New application of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione

Abstract

The present invention discloses a new use of androsterone-4,6,8(9),13(14)-tetraene-3,11,16-trione, namely, its use in preparing a drug for treating acute lymphocytic leukemia. The present invention adopts an acute lymphocytic leukemia cell line (T line, B line) and detects the IC value of androsterone-4,6,8(9),13(14)-tetraene-3,11,16-trione in an ALL cell line by a CCK-8 method. 50 The present invention studies the cytotoxicity of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione to human umbilical vein fusion cells Eahy.926 and rat normal liver cells BRL-3A. Flow cytometry technology shows that the compound mainly inhibits the growth of leukemia cells by inducing apoptosis and blocking the cell cycle. The present invention not only increases the diversity of the biological activity of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione, but also provides a new direction for the research and development of new drugs for the treatment of leukemia.

Description

Technical Field

[0001] The invention relates to application of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione in preparing medicine for treating acute lymphocytic leukemia, belonging to the technical field of medicine. Background Art

[0002] Acute lymphoblastic leukemia (ALL) is a malignant proliferation of lymphoid cells that is blocked in the early stages of differentiation and can invade the bone marrow, blood, and extramedullary sites. During the development of lymphoid cells, abnormal activation of oncogenes and inactivation of tumor suppressor genes can lead to differentiation arrest and uncontrolled proliferation of immature lymphocytes, ultimately leading to the development of acute lymphoblastic leukemia. Acute lymphoblastic leukemia can occur in all age groups and is one of the most common cancers in children. The long-term survival rate of children is reported to be approximately 80%, while the relapse-free survival rate of adults is less than 30%. However, for older patients (>60 years old), the outcome remains poor, with a 5-year overall survival rate of less than 20%.

[0003] Dexamethasone (DEX), a type of glucocorticoid, is a core drug for the treatment of ALL and can achieve clinical remission when used in combination with other chemotherapy drugs. However, when ALL patients are given large doses of steroids, especially with DEX, the risk of side effects increases. Side effects include osteoporosis, cardiovascular disease, Cushing's syndrome, psychiatric illness and mental disorders, and endocrine and metabolic dysfunction. Therefore, it is very important to develop an alternative drug and replace dexamethasone for the treatment of acute lymphoblastic leukemia.

[0004] Androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione is a natural small molecule compound derived from the plant Simiaki truncatula. Its molecular formula is C 19 H 18 O3, with a molecular weight of 294.13, belongs to the class of androstenoid compounds. Epigynumauritum is a species of the Apocynaceae family, distributed in Myanmar and Malaysia. There is only one species of Epigynumauritum in my country, which is native to southern Yunnan. It is a plant with heat-clearing, detoxifying and analgesic effects. As a medicinal plant of the Dai people, it has been recorded as a folk medicine. However, there are few studies on the pharmacological activity of androsten-4,6,8(9),13(14)-tetraene-3,11,16-trione, and no one has proposed its use in the treatment of leukemia so far. Summary of the invention

[0005] Dexamethasone (DEX) is a drug currently used in the clinic to treat acute lymphoblastic leukemia. However, it has certain toxic side effects and is prone to drug resistance. The prevalence and recurrence rates of acute lymphoblastic leukemia (ALL) are very high. Therefore, the development of new safe ALL drugs from natural products has considerable market demand and prospects, and contains huge social and economic effects.

[0006] The present invention provides a new use of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione, namely, use of the same in preparing a drug for treating acute lymphocytic leukemia.

[0007] The chemical structure of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione (DEP-1) is as follows:

[0008]

[0009] The drug component (or active ingredient) for treating acute lymphocytic leukemia described in the present invention is androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione, and one or more pharmaceutically acceptable excipients may be added to improve the absorption effect of the drug or facilitate use, so as to prepare a suitable dosage form, such as capsules or pills, powders, tablets, granules, oral liquids and injections.

[0010] The invention uses an in vitro experiment to first detect the cytotoxicity of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione on human umbilical vein fusion cells Eahy.926 and rat normal liver cells BRL-3A by CCK-8 method, and then detects the proliferation inhibition effect and half inhibitory concentration (IC50) of the compound on acute lymphoblastic leukemia cell lines (T lines: CEM / C1, MOLT-4, Jurkat; B lines: BALL-1, Nalm-6, RS4; 11) at a safe concentration after confirming that the compound has no toxic effect on normal cells at a concentration of 120 μmol / L. 50 ), and then through flow cytometry, it was found that the compound mainly inhibited the growth of leukemia cells by inducing apoptosis of leukemia cells and blocking the cell cycle. Based on the above test results, it is shown that the compound can be used as a candidate drug for the preparation of treatment of acute lymphoblastic leukemia.

