Use of camptothecin A in the preparation of a medicament for treating ovarian cancer

Abstract

The application discloses application of a bruceine A in preparation of a medicine for treating ovarian cancer and belongs to the technical field of cancer medicines. The embodiment of the application proves the application of the bruceine A in preparation of the medicine for treating the ovarian cancer, and the dosage of the bruceine A is 200 mg / kg. The embodiment of the application proves that the bruceine A has a significant proliferation inhibiting effect on human ovarian cancer cells; the soft agar colony formation of the human ovarian cancer cells is significantly lower than that of a control group; the bruceine A significantly inhibits the invasion ability of the human ovarian cancer cells; the bruceine A can significantly reduce the volume and weight of the ovarian cancer tumor after being administered at a dosage of 200 mg / kg, has a significant inhibiting effect on the ovarian cancer tumor, and can be applied in preparation of the medicine for treating the ovarian cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cancer drugs, and particularly relates to application of bruceine A in preparation of a drug for treating ovarian cancer. BACKGROUND

[0002] Ovarian cancer refers to a malignant tumor disease occurring in the ovary, and a malignant tumor originating from the fallopian tube and peritoneum has similar clinical characteristics and treatment modes to ovarian cancer and is often also collectively referred to as ovarian cancer. Ovarian cancer has various histological types, mainly of epithelial origin. Ovarian cancer can occur at any age, and different histological types of tumors have different age groups, for example, germ cell tumors are most common in women under 20 years old, borderline tumors are most common in women aged 30-40 years old, and overall most ovarian cancers occur in women over 50 years old. Early lesions of ovarian cancer are often asymptomatic and difficult to detect, and the symptoms of late lesions are atypical. The treatment adopts a comprehensive treatment scheme mainly based on surgery, and the recurrence rate after treatment is high. The prognosis is related to tumor pathological types, stages, treatment schemes, patient individuals and various factors. Ovarian cancer is prone to metastasis and recurrence, and the factors affecting the recurrence of patients are various, including: tumor stage, histological type, size of residual lesions after surgery, chemotherapy scheme, age, general physical condition and the like. At present, the overall prognosis of ovarian cancer is poor, and mainly relies on comprehensive treatment such as postoperative radiotherapy, chemotherapy and targeted therapy. In recent years, researchers have tried to find active ingredients that can effectively inhibit malignant tumors from natural products for the treatment of ovarian cancer, but no suitable natural product ingredient that can effectively inhibit ovarian cancer has been found.

[0003] Bruceine A (BRA) is also called bruceine A, which is a quassinoid compound extracted and separated from the fruits of the plant Bruceine javanica (Linnaeus) Merrill. According to traditional Chinese medicine, bruceine is bitter and cold in nature, has small toxicity, belongs to the large intestine and liver meridian, and has the effects of clearing heat, detoxifying, stopping malaria, stopping diarrhea and corroding tumors. In some areas, bruceine is often used as an antimalarial drug, and bruceine oil emulsion is often used for the adjuvant treatment of lung cancer and lung cancer brain metastasis. At present, the research on the monomer components in bruceine mainly focuses on bruceine D, but there is no experimental data on whether the monomer component BRA in bruceine has an anti-tumor effect. SUMMARY

[0004] The application provides application of bruceine A in preparation of a drug for treating ovarian cancer, and aims to solve the problem of lack of effective natural product drug treatment for ovarian cancer, and researches a new natural product drug with high activity and small toxicity for treating ovarian cancer.

[0005] The embodiment of the application is implemented as follows:

[0006] The human ovarian cancer cell experiment result of the embodiment of the present application shows that the camptothecin A has significant proliferation inhibition effect on human ovarian cancer SKOV3, A2780 and Anglne cells; the soft agar colony formation of SKOV3 cells in each dose group (50nM / L and 200nM / L) of the camptothecin A is significantly lower than that of the solvent (DMSO) control group; each dose group (50nM / L and 200nM / L) of the camptothecin A significantly inhibits the invasion ability of SKOV3 cells.

