阅读说明：本技术 Gl-v9与蒽环类抗生素在制备白血病治疗药物中的应用 (Application of GL-V9 and anthracycline antibiotics in preparation of leukemia treatment drug ) 是由 郭青龙 惠慧 李慧 于 2021-02-07 设计创作，主要内容包括：本发明公开了黄酮类衍生物GL-V9与蒽环类抗生素在制备白血病治疗药物中的应用,属于医药技术领域。本发明发现黄酮类衍生物GL-V9联合低剂量的蒽环类抗生素能显著抑制急性髓系白血病(AML)细胞的生长,通过MTT实验以及U937细胞裸鼠移植瘤模型验证了GL-V9与低剂量的蒽环类抗生素可发挥协同抗AML作用,提示这种联合给药方式在治疗AML中的应用潜力。(The invention discloses application of a flavonoid derivative GL-V9 and an anthracycline antibiotic in preparation of a leukemia treatment drug, belonging to the technical field of medicines. The invention discovers that the growth of Acute Myeloid Leukemia (AML) cells can be remarkably inhibited by combining flavonoid derivative GL-V9 with low-dose anthracycline, and the GL-V9 and the low-dose anthracycline can play a synergistic AML-resisting effect through an MTT (methyl thiazolyl tetrazolium) experiment and a U937 cell nude mouse transplantation tumor model, thereby prompting the application potential of the combined administration mode in the treatment of AML.)

Application of GL-V9 and anthracycline antibiotics in preparation of leukemia treatment medicines.

2. Use according to claim 1, characterized in that: the leukemia is acute myelocytic leukemia AML.

3. Use according to claim 1, characterized in that: the anthracycline antibiotic is daunorubicin.

4. A leukemia treating drug, which is characterized in that: the medicine comprises GL-V9 and an anthracycline, wherein the anthracycline is daunorubicin.

5. The leukemia therapy drug according to claim 4, wherein: the leukemia is acute myelocytic leukemia AML.

6. The leukemia therapy drug according to claim 4, wherein: the concentration of GL-V9 in the treatment drug is 2-4 [ mu ] M, and the concentration of daunorubicin is 0.1 nM.

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of GL-V9 and anthracycline antibiotics in preparation of a leukemia treatment medicine.

Background

Leukemia is a malignant clonal disease of hematopoietic stem cells, commonly known as leukemia. Leukemia cells have the characteristics of uncontrolled proliferation, abnormal differentiation, blocked apoptosis and the like, so that the leukemia cells can proliferate and accumulate in bone marrow and other hematopoietic tissues without limit, gradually replace the normal hematopoietic function, lead erythrocytes, leukocytes and the like not to generate and play a role normally, and infiltrate other non-hematopoietic tissues and organs, thereby causing a series of symptoms, multi-organ failure and even death. Leukemia patients clinically see different degrees of anemia, hemorrhage, enlargement of liver, spleen, lymph nodes, fever due to infection and bone pain.

The flavonoid GL-V9 is a derivative of structurally optimized wogonin, and is prepared by two-step reaction of wogonin in acetone and K₂CO₃Under the reaction condition, 1, 4-dibromobutane is used for replacing phenolic hydroxyl on C7 position of wogonin, and then pyrrolidine is used for replacing bromine in the product in the previous step, so that GL-V9 is finally obtained.

At present, the leukemia therapy mainly comprises chemotherapy, radiotherapy, immunotherapy, hematopoietic stem cell transplantation and the like, and the combined application of GL-V9 and other medicines for treating the leukemia has not been reported.

Disclosure of Invention

The invention aims to provide application of GL-V9 and anthracycline antibiotics in preparation of leukemia treatment medicines.

Application of GL-V9 and anthracycline antibiotics in preparing medicines for treating leukemia.

Further, the anthracycline antibiotic is daunorubicin.

Further, the leukemia is acute myeloid leukemia AML.

A leukemia treating medicine comprises GL-V9 and anthracycline, wherein the anthracycline is daunorubicin.

Further, the leukemia is acute myeloid leukemia AML.