[0011] Compared with the prior art, the present invention has the following advantages:

[0012] 1. The present invention has discovered new medical uses for the monomer compound of Simiao Caulis androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione and has opened up a new research field;

[0013] 2. The present invention has found through experiments that the monomer compound of Simiao Caulis androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione can effectively inhibit the proliferation of ALL cell lines at a relatively low dose; the raw materials are derived from natural products and are highly safe;

[0014] 3. The inhibitory effect of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione on the proliferation of ALL cell lines is better than or equivalent to that of DEX. In addition, the separation and purification process of this compound is simple and low-cost, making it suitable for industrial production and market promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The survival rate of normal cells (BRL-3A and Eahy.926) after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0016] Figure 2 The survival rate of T-cell acute lymphoblastic leukemia cell lines (CEM / C1, MOLT-4, Jurkat) after 48 hours of culture in different concentrations of androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone;

[0017] Figure 3 The survival rate of B-lineage acute lymphoblastic leukemia cell lines (BALL-1, Nalm-6, RS4; 11) after 48 hours of culture in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone;

[0018] Figure 4 The changes in mitochondrial membrane potential of the acute lymphoblastic leukemia cell line CEM / C1 after being cultured in different concentrations of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours. The concentrations from left to right are 10, 20, and 40 μmol / L.

[0019] Figure 5 The mitochondrial membrane potential changes of acute lymphoblastic leukemia cell line MOLT-4 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours. The concentrations from left to right are 10, 20, and 40 μmol / L.

[0020] Figure 6The changes in mitochondrial membrane potential of Jurkat acute lymphoblastic leukemia cell line after 48 hours of culture in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone. The concentrations from left to right in the figure are 10, 20, and 40 μmol / L.

[0021] Figure 7 The changes in mitochondrial membrane potential of acute lymphoblastic leukemia cell line BALL-1 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours. The concentrations from left to right are 15, 30, and 60 μmol / L.

[0022] Figure 8 The changes in mitochondrial membrane potential of the acute lymphoblastic leukemia cell line Nalm-6 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours. The concentrations from left to right are 15, 30, and 60 μmol / L.

[0023] Fig. 9 The figure shows the changes in mitochondrial membrane potential of acute lymphoblastic leukemia cell line RS4; 11 was cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours. The concentrations from left to right are 15, 30, and 60 μmol / L.

[0024] Fig.10 This is the cell cycle distribution diagram of the acute lymphoblastic leukemia cell line CEM / C1 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0025] Fig.11 This is a bar graph of cell cycle distribution of acute lymphoblastic leukemia cell line CEM / C1 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0026] Fig.12 This is the cell cycle distribution diagram of acute lymphoblastic leukemia cell line MOLT-4 after being cultured in different concentrations of androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0027] Fig.13 This is a bar graph of cell cycle distribution of acute lymphoblastic leukemia cell line MOLT-4 after being cultured in different concentrations of androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0028] Fig.14 This is the cell cycle distribution diagram of the acute lymphoblastic leukemia cell line Jurkat after being cultured in different concentrations of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0029] Fig.15 This is a bar graph of cell cycle distribution of acute lymphoblastic leukemia cell line Jurkat after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0030] Fig.16 This is the cell cycle distribution diagram of the acute lymphoblastic leukemia cell line BALL-1 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0031] Fig.17 This is a bar graph of cell cycle distribution of acute lymphoblastic leukemia cell line BALL-1 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0032] Fig.18 This is the cell cycle distribution diagram of the acute lymphoblastic leukemia cell line Nalm-6 after being cultured in different concentrations of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0033] Fig.19 The cell cycle distribution bar graph of the acute lymphoblastic leukemia cell line Nalm-6 after being cultured in different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0034] Fig. 20 This is the cell cycle distribution diagram of acute lymphoblastic leukemia cell line RS4;11 after being cultured in different concentrations of androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0035] Fig.21 This is a bar graph of the cell cycle distribution of the acute lymphoblastic leukemia cell line RS4; 11 after being cultured in different concentrations of androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone for 48 hours;

[0036] In the figure, Compound or DEP-1 is androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione, and DEX is dexamethasone. DETAILED DESCRIPTION

[0037] The androstane-4,6,8(9),13(14)-tetraene-3,11,16-trione used in the examples of the present invention is prepared according to the existing plant chemical separation and purification technology. The present invention is further described below in combination with the data and drawings of the experimental examples. These experimental examples are only used to illustrate the present invention and are not used to limit the scope of application of the present invention. After reading the description of the present invention, various equivalent changes, modifications and modifications made by scientific and technological personnel in this field are all within the scope defined by the claims of the present invention. The reagents used in the examples, unless otherwise specified, are all conventional commercial products or reagents prepared by conventional methods. The methods in the examples, unless otherwise specified, are all conventional experimental methods.

