Application of camptothecin, mycobacterium tuberculosis inhibitor, pharmaceutical composition and application of mycobacterium tuberculosis inhibitor

Abstract

The invention discloses application of camptothecin, a mycobacterium tuberculosis inhibitor, a pharmaceutical composition and application of the mycobacterium tuberculosis inhibitor and the pharmaceutical composition, and belongs to the technical field of biological medicine. The invention finds that the drug inhibits the survival of mycobacterium tuberculosis and mycobacterium abscessus by regulating the immune response of host cells, so that diseases caused by mycobacterium tuberculosis and mycobacterium abscessus infection can be effectively inhibited, and therefore, the camptothecin can be used as a novel drug for resisting mycobacterium tuberculosis and mycobacterium abscessus.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of camptothecin, a Mycobacterium tuberculosis inhibitor, a pharmaceutical composition and the application thereof. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis complex. 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. However, the efficacy of drug-resistant tuberculosis is less than 50%, and it can develop into a progressive, incurable disease to a certain extent. Other co-infections and / or complications that impair immunity require more complex prevention and treatment methods.

[0003] However, the diagnosis and treatment of PNTM in clinical practice are still lacking in standardization. Among them, Mycobacterium abscessus is one of the main mycobacterium pathogens that cause non-tuberculous pulmonary infections. It is a highly resistant opportunistic pathogen that is resistant to multiple antibiotics and difficult to treat. It is currently believed that the body's low immune function and inability to effectively kill tuberculosis bacteria are the main reasons for latent infection, chronic protracted tuberculosis, and poor BCG vaccination results. Therefore, regulating the body's immune status is the most promising measure for the treatment of tuberculosis. In the face of growing clinical needs, screening anti-tuberculosis drugs with immune regulatory effects is an important direction for the treatment of tuberculosis. Summary of the invention

[0004] The main purpose of the present invention is to propose the application of camptothecin, a Mycobacterium tuberculosis inhibitor, a pharmaceutical composition and its application, aiming to provide a new anti-tuberculosis drug for treating diseases caused by Mycobacterium tuberculosis infection.

[0005] To achieve the above object, the present invention proposes the use of camptothecin in the preparation of a preparation for inhibiting diseases caused by infection with Mycobacterium tuberculosis and / or non-tuberculous mycobacteria.

[0006] In one embodiment, the camptothecin affects the survival of Mycobacterium tuberculosis by regulating the host immune status.

[0007] In one embodiment, the nontuberculous mycobacterium comprises Mycobacterium abscessus.

[0008] The present invention provides a Mycobacterium tuberculosis inhibitor, which comprises camptothecin.

[0009] The present invention provides application of camptothecin in preparing medicine for treating diseases caused by infection of Mycobacterium tuberculosis.

[0010] In one embodiment, the disease comprises tuberculosis.

[0011] In one embodiment, the medicament is for inhibiting infection by Mycobacterium tuberculosis comprising H37Rv.

[0012] The invention provides a pharmaceutical composition, which comprises camptothecin and isoniazid.

[0013] In one embodiment, the medicament further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

[0014] In the technical scheme of the present invention, the present invention first discovered that camptothecin has an anti-tuberculosis effect, and uses camptothecin in the preparation of a preparation for inhibiting diseases caused by Mycobacterium tuberculosis and / or non-tuberculous mycobacteria infection. The present invention discovered that the drug inhibits the survival of Mycobacterium tuberculosis by regulating the host cell immune response, thereby being able to effectively inhibit diseases caused by Mycobacterium tuberculosis infection, and verified that Mycobacterium tuberculosis can be effectively inhibited at the cellular level. Therefore, camptothecin can be studied and developed as a new anti-tuberculosis drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] 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 drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0016] Figure 1 A schematic diagram of the effects of different concentrations of camptothecin on THP-1 cell viability provided by an embodiment of the present invention; Figure 2 A schematic diagram showing the results of the inhibition of Mycobacterium tuberculosis by camptothecin at the macrophage level provided by an embodiment of the present invention; Figure 3 A schematic diagram showing the results of the inhibition of Mycobacterium tuberculosis at the macrophage level by camptothecin combined with isoniazid provided in an embodiment of the present invention; Figure 4 This is a schematic diagram of the results of the inhibition of non-tuberculous mycobacteria by camptothecin at the macrophage level provided by the embodiment of the present invention.

[0017] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0018] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at 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 technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions 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 belong to the scope of protection of the present invention.

