Application of etoposide in preparation of immunomodulator and medicine

Abstract

The invention provides an application of etoposide in preparation of an immunomodulator and a medicine, and relates to the technical field of biology, and the medicine is used for treating diseases caused by mycobacterium tuberculosis infection. According to the technical scheme, etoposide is adopted and applied to preparation of the immunomodulator, the biological activity of mycobacterium tuberculosis in cells can be inhibited, and therefore the anti-tuberculosis level in the cells can be adjusted. According to the scheme, the research for the first time finds that etoposide has a human macrophage level anti-tuberculosis effect and has a good clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of etoposide in the preparation of immunomodulators and medicines. Background Art

[0002] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been among the world's leading infectious diseases in terms of morbidity and mortality for several consecutive years.

[0003] With the widespread use of antibiotics and the characteristics of tuberculosis treatment and transmission, the number of patients with multidrug-resistant tuberculosis is increasing day by day, but the efficacy of drug-resistant tuberculosis is less than 50%, and to a certain extent it can develop into a progressive, incurable disease. Other co-infections and / or complications that impair immunity require more complex prevention and treatment methods, which brings a huge economic burden to people's lives and national health services. Summary of the invention

[0004] The main purpose of the present invention is to propose an application of etoposide in the preparation of immunomodulators and drugs, aiming to solve the problem that existing antibacterial agents may cause Mycobacterium tuberculosis to develop drug resistance.

[0005] To achieve the above object, the present invention proposes a use of etoposide in the preparation of an immunomodulator.

[0006] In one embodiment, the immunomodulator is used to inhibit Mycobacterium tuberculosis.

[0007] In one embodiment, the immunomodulator is used to inhibit Mycobacterium tuberculosis H37Rv.

[0008] In one embodiment, the immunomodulator is used to regulate the immune response of macrophages.

[0009] In one embodiment, the concentration of etoposide in the immunomodulator is no higher than 80 μM.

[0010] The present invention also provides an application of etoposide in preparing a medicine for treating diseases caused by Mycobacterium tuberculosis.

[0011] In one embodiment, the disease comprises tuberculosis.

[0012] In one embodiment, the medicament further comprises at least one of a pharmaceutically acceptable excipient, a carrier and a diluent.

[0013] The technical solution of the present invention adopts etoposide and is applied to the preparation of immunomodulators, which can inhibit the biological activity of Mycobacterium tuberculosis in cells, thereby regulating the anti-tuberculosis level in cells. This solution is the first to study and find that etoposide has anti-tuberculosis effects at the level of human macrophages, and has good clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0015] Figure 1 This is a graph showing the test results of the effect of etoposide on THP-1 cell viability in Example 1 of the present invention; Figure 2 This is a graph showing the inhibition of Mycobacterium tuberculosis H37Rv by etoposide at the THP-1 macrophage level in Example 2 of the present invention.

[0016] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0017] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0018] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0019] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been at the forefront of the world's infectious disease incidence and mortality for several consecutive years. Among them, China is one of the countries with the heaviest tuberculosis burden in the world. With the widespread use of antibiotics and the characteristics of tuberculosis treatment and transmission, the number of patients with multidrug-resistant tuberculosis is increasing day by day, but the efficacy of drug-resistant tuberculosis is less than 50%, and to a certain extent, it can develop into a progressive and incurable disease. Other co-infections and / or complications that impair immunity require more complex prevention and treatment methods, which impose a huge economic burden on people's lives and national health services. It is currently believed that low immune function and inability to effectively kill tuberculosis bacteria are the main reasons for latent infection, chronic and protracted tuberculosis, and poor BCG vaccination effects. Regulating the body's immune status is the most promising measure for the treatment of tuberculosis. For this reason, in recent years, a new type of immunotherapy measure, host-directed therapy (HDT), has been considered an effective measure for the treatment of drug-resistant tuberculosis. HDT refers to the use of immunomodulators to regulate the body's immune response to tuberculosis infection in order to kill tuberculosis and control tuberculosis. The therapeutic target of HDT is the immune response mediated by immune cells, and its measures are to use immunomodulators to optimize the bactericidal activity of immune cells and control tissue damage caused by inflammatory responses. Therefore, HDT is not affected by tuberculosis drug resistance, and will also have a good effect on patients with concurrent tuberculosis infection such as AIDS, diabetes, and rheumatism. However, how to choose the immune response target and immunomodulator is the key to HDT.