[0038] Dexamethasone in the examples was purchased from Dalian Meilun Biotechnology Co., Ltd.; cell counting (CCK-8) and JC-1 mitochondrial membrane potential detection kits were purchased from Shanghai Baisai Biotechnology Co., Ltd.; cell cycle detection kits were purchased from Beijing Sizhengbai Biotechnology Co., Ltd. RPMI1640 medium, DMEM medium, fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin and streptomycin were all purchased from Gibco; DMSO was purchased from Beijing Solaibao Biotechnology Co., Ltd.

[0039] Example 1: Cell activity detection of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione on acute lymphoblastic leukemia cell lines

[0040] 1. Preparation of experimental materials

[0041] Solution preparation: Androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone were prepared into 30 mM stock solution with cell-grade DMSO under sterile conditions, sealed and stored in a -20°C refrigerator away from light for later use. In the experiment, the stock solution was diluted into sample solutions of different concentrations according to the final concentration required after drug addition.

[0042] Cell lines: human acute lymphoblastic leukemia cell lines (T lines: CEM / C1, MOLT-4, Jurkat; B lines: BALL-1, Nalm-6, RS4; 11), rat normal liver cells BRL-3A and human umbilical vein fusion cells Eahy.926;

[0043] CEM / C1, MOLT-4, Jurkat, BALL-1, BRL-3A and Eahy.926 were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences, and NALM-6 and RS4;11 cell lines were purchased from Guangzhou Saiku Biotechnology Co., Ltd. Human acute lymphoblastic leukemia cell lines were cultured in RPMI1640 medium containing 10% fetal bovine serum + 1% penicillin and streptomycin, and normal cell lines were cultured in DMEM medium containing 10% fetal bovine serum + 1% penicillin and streptomycin in a 37°C, 5% CO2 incubator for later use.

[0044] 2. Experimental methods

[0045] Normal cells in the logarithmic growth phase were taken and prepared with complete culture medium at a concentration of 1×10 5 The cell suspension of 100 μg / mL was inoculated in a 96-well plate, 200 μL per well. The experiment was divided into a drug group and a negative control group, and 5 replicate wells were set for each group (or each drug concentration). After 24 hours of culture, the drug group replaced the original culture medium with 200 μL of culture medium containing different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone (drug solution concentrations of 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L and 120 μmol / L), and the negative control group replaced the original culture medium with 200 μL of fresh complete culture medium. After 22 hours of continuous culture, 20 μL of CCK-8 reagent was added to each well, and the cells were returned to the incubator for further incubation for 4 hours. The absorbance value (OD value) of each well was measured at a wavelength of 490 nm using an ELISA reader, and the survival rate of tumor cells at different concentrations of drugs was calculated according to the formula: survival rate = (OD value of drug group / OD value of negative control group) × 100%. The results are shown in Figure 1 ;

[0046] Different ALL cell lines in exponential growth phase were cultured at 5×10 4 The cells were inoculated at a density of 10 μmol / L, 20 μmol / L, 40 μmol / L, 80 μmol / L and 120 μmol / L in the drug treatment group, and an equal volume of complete culture medium was added to the negative control group. The total volume of each well was 200 μL. Five replicate wells were set for each group (or each concentration). After 44 h of culture, 20 μL of CCK-8 solution was added to each well. After further culture for 4 h, the absorbance of each well at a wavelength of 490 nm was detected by an enzyme reader, and the survival rate and IC 50 Value, survival rate = (OD value of drug group / OD value of negative control group) × 100%. Graphpad Prism8.0 software was used to calculate the half inhibitory concentration (IC 50 ), IC 50 The values ​​are shown in the table below, and the survival rates are shown in Figure 2-3 ;

[0047]

[0048] The above results show that at a drug concentration of 120 μmol / L, androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone had no damaging effect on normal cells. In addition, androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione significantly reduced the viability of ALL cells in a dose-dependent manner within 48 h; the half inhibitory concentration (IC50) of CEM / C1, MOLT-4, Jurkat, BALL-1, NALM-6, RS4; 11 50 The IC values ​​of dexamethasone for all cells except RS4;11 cells were 20.65, 36.06, 23.45, 28.67, 33.12, and 32.92 μmol / L, respectively. 50 The values ​​were all higher than those of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione. In conclusion, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione was more sensitive to CEM / C1, MOLT-4, Jurkat, BALL-1 and NALM-6 cells than DEX, but was less sensitive to RS4;11 cells than DEX.

