote the chemical structure modification and structure-activity relationship research of this kind of compound, and help to improve the medicinal value of Baixian .

Contents of the invention

The purpose of the present invention is to provide a new pharmaceutical application of arketone.

The object of the present invention is to provide an antitumor drug sensitizer.

In one aspect, the present invention provides the use of arketone in the preparation of antitumor drug sensitizers. Among them, the full English name of arketone is Fraxinellone, and its structure is shown in Figure 1.

Said tumors include oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer or pancreatic cancer.

The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

Phenone can be used as a tumor multidrug resistance reversal agent.

In another aspect, the present invention provides an anti-tumor pharmaceutical composition, the active ingredients of which are anti-tumor drugs and alkalone.

The anti-tumor drugs are cell cycle specific drugs or cell cycle non-specific drugs.

The antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil.

The invention also provides a method for inhibiting the growth and proliferation of tumor cells in vitro, that is, adding alkone and antitumor drugs to the culture medium of tumor cells.

Wherein, the final concentration of adding alkone is 1-100 μ mol/L.

The tumor includes oral cancer, breast cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer.

When implementing the above method, first add arketone, and then add antineoplastic drugs. It is also possible to add arketone and antineoplastic drugs at the same time.

The invention provides a new application of arketone, that is, its application in the preparation of antitumor drug sensitizers. Ashenone is a natural product, which has the effect of reversing the multidrug resistance of tumor cells, and can be used as a reversal agent for tumor multidrug resistance; it also has the effect of increasing the sensitivity of tumor multidrug resistant cells to antitumor drugs, and can be used as a chemotherapeutic agent. Sensitizer use. The small molecule compound alkone in the present invention is developed as a new antitumor drug or its auxiliary component, has obvious tumor suppressing effect, is environmentally friendly, and will provide a new way and means for treating and curing tumors.

Description of drawings

Figure 1 is the structure of arketone.

detailed description

The invention provides an antitumor drug, the active ingredient of which is alkone. The tumor can be liver cancer cells, gastric cancer cells, cervical cancer cells or blood cancer cells.

The small molecule compounds of the present invention can be prepared by various conventional preparation methods. For example, the method of artificial chemical synthesis is adopted.

Utilizing the small molecular compound of the present invention, through various conventional screening methods, substances that interact with alkone, such as receptors, inhibitors or antagonists, can be screened out.

When the present invention and its inhibitors, antagonists, etc. are administered (administered) therapeutically, various effects can be provided. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, and the pH value can be formulated according to Depending on the nature of the substance and the condition being treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, subcutaneous, intradermal, or topical administration.

Taking the alkone of the present invention as an example, it can be used in combination with a suitable pharmaceutically acceptable carrier. Such pharmaceutical compositions contain a therapeutically effective amount of the compound and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should match the mode of administration. The alkone of the present invention can be made into an injection form, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example about 1 microgram/kg body weight to about 5 mg/kg body weight per day. In addition, the arketones of the present invention can also be used with other therapeutic agents. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

experimental method:

1. Cell recovery

1) Take out the cryotube from the liquid nitrogen tank, put it directly into warm water at 37°C, and shake it from time to time to melt it as soon as possible.

2) Take out the cryotube from the 37°C water bath, suck out the cell suspension with a pipette, pour into the centrifuge tube and add more than 10 times of culture medium, mix and centrifuge at low speed, discard the supernatant, and wash again with culture medium again.

3) After properly diluting with the culture medium, inoculate the culture bottle, place it in a 37°C incubator for static culture, replace the culture medium the next day, and continue the culture. Subculture when cultured to a certain concentration. MCF-7 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, KB and KB/VCR cells were cultured in MEM medium containing 10% fetal bovine serum, 2mM glutamine and 1mM sodium pyruvate, MCF -7/ADR cells were cultured in MEM medium containing 10% fetal bovine serum, 0.01mg/ml insulin and 1mM sodium pyruvate. The medium contains 100U/ml penicillin and 100μg/ml streptomycin.