[0007] The drug efficacy experiment result of the nude mouse subcutaneous transplantation of the ovarian cancer cell line SKOV3 tumor model of the embodiment of the present application shows that the camptothecin A can significantly reduce the volume and weight of the ovarian cancer tumor after being administered at a dose of 200mg / kg, and has significant in-vivo anti-tumor activity effect.

[0008] It is proved by the embodiment of the present application that the camptothecin A can be applied in the preparation of a drug for treating ovarian cancer.

[0009] The molecular formula of the camptothecin A is C 26 H 34 O 11 ; the molecular weight is 522.5416, and the structural formula is shown as formula I:

[0010]

[0011] The application of the above camptothecin A in the preparation of a drug for treating ovarian cancer can be administered alone or in combination therapy.

[0012] The combination therapy includes radiotherapy, chemotherapy and surgical treatment.

[0013] The camptothecin A can be in any pharmaceutically acceptable dosage form.

[0014] The camptothecin A can be in any pharmaceutically acceptable dose. Preferably, the amount of the camptothecin A is within 200mg / kg, preferably 200mg / kg.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] 1. The camptothecin A has significant proliferation inhibition effect on human ovarian cancer cells.

[0017] 2. The soft agar colony formation of the camptothecin A on human ovarian cancer cells is significantly lower than that of the control group, and the camptothecin A significantly inhibits the invasion ability of human ovarian cancer cells.

[0018] 3. The camptothecin A can significantly reduce the volume and weight of the ovarian cancer tumor after being administered at a dose of 200mg / kg, has significant inhibition effect on ovarian cancer tumor, and can be applied in the preparation of a drug for treating ovarian cancer. Attached Figure Description

[0019] Figure 1 This figure shows the cell activity assay results of the inhibitory effect of crotonin A on the proliferation of human ovarian cancer SKOV3, A2780, and Anglne cells. The data in the figure are expressed as "mean inhibition rate ± standard deviation at each effective concentration".

[0020] Figure 2 The graph shows the soft agar colony rates of SKOV3 cells after treatment with different doses of crotonin A. Data are expressed as mean ± standard deviation. One-way ANOVA and Dunnett's multiple comparisons test were used to compare the significant differences in apoptosis rates between the control group and each dose group of BRA. A p-value less than 0.05 was considered statistically significant.

[0021] Figure 3 The figure shows the cell invasion results of SKOV3 cells after treatment with different doses of crotonin A.

[0022] Data are expressed as mean ± standard deviation. One-way ANOVA and Dunnett's multiple comparisons test were used to compare the significant differences in apoptosis rates between the control group and each dose group. A p-value less than 0.05 was considered statistically significant.

[0023] Figure 4 This figure shows the tumor growth results of each experimental group in the SKOV3 subcutaneous transplantation tumor model of human ovarian cancer. Data are expressed as mean ± standard deviation. The relative tumor volume difference between the control and treatment groups was compared using an unpaired t-test (Welch's correction), with a p-value less than 0.05 considered statistically significant.

[0024] Figure 5 Photographs of SKOV3 tumor-bearing mice taken after drug administration (day 19).

[0025] Figure 6 The image shows the tumor weight results after administration of crotonin A to SKOV3 transplanted tumors in nude mice. Data are expressed as mean ± standard deviation. Unpaired t-tests (Welch's correction) were used to compare the significant differences in tumor weight between the solvent control group and the crotonin A administration group. A p-value less than 0.05 was considered statistically significant. Detailed Implementation

[0026] With reference to the drawings of the embodiments of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without creative effort belong to the scope of protection of the present application.

[0027] At present, the treatment of ovarian cancer lacks effective treatment of natural product drugs, in order to solve the above technical problems, the present application provides a use of bruceine A in the preparation of a drug for treating ovarian cancer.