[0019] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis complex. 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. However, the efficacy of drug-resistant tuberculosis is less than 50%, and it can develop into a progressive and incurable disease to a certain extent. Other co-infections and / or complications that damage immunity require more complex prevention and treatment methods. It is currently believed that low immune function and inability to effectively kill tuberculosis bacteria are the main reasons for latent infection, chronic protracted course of tuberculosis and poor effect of BCG vaccination. Therefore, regulating the body's immune status is the most promising measure for the treatment of tuberculosis. In the face of growing clinical needs, screening anti-tuberculosis drugs with immune regulation is an important direction for the treatment of tuberculosis.

[0020] In view of this, the present invention proposes the use of camptothecin in the preparation of a preparation for inhibiting diseases caused by infection with Mycobacterium tuberculosis and / or non-tuberculous mycobacteria.

[0021] It should be noted that the structural formula of camptothecin is as follows:

[0022] Camptothecin is a monoterpene indole alkaloid, mainly found in plants such as Camptotheca acuminata, Camptotheca sempervirens and Camptothecin. Camptothecin can selectively bind to DNA topoisomerase I and inhibit its activity, and has strong anti-tumor activity against colorectal cancer, breast cancer, lung cancer and ovarian cancer.

[0023] It is understandable that HDT, as a new type of immunotherapy measure, host-directed therapy (HDT), is considered to be 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 resistance, and it will also have a good effect on patients with concurrent tuberculosis infection such as AIDS, diabetes, and rheumatism. However, how to choose immune response targets and immunomodulators is the key to HDT.

[0024] In some embodiments of the present invention, the camptothecin affects the survival of Mycobacterium tuberculosis by regulating the host immune state, that is, it can be used as an HDT drug to inhibit the survival of Mycobacterium tuberculosis by regulating the host immune state, rather than as an antibiotic to kill Mycobacterium tuberculosis.

[0025] The present invention provides a Mycobacterium tuberculosis inhibitor, which includes camptothecin. Camptothecin, as a natural product, has a wide source, good safety and biocompatibility, and has been verified by experiments at the cell level to be able to effectively inhibit Mycobacterium tuberculosis. Therefore, camptothecin can be used as a Mycobacterium tuberculosis inhibitor to develop new anti-tuberculosis inhibitors.

[0026] In some embodiments of the present invention, the amount of camptothecin used to inhibit Mycobacterium tuberculosis H37Rv is not less than 100 μM.

[0027] It should be noted that MIC (Minimum Inhibitory Concentration) refers to the lowest concentration at which a drug can inhibit bacterial growth. For example, the MIC value of camptothecin against Mycobacterium tuberculosis H37Rv strain is 100 μM, which means that when the concentration of camptothecin reaches or exceeds 100 μM, it can effectively inhibit the growth of H37Rv.

[0028] Within the above range, the effect of inhibiting Mycobacterium tuberculosis H37Rv is better, which provides a scientific basis for the preparation of subsequent Mycobacterium tuberculosis inhibitors or drugs and avoids the risk of drug resistance or side effects due to unreasonable dosage.

[0029] The present invention also provides the use of camptothecin in preparing medicines for treating diseases caused by Mycobacterium tuberculosis infection.

[0030] In the technical solution of the present invention, the present invention first discovered the use of camptothecin in the preparation of a drug for treating a disease caused by Mycobacterium tuberculosis infection, and the drug is used for treating a disease caused by Mycobacterium tuberculosis infection.

[0031] In the embodiment of the present invention, the disease includes tuberculosis. Since Mycobacterium tuberculosis mainly causes tuberculosis (TB), camptothecin can be used to treat tuberculosis caused by Mycobacterium tuberculosis.

[0032] In an embodiment of the present invention, the drug is used to inhibit infection by Mycobacterium tuberculosis, wherein the Mycobacterium tuberculosis includes H37Rv.

[0033] It is understandable that Mycobacterium tuberculosis is the main pathogen that causes tuberculosis, and H37Rv is an important tool for studying tuberculosis and its treatment. The activity of H37Rv indicates its potential in the treatment of tuberculosis; H37Ra is a weak strain of H37Rv, which loses most of its pathogenicity after cultivation. Because of its higher safety, it is often used in research.

[0034] It should be noted that camptothecin can inhibit Mycobacterium tuberculosis by regulating macrophage immunity without causing drug resistance problems.

[0035] Tuberculosis is a highly contagious chronic disease. The use of camptothecin can prevent bacterial infection in the early stages and help control bacterial reproduction during treatment, slowing the progression of the disease, thereby achieving the effect of treating and preventing tuberculosis.

[0036] The present invention provides a pharmaceutical composition, which comprises camptothecin and isoniazid, that is, camptothecin and isoniazid are used in combination to produce a synergistic anti-tuberculosis effect and improve the inhibitory effect on Mycobacterium tuberculosis.