2. Subculture of cells

Observe the growth of the cells every day. When the cells grow to about 90% confluence in the culture flask, they should be subcultured every 2-4 days. Passage one bottle into three flasks, or one 25 cm ² into one 75 cm ² culture flask. method:

1) Wash the cells once with 1X phosphate buffer.

2) Add 2-3ml trypsin digestion solution for digestion, and place in a 37°C incubator for several minutes. Tap the cell culture flask with your hands to detach the cells.

3) Terminate trypsinization with appropriate medium containing 10-15% Gibico fetal bovine serum. Aliquot the cells into new culture flasks and continue culturing.

When the suspension cells are subcultured, they are directly collected in a centrifuge tube and centrifuged, and the old medium is discarded. Generally, the ratio of one bottle to three bottles is divided into new culture bottles, and fresh medium is added to continue the culture.

3. Cell cryopreservation

1) Digest the cells cultured to the logarithmic growth phase with trypsin, collect them in a centrifuge tube, count them, and centrifuge.

2) Discard the trypsin and the old culture medium, add the prepared frozen culture medium (containing 10% DMSO, 40% DMEM and 50% Gibico fetal bovine serum), the final concentration of cells in the frozen medium is 0.5-1 ×10 ⁷ /ml. Pipette gently with a pipette to make the cells uniform, then divide into sterile cryopreservation tubes, add 1-1.5ml to each tube.

3) Put the cryopreservation tube into the freezer box and place it at -80°C for quick freezing, and transfer it to a liquid nitrogen tank for storage after 5 hours.

4. Experimental materials

Drug preparation:

Ash ketone was dissolved in DMSO (dimethyl sulfoxide), and prepared into 100mM or 50mM mother solution for later use.

Vincristine (VCR) and doxorubicin (ADR) were purchased from Roche Chemical Company with a purity greater than 99%.

Cell source:

The human oral cancer cell line KB and its drug-resistant cell line KB/VCR were provided by the Chinese Academy of Sciences, and the human breast cancer cell line MCF-7 and its drug-resistant cell line MCF-7/ADR were purchased from Nanjing Kaiji Biotechnology Company.

Reagent test kit:

CCK-8 kit was purchased from Tongren Company.

CCK-8 experiment

KB and KB/VCR cells as well as MCF-7 and MCF-7/ADR cells were seeded into 96-well plates at a density of 3500/well, and after 24 hours, different concentrations of ADR, VCR, alkone, and VCR and alkone were used together α-MEM containing 10% fetal bovine serum was prepared and added to each well. After culturing for 48 h, the culture medium was discarded, and 90 μL of serum-free medium and 10 μL of CCK-8 reagent were added to each well. After reacting at 37°C for 2 h, the absorbance value (OD450) at a wavelength of 450 nm was read with a microplate reader. The cell proliferation ratio of the medication group relative to the control group was obtained by calculation. In the blank group, only the medium was added without cells, and in the control group, DMSO with the same volume as the drug was added, and the cell survival rate = (OD450 of the experimental group-OD450 of the blank group)/(OD450 of the control group-OD450 of the blank group). The resistance fold (Resistance Fold, RF) was calculated based on the IC ₅₀ value.

RF = IC ₅₀ value of drug-resistant cell line/IC ₅₀ value of parental cell line. Three replicate wells were set up for each concentration, and the experiment was repeated 3 times.

All drug-resistant cell lines were grown in drug-free medium for 3 days prior to growth inhibition experiments. Each value is the result of 3 independent experiments, and IC ₅₀ is expressed in the form of "mean ± standard deviation". VCR, vincristine; ADR, daunorubicin; RF, resistance fold.