[0028] The experimental materials used in the following examples are as follows: cell strains: human ovarian cancer cell lines SKOV3, A2780 and Anglne were provided by the Henan Industrial Microorganism Strain Engineering Technology Research Center; 96-well plates were purchased from Costar Company (catalog number: 3599); RPMI1640 medium was purchased from Gibco Company (catalog number: C11995500BT); fetal bovine serum was purchased from Gibco Company (catalog number: 10270-106); CCK-8 kit was purchased from Meilunbio (catalog number: MA0218); DMSO was purchased from Beijing Solabio Technology Co., Ltd. (catalog number: D8370); Bruceine A (BRA) was purchased from Hubei Yongkong Technology Co., Ltd. (catalog number JOT-11226, batch 22061309), HPLC purity 98.73%, stored at -20℃.

[0029] The experimental instruments used in the following examples are as follows:

[0030] Experimental instruments: clean bench (Suzhou purification model BHC-1000IIA2); CO2 incubator (Thermo model: 3111); enzyme marker (Thermo Multiscan MK3).

[0031] Example 1: Effect of Bruceine A on the Proliferation of Human Ovarian Cancer Cells

[0032] Test sample: weigh 2 mg of Bruceine A, add 0.38 mL of DMSO to dissolve, and prepare a 10 mM / L solution.

[0033] Treatment method: CCK-8 method was used to evaluate the proliferation inhibition effect of Bruceine A on human ovarian cancer cells: human ovarian cancer cells (SKOV3, A2780 and Anglne) were digested with trypsin, counted, and mixed with complete culture solution (10% fetal bovine serum + 90% RPMI1640 medium) to prepare a cell suspension with a concentration of 2×10 4 μL of cell suspension was added to each well of the 96-well plate (2×103 The cells were then incubated at 37°C in a 5% CO2 incubator for 16 hours. The drug was diluted with complete culture medium to the desired concentration (3.9 nM / L-4000 nM / L), 100 μL of the drug-containing medium was added to each well, and a DMSO control group and a cell-free culture medium control group were set up. The 96-well plate was then incubated at 37°C in a 5% CO2 incubator for 72 hours, 10 μL of CCK-8 solution was then added to each well, and the plate was incubated for another 2 hours. The absorbance (OD value) of each well was then measured at a wavelength of 450 nm using an enzyme-labeled instrument. The average OD value of the wells in each group was calculated by subtracting the average OD value of the cell-free culture medium control group from the average OD value of the wells in each group, and the inhibition rate of the drug was calculated.

[0034] The results are shown in Table 1. Figure 1 As shown in Table 1, bruceine A had a significant inhibitory effect on the proliferation of human ovarian cancer SKOV3, A2780 and Anglne cells, and the half-maximal inhibitory concentration (IC 50 ) was less than 100 nM / L, and was 59.19 nM / L, 38.86 nM / L and 56.83 nM / L, respectively.

[0035] Example 2: Inhibitory effect of bruceine A on soft agar colony formation of human ovarian cancer SKOV3 cells

[0036] Test sample: The bruceine A solution was prepared by the method described in Example 1.

[0037] Treatment method: Completely dissolved 5% agar and pre-warmed complete culture medium (10% fetal bovine serum + 90% RPMI1640 medium) were uniformly mixed and added to a 6-well cell culture plate, each well containing 2 mL of 0.5% agar medium. The plate was incubated at room temperature until the agar was completely solidified. Human ovarian cancer cells SKOV3 were digested with trypsin, counted, and mixed with complete culture medium to prepare a cell suspension with a concentration of 1 x 10 3 The cells were then incubated at 37°C in a 5% CO2 incubator for 16 hours. The drug was diluted with complete culture medium to the desired concentration (3.9 nM / L-4000 nM / L), 100 μL of the drug-containing medium was added to each well, and a DMSO control group and a cell-free culture medium control group were set up. The 96-well plate was then incubated at 37°C in a 5% CO2 incubator for 72 hours, 10 μL of CCK-8 solution was then added to each well, and the plate was incubated for another 2 hours. The absorbance (OD value) of each well was then measured at a wavelength of 450 nm using an enzyme-labeled instrument. The average OD value of the wells in each group was calculated by subtracting the average OD value of the cell-free culture medium control group from the average OD value of the wells in each group, and the inhibition rate of the drug was calculated.