[0020] Etoposide is derived from podophyllotoxin. It was first synthesized in 1966 and approved by the U.S. Food and Drug Administration for cancer treatment in 1983. It is a widely used clinical anti-tumor drug and a cell cycle-specific drug. It mainly inhibits DNA synthesis and replication by interfering with the function of DNA topoisomerase II, thereby blocking the proliferation of tumor cells. Etoposide can be used to treat a variety of solid tumors, such as small cell lung cancer, ovarian cancer, testicular tumors, lymphomas, etc. However, its role in the anti-tuberculosis infection process is still unknown. The present invention finds that etoposide has the effect of inhibiting the intracellular survival of Mycobacterium tuberculosis at the macrophage level, which shows that etoposide has application value as an anti-tuberculosis drug.

[0021] In view of this, the present invention proposes a use of etoposide in the preparation of an immunomodulator.

[0022] The technical solution of the present invention adopts etoposide and is applied to the preparation of immunomodulators, which can inhibit the biological activity of Mycobacterium tuberculosis in cells, thereby regulating the anti-tuberculosis level in cells. This solution is the first to study and find that etoposide has anti-tuberculosis effects at the level of human macrophages, and has good clinical application value.

[0023] It should be noted that etoposide (Eupatilin) ​​is an important flavonoid active ingredient in the traditional Chinese medicine Artemisia argyi. Studies have reported that Eupatilin (10, 30, 100 μM) can inhibit the expression and degranulation of IL-4 in RBL-2H3 cells. Eupatilin (10, 30, 50, 100 μM) can increase the transactivation and expression of PPARα in HaCaT cells. Eupatilin (10, 30, 50 μM) can also inhibit TNFα-induced MMP-2 / -9 expression in HaCaT cells. In addition, Eupatilin can inhibit TNFα-induced p65 translocation, IκBα phosphorylation, AP-1 and MAPK signal transduction through PPARα. Eupatilin (10-50 μM) has no cytotoxic effect on ARPE19 cells. Eupatilin (10, 25, 50 μM) improves cell viability from oxidative stress and inhibits H2O2-induced ROS production in ARPE19 cells. In addition, Eupatilin (50 μM) inhibited H2O2-induced apoptosis and promoted the activation of PI3K / Akt pathway in RPE cells.

[0024] The structural formula of etoposide is shown below:

[0025] In one embodiment, the immunomodulator is used to inhibit Mycobacterium tuberculosis. The technical solution of the present invention uses etoposide as an immunomodulator to enhance the host's immune response to fight against Mycobacterium tuberculosis infection.

[0026] In one embodiment, the immunomodulator is used to inhibit Mycobacterium tuberculosis H37Rv. The technical solution of the present invention uses etoposide as an immunomodulator to enhance the host's immune response to specifically inhibit Mycobacterium tuberculosis H37Rv and reduce the biological activity of Mycobacterium tuberculosis H37Rv.

[0027] In one embodiment, the immunomodulator is used to regulate the immune response of macrophages.

[0028] In one embodiment, the concentration of etoposide in the immunomodulator is no more than 80 μM. The technical solution of the present invention can reduce the side effects of etoposide on macrophages by using etoposide with a concentration no more than 80 μM as an immunomodulator, thereby reducing the negative effects of etoposide on the growth and development of macrophages.

[0029] The present invention also provides an application of etoposide in preparing a medicine for treating diseases caused by Mycobacterium tuberculosis.

[0030] In one embodiment, the disease comprises tuberculosis.

[0031] In one embodiment, the medicament further comprises at least one of a pharmaceutically acceptable excipient, a carrier and a diluent.

[0032] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0033] Experimental Materials THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0034] Mycobacterium tuberculosis H37Rv (ATCC 27294) was preserved by Shenzhen Third People's Hospital.

[0035] Embodiment 1: Differentiation of THP-1 macrophages: THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in 1640 medium containing 10% fetal bovine serum at 37°C and 5% CO 2 The cells were cultured in a cell culture incubator, with 5 × 10 4 The cells were plated with 100 ng / ml of PMA for overnight stimulation to differentiate them into macrophages, and the culture medium was replaced with complete medium after 24 h.

[0036] Determination of cell survival rate: After the above-mentioned induced differentiated macrophages were cultured for 24 h, etoposide was added. The concentrations of etoposide were 5 μM, 10 μM, 30 μM, 50 μM, 70 μM, 80 μM, 90 μM, 100 μM, and 150 μM, respectively. Three replicates were performed for each concentration. Dimethyl sulfoxide (DMSO) was used as the control well and the blank well was without cell culture medium. The cells were incubated at 37 °C and 5% CO 2 The cells were cultured for 48 h, and the cell activity was detected using a CCK-8 kit. 10 μL of CCK-8 was added to each well. After culturing in an incubator for 2 h, the absorbance at OD450 nm was measured using an enzyme-labeled instrument.