Further, the concentration of GL-V9 in the therapeutic agent is 2-4 μ M, and the concentration of daunorubicin is 0.1 nM.

The leukemia treating medicine can also comprise acceptable auxiliary materials.

The acceptable adjuvant can be excipient, filler, adhesive, humectant, absorption enhancer or lubricant.

The leukemia treating medicine can be prepared into various dosage forms by adopting the conventional method in the field, including decoction, tablets, capsules, injection, powder injection, granules, medicinal granules, oral liquid, syrup and the like.

The invention finds that the flavonoid derivative GL-V9 can obviously inhibit the growth of Acute Myeloid Leukemia (AML) cells by combining with low-dose anthracycline antibiotics. The application potential of the combined administration mode in treating AML is suggested by verifying that GL-V9 and low-dose anthracycline antibiotics can play a synergistic effect on AML through an MTT (methyl thiazolyl tetrazolium) experiment and a U937 cell nude mouse transplantation tumor model.

Drawings

FIG. 1 shows the effect of GL-V9 in combination with daunorubicin on the growth of AML cell line Kasumi-1 in example 1.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

The structural formula of the flavonoid derivative GL-V9 is shown as the following formula:

example 1

Effect of GL-V9 in combination with daunorubicin on human AML cell growth

1. Reagent

(1)GL-V9(C₂₄H₂₇O₅N, molecular weight: 409.47) was supplied by university of Chinese pharmacy in the form of pale yellow powder with a purity of greater than 99%, and the powder was prepared as a 0.02M stock solution with dimethyl sulfoxide (DMSO) prior to use and stored at-20 ℃. The daunorubicin injection is provided by Nanjing drugstore hospital and is preserved in dark. All reagents were prepared to the desired concentration just before use in RPMI-1640 medium containing 10% fetal bovine serum.

(2) Cell culture reagent

Culture solution: RPMI-1640 medium, purchased from GIBCO, USA. Dissolving RPMI-1640 powder 10.39g in 1000mL of sterilized triple distilled water, and 2.0g of NaHCO was added₃Adjusting pH to 7.0 with 1M hydrochloric acid, filtering with cylindrical filter, sterilizing, packaging, and storing in refrigerator at 4 deg.C. Before use, 100U/mL penicillin and 100U/mL streptomycin are added.

② fetal bovine serum: product of GIBCO, usa. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.

③ PBS buffer solution: weighing 8.0g of NaCl, 0.20g of KCl and Na₂HPO₄·H₂O 1.56g、KH₂PO4.2.0g, dissolved in 1000mL of triple distilled water, autoclaved, and stored in a refrigerator at 4 ℃.

(3) Cell growth activity inhibition detection related reagent

MTT powder was purchased from Sigma-Aldrich.

2. Cell line

Human AML cell line Kasumi-1 was purchased from Shanghai national academy of sciences cell. Cells were cultured in RPMI1640 medium containing 100U/mL penicillin, 100mg/mL streptomycin and 10% fetal bovine serum.

3. Experimental methods

MTT solution can be reduced into blue-violet crystal formazan by mitochondrial dehydrogenase in living cells, and the DMSO can dissolve the formazan, and the color shade of the formed solution is in direct proportion to the cell activity, so that the cell activity can be detected. Culturing cells in a 96-well enzyme label plate according to a proper cell density, wherein the volume of the cells in each well is 100 mu L, and meanwhile, 100 mu L of GL-V9 and daunorubicin with specified concentration are added into the cells; continuously placing the cells to which the medicine is administered in an incubator for 24 hours, and adding 20 mu L of MTT solution into each hole; after further incubation for 4h, the supernatant was removed, 100. mu.L DMSO was added to each well, the mixture was shaken in a micro-shaker, and after the crystals were completely dissolved, the absorbance was measured at 570 nm. And (3) after the detected light absorption value is finished, calculating the growth inhibition rate of the drug to the cells according to the following formula:

the inhibitory effect of the natural product derivative GL-V9 combined with daunorubicin on the growth of the AML cell line Kasumi-1 at the 24h time point was determined by MTT assay. As shown in FIG. 1, GL-V9 combined with daunorubicin significantly inhibited the growth of AML cells, Kasumi-1 cells, as compared to the daunorubicin alone group, and this result preliminarily confirmed the effect of GL-V9 combined with chemotherapeutic agents on AML.