[0037] In some embodiments, the drug further comprises a pharmaceutically acceptable excipient, carrier and / or diluent. The selection of the above-mentioned carrier is related to the mode of administration and can be selected according to actual needs, while the diluent or excipient can be conventionally used in the pharmaceutical field.

[0038] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. 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.

[0039] 1. Experimental Materials 1. Strains and reagents Mycobacterium tuberculosis H37Rv strain (ATCC 27294) was stored in Shenzhen Third People's Hospital; Mycobacterium abscessus ATCC19977 strain was purchased from Beijing Bio-Bowei Company; THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences; Middlebrook 7H9 Broth was purchased from BD (Cat. No. 271310); Middlebrook OADC enrichment broth was purchased from BD (Cat. No. 212351); CCK-8 kit was purchased from Bio-Tech Biotech Co., Ltd.; The complete medium is a prepared medium. In the experiment, the 1640 complete medium is 1640+10% FBS, and the complete medium for bacteria is 7H9+OADC.

[0040] 2. 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 a cell culture incubator at 37°C and 5% CO2 using 1640 medium containing 10% fetal bovine serum. 5 × 104 cells were plated per well of a 96-well plate and stimulated overnight with PMA at a final concentration of 100 ng / ml to differentiate into macrophages. The culture medium was replaced with complete medium after 24 h.

[0041] Determination of cell viability: After culturing the above-mentioned induced differentiated macrophages for another 24 hours, camptothecin was added as the experimental well. The camptothecin concentrations were 0.1μM, 0.25μM, 0.5μM, and 1μM, respectively. Three replicates were performed for each concentration. DMSO was used as the control well and the cell-free culture medium was used as the blank well. The cells were cultured for another 48 hours at 37°C and 5% CO2. The cell activity was detected using the CCK-8 kit. 10μl CCK-8 was added to each well. After culturing in the incubator for 2 hours, the absorbance at OD450nm was determined using a microplate reader. The higher the absorbance, the stronger the cell activity. Conversely, a lower absorbance means weaker cell activity or more dead cells.

[0042] The cell viability was calculated according to the following formula: Cell survival rate = [(As-A b ) / (A c -A b )]×100% As: absorbance of experimental wells (culture medium containing cells, CCK-8, and different concentrations of camptothecin); Ac: absorbance of control wells (medium containing cells, CCK-8, DMSO); Ab: absorbance of blank wells (culture medium without cells, CCK-8).

[0043] After calculating the cell survival rates of different concentrations of camptothecin, a bar graph was drawn using GraphPad software. The results are shown in Figure 1 As shown: Figure 1The results of the effects of different concentrations of camptothecin on THP-1 cell viability are shown in Figure 1. The horizontal axis represents different concentrations of camptothecin, and the vertical axis represents cell viability. The results show that compared with the control, treatment with 0.1μM, 0.25μM, 0.5μM, and 1μM camptothecin has no effect on cell viability.

[0044] Example 2 Infection with Mycobacterium tuberculosis: The THP-1 cells of Example 1 were plated at 5×10 cells per well in a 24-well plate. 5 The number of cells was induced to become macrophages according to the above method, and 1 μM camptothecin was added for pretreatment 1 h before infection, and the solvent DMSO was added as a control. The cells were infected with Mycobacterium tuberculosis H37Rv at MOI=10, and washed three times with PBS after 4 h. 1640 complete medium was added and cultured in an incubator at 37°C and 5% CO2. Camptothecin was added during this process.

[0045] Count CFU on plates: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and plated at 10 2 , 10 3 The dilution was plated and cultured in a 37°C bacterial incubator for about three weeks. The CFU was counted and the results were calculated as follows: Figure 2 As shown, Figure 2 DMSO is dimethyl sulfoxide, which serves as the control group, and Camptothecin is camptothecin. The CFU of the two groups was calculated. The results showed that compared with the DMSO control group, the survival rate of Mycobacterium tuberculosis in THP1 macrophages was significantly decreased after the addition of camptothecin.

[0046] Example 3 Infection with Mycobacterium tuberculosis: THP-1 cells prepared in Example 1 were plated at 2.5 × 10 cells per well in a 24-well plate. 5 The number of cells was induced to become macrophages according to the above method, and camptothecin was added for pretreatment 1 hour before infection. The cells were infected with Mycobacterium tuberculosis H37Rv at MOI=10. After 4 hours, they were washed three times with PBS and added with 1640 complete medium. The cells were cultured in an incubator at 37°C and 5% CO2. During this process, camptothecin / isoniazid were always added.