[0037] The cell viability was calculated according to the following formula: Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%; As: experimental wells (culture medium containing cells, CCK-8, and different concentrations of etoposide); Ac: control (culture medium containing cells, CCK-8, and DMSO); Ab: Blank wells (culture medium without cells, CCK-8).

[0038] After calculating the cell viability at different concentrations of etoposide, a bar graph was drawn using GraphPad software.

[0039] The results of the effects of different concentrations of etoposide on THP-1 cell viability are shown in Figure 1 As shown, the results showed that compared with the control, 5μM, 10μM, 30μM, 50μM, 70μM, and 80μM etoposide treatment had no significant difference in cell survival rate, while 90μM, 100μM, and 150μM etoposide treatment significantly reduced cell survival rate. The IC50 concentration of etoposide for THP-1 cells was calculated and analyzed to be 380.7μM.

[0040] Embodiment 2: Infection of Mycobacterium tuberculosis: THP-1 cells were plated at 5 × 10 cells per well in a 24-well plate. 5 The number of cells was induced into macrophages according to the above method, and 10 μM etoposide was added for pretreatment 1 h before infection. The solvent DMSO was used as a control. The cells were infected with Mycobacterium tuberculosis H37Rv at an MOI of 10. After 4 h, the cells were washed three times with phosphate buffered saline (PBS) and cultured in 1640 complete medium at 37°C and 5% CO 2 The cells were cultured in an incubator at 4 °C and etoposide was added throughout the process.

[0041] Counting CFU on plates: 4 and 72 hours after infection, cells were lysed with 0.025% sodium dodecyl sulfate (SDS) and the cells were counted at 10 2 , 10 3 The dilution multiples were plated and cultured in a 37°C bacterial incubator for about three weeks to count the CFU.

[0042] Etoposide inhibits Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level Figure 2 As shown, the results showed that the survival rate of Mycobacterium tuberculosis was significantly decreased after the addition of etoposide compared with the DMSO control group.

[0043] Embodiment 3: Minimum inhibitory concentration (MIC) test: Take a 96-well plate, add 100 μL ddH2O to each well at the outermost edge of the 96-well plate, add 98 μL 7H9-OADC full culture medium to each well of B2-G2, and add 50 μL 7H9-OADC full culture medium to each well of the remaining wells. Add 2 μL etoposide to each well of B2-D2, and add 2 μL isoniazid (INH) to each well of E2-G2 as a positive control. From B2-G2 to B10-G10, two-fold dilutions are made, and B11-G11 is a negative control well without drug treatment. Finally, each well (except the ddH2O well) is added with 2×10 5 CFU / 50μL of Mycobacterium tuberculosis H37Rv bacterial solution was sealed with sealing film and placed in a 37℃ bacterial incubator for 10-14 days. After taking out, the bacterial inhibition of drugs with different concentrations was observed and the MIC value was read. The test results are shown in Table 1.

[0044] Table 1 Minimum inhibitory concentrations of etoposide and isoniazid against Mycobacterium tuberculosis H37Rv

[0045] The results showed that the minimum inhibitory concentration of etoposide against Mycobacterium tuberculosis in vitro was greater than 50 μM, which was much greater than the concentration used at the cellular level. It can be seen that the anti-tuberculosis effect of etoposide at the macrophage level is not directly acting on bacteria as an antibiotic, but inhibiting the intracellular survival of Mycobacterium tuberculosis by targeting the host to regulate the host immune response. The minimum inhibitory concentration of INH against Mycobacterium tuberculosis is 0.02 μg / mL, which is consistent with the concentration range reported in the literature, and serves as a positive control to illustrate the reliability of the experimental data.

[0046] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An application of etoposide in the preparation of an immunomodulator.

2. The use of etoposide in the preparation of an immunomodulator according to claim 1, characterized in that: The immunomodulator is used to inhibit Mycobacterium tuberculosis.

3. The use of etoposide in the preparation of an immunomodulator according to claim 1, characterized in that: The immunomodulator is used for inhibiting Mycobacterium tuberculosis H37Rv.

4. The use of etoposide in the preparation of an immunomodulator as claimed in claim 3, characterized in that: The immunomodulator is used to regulate the immune response of macrophages.

5. Use of etoposide in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis.

6. Use of etoposide according to claim 5 in the preparation of a medicament for treating a disease caused by Mycobacterium tuberculosis, characterized in that: The diseases include tuberculosis.

7. Use of etoposide according to claim 6 in the preparation of a medicament for treating a disease caused by Mycobacterium tuberculosis, characterized in that: The medicament further comprises at least one of a pharmaceutically acceptable excipient, a carrier and a diluent.

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