[0049] Example 2: Flow cytometry detection of the effects of different concentrations of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone on mitochondrial membrane potential and cell cycle distribution of acute lymphoblastic leukemia cell lines

[0050] 1. Mitochondrial membrane potential detection

[0051] ALL cell lines in exponential growth phase were cultured at 5×10 5 The number of cells was inoculated in a 6-well plate, and different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone solution were added (T-ALL: 10, 20, 40 μmol / L; B-ALL: 15, 30, 60 μmol / L). The negative control group was added with an equal volume of culture medium. After 48 hours of culture at 37°C and 5% CO2 incubator, the culture was terminated, and the cells were collected by centrifugation at 3000 rpm for 5 minutes. The cell pellet was washed once with cold PBS. 0.5 ml of JC-1 dye solution was added and incubated at 37°C for 15 minutes. After that, the cells were collected by centrifugation at 3000 rpm for 5 minutes, and the cell pellet was washed twice with cold PBS, and then gently blown with 0.5 ml of PBS. The mitochondrial membrane potential of ALL cells was analyzed by flow cytometry.

[0052] The results are as follows Figure 4-9As shown in the figure, the mitochondrial membrane potential was detected by JC-1 fluorescent probe. When the membrane potential was high, it was red fluorescence (R2), and when the membrane potential was low, it produced green fluorescence (R3). The decrease in the red / green fluorescence ratio (R2 / R3) reflects the decrease in mitochondrial membrane potential, which is a manifestation of early cell apoptosis. Compared with the control group, the green fluorescence (R3) of BALL-1 and NALM-6 cells increased, and the red / green fluorescence ratio (R2 / R3) was significantly reduced in a dose-dependent manner. In addition, the dexamethasone treatment group significantly reduced the red / green fluorescence ratio of CEM / C1 and RS4;11 cells. However, for MOLT-4 cells and Jurkat cells, the flow cytometry results showed that the compounds and dexamethasone had no obvious effect on the change of the red / green ratio. The results showed that the toxicity of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione to B-lineage acute lymphoblastic leukemia cells may be related to the apoptosis mediated by the mitochondrial pathway, while its toxicity to T-lineage acute lymphoblastic leukemia cells was not related to the apoptotic pathway.

[0053] 2. Cell cycle distribution

[0054] ALL cell lines in exponential growth phase were cultured at 5×10 5 Cells were seeded in 6-well plates, and different concentrations of androst-4,6,8(9),13(14)-tetraene-3,11,16-trione and dexamethasone solution (T-ALL: 10, 20, 40 μmol / L; B-ALL: 15, 30, 60 μmol / L) were added. The negative control group was added with an equal volume of culture medium. After 48 hours of culture in a 37°C, 5% CO2 incubator, the culture was terminated, and the cells were collected by centrifugation at 3000 rpm for 5 minutes. The cell pellets were washed twice with cold PBS and fixed with 75% ethanol at 4°C for 24 hours; the cells were collected by centrifugation at 3000 rpm for 5 minutes, and the cell pellets were washed with pre-cooled PBS. The cells were suspended in PI+RNase A staining buffer and stained at 37°C for 30 minutes. The cells were collected by centrifugation at 3000 rpm for 5 minutes, and the cell pellets were gently blown with 0.5 mL PBS, and then the cell cycle was analyzed by flow cytometry.

[0055] The results are as follows Figure 10-21As shown in the results, androst-4,6,8(9),13(14)-tetraene-3,11,16-trione caused different cell cycle arrests in different cells. For MOLT-4 cells, at 40 μmol / L, the cell cycle was arrested in the G0 / G1 phase and the G2 / M phase was reduced. For BALL-1 cells, the percentage of cells in the G2 / M phase increased, while the percentage of cells in the G0 / G1 phase decreased in a dose-dependent manner. After CEM / C1, MOLT-4, NALM-6 and RS4;11 cells were treated with dexamethasone, the number of cells in the G0 / G1 phase increased significantly, while the number of cells in the G2 / M phase decreased significantly. The results indicate that the inhibition of MOLT-4 and BALL-1 cell proliferation by androst-4,6,8(9),13(14)-tetraene-3,11,16-trione may be related to cell cycle arrest.

[0056] In summary, androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione has an inhibitory effect on acute lymphoblastic leukemia cell lines, and the higher the concentration, the better the inhibitory effect, indicating that it has potential value in the preparation of new drugs for the treatment of leukemia.

Claims

1. Application of androsta-4,6,8(9),13(14)-tetraene-3,11,16-trione in the preparation of drugs for the treatment of acute lymphocytic leukemia.

Images