[0047] Count CFU on plates: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and plated 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.

[0048] Figure 3This is the inhibition of camptothecin combined with isoniazid on Mycobacterium tuberculosis H37Rv, where DMSO is dimethyl sulfoxide as the control group, Camptothecin is camptothecin, INH is isoniazid, and INH+Camptothecin is isoniazid combined with camptothecin. Figure 3 The results showed that isoniazid or camptothecin alone can effectively inhibit or kill Mycobacterium tuberculosis in macrophages, and the killing effect of isoniazid combined with camptothecin is significantly improved compared with either use alone.

[0049] Example 4 Minimum inhibitory concentration (MIC) test: Take a 96-well plate, add 100μL ddH2O to each well at the outermost edge of the 96-well plate, add 98μL 7H9-OADC full culture medium to each well of B2-G2, and add 50μL 7H9-OADC full culture medium to each well of the remaining wells. Add 2μL camptothecin to each well of B2-D2, and add 2μL INH (isoniazid) to each well of E2-G2 as a positive control. From B2-G2 to B10-G10, two-fold dilution was performed, and B11-G11 was a negative control well without drug treatment. Finally, each well (except the ddH2O well) was added with 2×10 5 CFU / 50μL of Mycobacterium tuberculosis H37Rv bacterial solution was sealed with sealing film and placed in a 37°C bacterial incubator for culture for 10-14 days.

[0050] After taking out, observe the bacterial inhibition of drugs with different concentrations, read the MIC value, and the results are shown in Table 1: Table 1

[0051] Table 1 shows the MIC of camptothecin and INH against Mycobacterium tuberculosis H37Rv. The results show that the minimum inhibitory concentration of camptothecin against Mycobacterium tuberculosis in vitro is greater than 100 μM, which is much greater than the concentration used at the cellular level. It can be seen that the anti-tuberculosis effect of camptothecin 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. It is used as a positive control to illustrate the reliability of the experimental data.

[0052] Experimental verification showed that compared with the control group, the survival rate of Mycobacterium tuberculosis in macrophages was significantly reduced after adding 1μM camptothecin, and the MIC value of camptothecin to Mycobacterium tuberculosis was greater than 100μM. It can be seen that camptothecin inhibits the intracellular survival of Mycobacterium tuberculosis by targeting the host to regulate the host immune response. Therefore, camptothecin can be used in the preparation of drugs for the treatment of diseases caused by Mycobacterium tuberculosis infection, which broadens the application scenarios of camptothecin.

[0053] Example 5 Infection with Mycobacterium abscessus: The THP-1 cells of Example 1 were plated at 5×10 cells per well in a 24-well plate. 5 The number of cells was induced to become macrophages according to the above method, and 1 μM camptothecin was added for pretreatment 1 hour before infection, and the solvent DMSO was added as a control. The cells were infected with Mycobacterium abscessus ATCC19977 strain at MOI=10. After 4 hours, the cells were washed three times with PBS, added with 1640 complete medium, and continued to be cultured in an incubator at 37°C and 5% CO2. During this process, the addition of camptothecin was maintained.

[0054] Count CFU on plates: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and plated at 10 2 , 10 3 The dilution was plated and cultured in a 37°C bacterial incubator for about three weeks. The CFU was counted and the results were calculated as follows: Figure 4 As shown, Figure 4 DMSO was used as the control group and Camptothecin was used as the camptothecin. The CFU of the two groups was calculated. The results showed that the survival rate of Mycobacterium abscessus in THP1 macrophages was significantly decreased after the addition of camptothecin compared with that of the DMSO control group.

[0055] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. Use of camptothecin in the preparation of a preparation for inhibiting diseases caused by infection with Mycobacterium tuberculosis and / or non-tuberculous mycobacteria.

2. The use according to claim 1, characterized in that The camptothecin affects the survival of tuberculosis bacteria by regulating the host immune status.

3. The use according to claim 1, characterized in that The nontuberculous mycobacteria include Mycobacterium abscessus.

4. A Mycobacterium tuberculosis inhibitor, characterized in that The Mycobacterium tuberculosis inhibitors include camptothecin.

5. Use of camptothecin in the preparation of drugs for treating diseases caused by Mycobacterium tuberculosis infection.

6. The use according to claim 5, characterized in that The diseases include tuberculosis.

7. The use according to claim 5, characterized in that The medicament is used for inhibiting infection by Mycobacterium tuberculosis, wherein the Mycobacterium tuberculosis includes H37Rv.

8. A pharmaceutical composition, characterized in that The drug combination includes camptothecin and isoniazid.

9. The pharmaceutical composition according to claim 8, characterized in that The medicament further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

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