Example 2

Effect of GL-V9 in combination with daunorubicin on U937 cell nude mouse transplantable tumors

1. Test drug

(1)GL-V9(C₂₄H₂₇O₅N, molecular weight: 409.47) is provided by university of Chinese pharmacy, and has light yellow powder with purity of more than 99%, and is stored at room temperature after jet milling. When in administration, the medicine powder is weighed, suspended by 0.5 percent CMC-Na solution for use, and is administrated by intragastric administration according to the weight of 0.2mL/20 g.

(2) Daunorubicin hydrochloride for injection is provided by Nanjing drugstore Hospital and is stored in the dark. Before use, the required concentration is prepared by normal saline, and the injection is administered by tail vein.

2. Cell line

Human AML cell line U937 was purchased from shanghai academy of sciences cell. The cells used were cultured in RPMI1640 medium containing 100U/mL penicillin, 100mg/mL streptomycin and 10% fetal bovine serum.

3. Laboratory animal

Source, species, strain: BALB/c nude mice, obtained from Kyowa Kavens laboratory animals Co., Ltd. (laboratory animal production license: SCXK (threo) 2016-0010). The animal is bred in SPF animal house of the center of Chinese university of pharmacy: the temperature is 23 +/-2 ℃, and the relative humidity is 55 +/-5%.

The week age is as follows: 6w

Weight: 16-18g

Sex: and (4) female.

4. Experimental methods

Establishment of U937 cell nude mouse subcutaneous transplantation tumor model

Taking a human AML cell U937 cell strain in logarithmic growth phase, preparing into 5 × 10 cells under aseptic condition⁶Per mL cell suspension, mixed with Matrigel gel 1: 1, and then inoculated subcutaneously into the right armpit of a nude mouse at 0.1 mL.Administration was started on day 6 post-inoculation, and nude mice were randomly divided into 4 groups of 6 mice each: (1) a saline control group; (2) the positive medicine daunorubicin group (1.5mg/kg) is administrated 1 time every 2 days; (3) GL-V9 group (300mg/kg), administered by intragastric administration every day; (4) GL-V9(300mg/kg) in combination with daunorubicin (1.5 mg/kg). The dosing volume was 0.2mL/20g, the dosing cycle was 14 days, the mice were sacrificed and the tumor mass was surgically removed and weighed.

The tumor inhibition rate calculation formula is as follows:

T_weight: mean tumor weight in treatment group; c_weight: negative control group mean tumor weight.

The results of experimental treatment of the drug GL-V9, the low dose positive drug daunorubicin, and combinations of the two on human AML cell U937 nude mouse transplantable tumors are shown in Table 1. The experimental results are as follows, the drug GL-V9 is administrated by gavage at 300mg/kg, and the administration is carried out every day, and after 14 days of administration, the tumor inhibition rate of the transplanted tumor of the human AML cell U937 nude mouse reaches 60.12%. Daunorubicin is administered by intravenous injection at 1.5mg/kg 1 time every 3 days, and after 5 times of total administration, the tumor inhibition rate of the transplanted tumor of human AML cell U937 nude mouse reaches 48.87%. GL-V9 combined with daunorubicin significantly inhibited the growth of AML cell U937, and the tumor inhibition rate of human AML cell U937 nude mouse transplanted tumor reached 73.96%. This result preliminarily confirms the effect of GL-V9 in combination with chemotherapeutic drugs on AML.

TABLE 1 inhibitory Effect of the drug GL-V9 on the growth of human AML cell U937 nude mouse xenograft tumors (X + -SD)

p₁: comparing with a blank control group; p is a radical of₂: comparison with GL-V9 alone; p is a radical of₃: compared with daunorubicin alone.