[0038] The results are shown in Table 1. Figure 2As shown, the soft agar colony formation assay results indicated that the soft agar colony formation of SKOV3 cells in each dose group of crocin A (50 nM / L and 200 nM / L) was significantly lower than that in the solvent (DMSO) control group (BRA (50 nM / L) vs. solvent, P<0.0001; BRA (200 nM / L) vs. solvent, P<0.0001).

[0039] Example 3: Inhibitory effect of crotonin A on the invasion of human ovarian cancer SKOV3 cells

[0040] Test sample: The crotonin A solution was prepared by the method described in Example 1.

[0041] Treatment method: Human ovarian cancer cells SKOV3 were digested with trypsin, counted, and mixed with RPMI-1640 medium to prepare a concentration of 1×10⁻⁶. 5 Cell suspension at 100 cells / mL. Add 600 μL of complete culture medium to the lower chamber of a 24-well transwell plate. Set up a DMSO control and crotonin A treatment groups (50 nM / L and 200 nM / L), with 4 parallel wells per group. Place the transwell chamber into the 24-well plate with forceps. Add 100 μL of cell suspension to the upper chamber of the transwell pre-coated with Matrigel, and incubate for 24 h. Remove the chamber, aspirate the culture medium, and gently wipe away the cells from the Matrigel and the upper chamber with a cotton swab. Add 600 μL of 4% paraformaldehyde to a new 24-well plate, place the chamber in, and fix for 20-30 minutes. Discard the fixative, stain with 0.1% crystal violet for 10 minutes, wash three times with deionized water, air dry appropriately, and photograph under a high-power microscope. After staining, add 600 μL of 33% acetic acid to each Transwell chamber to completely elute the crystal violet. After thorough shaking, take 100 μL of the eluent and measure the absorbance (OD) at 570 nm using a microplate reader. 570 ).

[0042] The results are as follows Figure 3 As shown in the Transwell cell invasion assay, compared with the solvent (DMSO) control group, all doses of crotonin A (50 nM / L and 200 nM / L) significantly inhibited the invasion ability of SKOV3 cells (BRA (50 nM / L) vs. solvent, P < 0.0001; BRA (200 nM / L) vs. solvent, P < 0.0001).

[0043] Example 4: Pharmacological experiment on a nude mouse subcutaneous transplantation model of SKOV3 ovarian cancer cell line tumor.

[0044] Experimental animals: BALB / c nude mice, female, 4 weeks old, body weight about 17 g, purchased from Hunan Slike Jingda Experimental Animal Co., Ltd., production license number: SCXK(Xiang)2019-0004, animal qualification certificate number: 430727221102297442. Raising environment: SPF level.

[0045] The experimental animals were raised in a constant temperature and humidity independent ventilation box, the temperature of the feeding room was 20-26℃, the humidity was 40-70%, the air exchange was 10-20 times / hour, the day and night light and dark alternating time was 12h / 12h; continuously supply of cobalt 60 radiation sterilization mouse full price granular feed, unlimited free intake, drink tap water (high pressure steam sterilization after use), drinking water bottle uninterrupted water supply, free intake. The mouse box is a polysulfone mouse box, which is used after high pressure sterilization, with a size of 325mmx210mmx180mm; the bedding is high pressure sterilized corn cob, 5 animals per box; the experimental animals are marked by ear tags.

[0046] Experimental materials: Bruceine A (BRA) was purchased from Hubei Yongkong Technology Co., Ltd. (Catalog No. JOT-11226, Batch 22061309), with a HPLC purity of 98.73%, stored at -20℃ in a sealed state. PEG300 was purchased from Jiangsu Hai'an Petroleum Chemical Factory; PBS was purchased from Tianjin Haoyang Huake Biological Technology Co., Ltd.; DMSO was purchased from Beijing Solaybao Technology Co., Ltd.

[0047] Drug preparation: Bruceine A powder was weighed and dissolved in solvent solution (10% DMSO+30% PEG300+60% PBS).