Example 1

Experimental results:

KB/VCR and MCF-7/ADR are two commonly used multidrug-resistant cell lines, and in this example, these two kinds of cells also exhibit the characteristic of multidrug resistance. As shown in Table 1, compared with KB cells, the multiples of drug resistance of KB/VCR cells to VCR and ADR were 81.9 times and 94.4 times, respectively, while MCF-7/ADR cells were more resistant to VCR and ADR than MCF-7 cells. The drug multiples were 38.1 times and 20.9 times respectively, showing that the drug-resistant cells used in the experiment had multidrug resistance, and the MDR activity was similar to the results reported in the literature.

The half maximal inhibitory concentration _IC50 of arketone to the parental cell lines KB and MCF-7 were 198.6 μM and 191.2 μM, respectively, and the _IC50 to the drug-resistant cell lines KB/VCR and MCF-7/ADR were 264.2 μM and 277.9 μM, respectively, There was no significant difference between the two, and the multidrug-resistant cell lines KB/VCR and MCF-7/ADR did not show cross-resistance to the compound arketone, indicating that arketone can escape the multidrug resistance of drug-resistant cells . At a concentration of 10 μM and below, alkone has no obvious inhibition on cell growth, and at a concentration higher than 100 μM, it can inhibit cell proliferation.

Conclusion: Ashenone can overcome the drug resistance of drug-resistant cell lines, and the sensitivity of drug-resistant cell lines to arketone is basically the same.

Example 2: CCK-8 method detects the reversal effect of alkone on the multidrug resistance activity of tumor cells

KB/VCR, MCF-7/ADR and HCT-8/VCR cells were inoculated into 96-well plates at a density of 3500/well. After 24 h, different concentrations of VCR, and different concentrations of VCR and alkone were used together with 10% α-MEM of fetal bovine serum was prepared and added to each well. After culturing for 48 h, the culture medium was discarded, and the CCK-8 kit was used to measure the activity of the drug-resistant cell line and its parental cells on VCR and VCR + alkone in the same manner as in Example 1, and a curve was drawn. Three replicate wells were set for each concentration, and the experiment was repeated 3 times.

Experimental results:

The multiplicity of drug resistance of KB/VCR cells to VCR was 81.9 times, and that of MCF-7/ADR cells to ADR was 20.9 times. When 10 μM alkone was added, the sensitivity of KB/VCR cells to VCR was not significantly increased, but the sensitivity of ADR to drug-resistant cells MCF-7/ADR was increased by 1.58 times. Phenone can reverse the drug resistance of tumor multidrug resistant cells to chemotherapy drugs. However, this effect was not obvious in the parental cells.

in conclusion:

Phenone can reverse the drug resistance of tumor multi-drug resistant cells KB/VCR and MCF-7/ADR, and can be used as a sensitizer for anti-tumor drugs, or made into a pharmaceutical composition with anti-tumor drugs.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. The application of arketone in the preparation of antitumor drugs, the application of which is the application of arketone in the preparation of antitumor drug sensitizers. 2. The application as claimed in claim 1, characterized in that alkone is a tumor multidrug resistance reversal agent. 3. The application according to claim 1, wherein the tumor comprises oral cavity cancer, nasopharyngeal cancer, breast cancer, liver cancer, lung cancer, cervical cancer, colon cancer or pancreatic cancer. 4. The application according to claim 1, wherein the antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil. 5. An anti-tumor pharmaceutical composition, characterized in that the active ingredients of the anti-tumor pharmaceutical composition are anti-tumor drugs and alkone. 6. The antitumor drug composition according to claim 5, wherein the antitumor drug is a cell cycle-specific drug or a cell cycle non-specific drug. 7. The antitumor drug composition according to claim 5, wherein the antitumor drug is vincristine, daunorubicin, paclitaxel or 5-fluorouracil. 8. A method for inhibiting the growth and proliferation of tumor cells in vitro, characterized in that alkalone and antitumor drugs are added to the medium of tumor cells. 9. The method according to claim 8, wherein the tumor comprises oral cancer, breast cancer, nasopharyngeal cancer, liver cancer, lung cancer, cervical cancer or pancreatic cancer. 10. The method according to claim 8, characterized in that first adding arketone and then adding antineoplastic drugs.