[0048] Experimental method: Human ovarian cancer SKOV3 cells were cultured in RPMI-1640 culture medium containing 10% fetal bovine serum (purchased from American Gibco Company). The SKOV3 cells in the exponential growth phase were collected, resuspended with PBS to a suitable concentration (2x10 7 / ml) of cell suspension for subcutaneous tumor inoculation in nude mice. Female mice were inoculated with 100 μl of cell suspension (containing 2x10 6 SKOV3 cells) in the right axillary. When the tumor grew to an average volume of about 50mm 3 , the mice were randomly divided into groups according to the tumor size, and the drug was administered on the same day. The drug administration volume was 100 μL per nude mouse, and the specific drug administration scheme is shown in Table 1. The long diameter a and short diameter b of the tumor were measured every other day with a vernier caliper. The subcutaneous SKOV3 tumor volume of nude mice in each experimental group during drug administration is shown in Table 2. Figure 4

[0049] Table 1. Specific drug administration scheme

[0050] ​

[0051]

[0052] Experimental results: No nude mice died during the drug administration period, and the mice tolerated the drug well. On day 19 of drug administration, the tumor volume in the solvent control group was 591.33 ± 101.73 mm. 3 The experiment concluded, and the tumor was harvested and weighed. The formula for calculating tumor volume is: Tumor volume (mm²) 3 )=1 / 2×(a×b 2 (Where a represents the major axis and b represents the minor axis). At the end of the trial, the mean tumor volume in the 200 mg / kg crotonin A treatment group was 263.37 ± 210.06 mm. 3 The results showed a statistically significant difference compared to the control group (P = 0.0179) (see results below). Figure 4 and Figure 5 (As shown).

[0053] Tumor samples were collected at the experimental endpoint, and tumor weight (g) was measured. The results were consistent with the tumor volume measurements. Tumor weights in each treatment group and control group were as follows: Figure 6 Compared with the tumor weight of the solvent control (0.554±0.051g), the tumor weight after administration of 200mg / kg of crotonin A was 0.176±0.139g, and the difference was statistically significant (P=0.0006). The results indicate that crotonin A has good in vivo antitumor activity in an ovarian cancer transplant model.

[0054] Compared with the prior art, the present invention has the following advantages:

[0055] 1. Chlorophytin A has a significant inhibitory effect on the proliferation of human ovarian cancer cells.

[0056] 2. The soft agar colony formation of human ovarian cancer cells was significantly lower than that of the control group by crocin A, and crocin A significantly inhibited the invasive ability of human ovarian cancer cells.

[0057] 3. When crotonin A is administered at a dose of 200 mg / kg, it can significantly reduce the volume and weight of ovarian cancer tumors, and has a significant inhibitory effect on ovarian cancer tumors. It can be used in the preparation of drugs for the treatment of ovarian cancer.

[0058] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of a bruceine A in the manufacture of a medicament for the treatment of ovarian cancer.

2. Use of a combination of compounds according to claim 1 for the manufacture of a medicament for the treatment of ovarian cancer, characterized in that: The molecular formula of the said bruceine A is: C 26 H 34 O 11 The structural formula is shown as formula I: ； Formula I.

3. Use of a combination of compounds according to claim 1 for the manufacture of a medicament for the treatment of ovarian cancer, characterized in that: It can be administered alone or in combination therapy.

4. Use of a combination of compounds according to claim 3 for the manufacture of a medicament for the treatment of ovarian cancer, characterized in that: The combination therapy includes radiotherapy, chemotherapy, and surgical treatment.

5. Use of a combination of compounds according to claim 1 for the manufacture of a medicament for the treatment of ovarian cancer, characterized in that: The bruceine A can be prepared into any pharmaceutically acceptable dosage form.

6. Use of a combination of compounds according to claim 1 for the manufacture of a medicament for the treatment of ovarian cancer. The bruceine A can be in any pharmaceutically acceptable dosage.

7. Use of a combination of compounds according to claim 1 for the manufacture of a medicament for the treatment of ovarian cancer. The dosage of the bruceine A is within 200 mg / kg.

8. Use of a combination of compounds according to claim 1 for the manufacture of a medicament for the treatment of ovarian cancer. The dosage of the bruceine A is 200 mg / kg.

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