Use of elvitegravir for the preparation of an inhibitor of mycobacterium tuberculosis or non-tuberculous mycobacteria

Abstract

This invention discloses the application of erteiravir in the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria, relating to the field of biomedical technology. This invention discloses for the first time that erteiravir has the effect of inhibiting the intracellular survival of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level, exhibiting anti-tuberculosis activity at the macrophage level, and can be applied to the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria. This invention also discloses that the anti-tuberculosis effect of erteiravir at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits the intracellular survival of Mycobacterium tuberculosis by targeting and regulating the host immune response. Therefore, erteiravir also has the potential as an immunomodulator, achieving anti-tuberculosis effects by regulating the body's immune response to Mycobacterium tuberculosis infection without being affected by drug resistance of Mycobacterium tuberculosis, and has a good therapeutic effect on multidrug-resistant tuberculosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of erteiravir in the preparation of inhibitors of Mycobacterium tuberculosis or non-tuberculous mycobacteria. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by the Mycobacterium tuberculosis complex. Currently, clinical treatment of TB mainly relies on anti-tuberculosis drugs such as isoniazid, rifampin, and pyrazinamide.

[0003] However, due to the long-term and widespread use of antibiotics, insufficient patient adherence, and the complex biological characteristics of Mycobacterium tuberculosis itself, the incidence of drug-resistant tuberculosis continues to rise. Multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that develops resistance to at least two of the most important anti-tuberculosis drugs, such as isoniazid and rifampin. Current anti-tuberculosis drugs are significantly less effective against MDR-TB, typically below 50%. Meanwhile, the infection rate of pulmonary nontuberculous mycobacteria (PNTM) is increasing year by year, and its clinical importance is receiving growing attention; however, the diagnosis and treatment of PNTM in clinical practice are currently lacking standardization. Among the many pathogenic nontuberculous mycobacteria, Mycobacterium abscessatum is one of the main mycobacterial pathogens causing pulmonary nontuberculous infections. It is a highly drug-resistant opportunistic pathogen, resistant to multiple antibiotics, making clinical treatment extremely difficult.

[0004] Therefore, both multidrug-resistant tuberculosis and PNTM infections caused by Mycobacterium abscessus face the challenge of lacking effective treatment strategies. Summary of the Invention

[0005] The main objective of this invention is to propose a new application of erteiravir in the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria, aiming to solve the problem of the lack of effective treatment strategies for multidrug-resistant tuberculosis and PNTM infection caused by Mycobacterium abscessus in the prior art.

[0006] To achieve the above objectives, this invention proposes the application of erteiravir in the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0007] In one embodiment, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv.

[0008] In one embodiment, the nontuberculous mycobacteria include Mycobacterium abscessus.

[0009] In one embodiment, the inhibitor is used to modulate the immune response of macrophages.

[0010] This invention proposes the use of erteiravir in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacterial infections.

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

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

[0013] This invention discloses for the first time that erteiravir inhibits the intracellular survival of Mycobacterium tuberculosis H37Rv and Mycobacterium abscessus at the macrophage level, exhibiting anti-tuberculosis activity at the macrophage level. It can be used to prepare inhibitors of Mycobacterium tuberculosis or non-tuberculous mycobacteria, serving as a novel anti-tuberculosis or anti-non-tuberculous mycobacteria drug. This invention also discloses that erteiravir's anti-tuberculosis effect at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits the intracellular survival of Mycobacterium tuberculosis by targeting and regulating the host's immune response. Therefore, erteiravir has the potential to act as an immunomodulator, achieving its anti-tuberculosis effect by modulating the body's immune response to Mycobacterium tuberculosis infection, unaffected by Mycobacterium tuberculosis drug resistance, and showing good therapeutic efficacy against multidrug-resistant tuberculosis. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0015] Figure 1 This is a graph showing the effect of different concentrations of erteiravir on the cell viability of THP-1 macrophages in Example 1; Figure 2 This is a graph showing the results of erteiravir's inhibition of Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level in Example 2; Figure 3 This is a graph showing the results of erteiravir's inhibition of Mycobacterium abscessus at the THP-1 macrophage level in Example 4.

[0016] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0017] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0018] It should be noted that if the embodiments of the present invention involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0019] Furthermore, if the embodiments of this invention involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution where both A and B are satisfied simultaneously. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention.

[0020] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has ranked among the top infectious diseases in terms of incidence and mortality worldwide for several consecutive years. The combined use of some first-line anti-TB drugs, such as isoniazid (INH), ethambutol (EMB), rifampin (RIF), and pyrazinamide (PZA), can exert a good anti-TB effect. However, drug abuse makes it easy for pathogens to develop drug resistance, and the number of patients with multidrug-resistant TB is increasing daily, and to some extent, it can develop into a progressive and incurable disease.

[0021] Multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that has developed resistance to at least two of the most important anti-tuberculosis drugs, such as isoniazid and rifampin. Current anti-tuberculosis drugs are significantly less effective against MDR-TB, typically below 50%. Meanwhile, the infection rate of pulmonary nontuberculous mycobacteria (PNTM) is increasing year by year, and its clinical importance is receiving growing attention. However, the diagnosis and treatment of PNTM in clinical practice are currently lacking standardization. Among the many pathogenic nontuberculous mycobacteria, *Mycobacterium abscessum* is one of the main mycobacterial pathogens causing pulmonary nontuberculosis infections. It is a highly drug-resistant opportunistic pathogen, resistant to multiple antibiotics, making clinical treatment extremely difficult. Therefore, both MDR-TB and PNTM infections caused by *Mycobacterium abscessum* face the challenge of lacking effective treatment strategies.

[0022] A weakened immune system, unable to effectively eradicate Mycobacterium tuberculosis, is a major cause of latent infection, chronic tuberculosis, and poor efficacy of BCG vaccination. Modulating the body's immune status is a promising approach to treating tuberculosis. Therefore, in recent years, a novel immunotherapy—host-directed therapy (HDT)—has been considered an effective treatment for drug-resistant tuberculosis. HDT uses immunomodulators to regulate the body's immune response to Mycobacterium tuberculosis infection, aiming to kill the bacteria and control the disease. The therapeutic target of HDT is the immune cell-mediated immune response. It optimizes the bactericidal activity of immune cells and controls tissue damage caused by inflammation using immunomodulators. Therefore, HDT is not affected by Mycobacterium tuberculosis resistance and also shows good efficacy in patients with concurrent Mycobacterium tuberculosis infection, such as those with AIDS, diabetes, or rheumatic diseases. Therefore, the selection of immune response targets and immunomodulators is crucial for HDT.

[0023] Based on the above background, this invention proposes the application of erteiravir in the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0024] Elvitegravir (EVG) is an HIV integrase inhibitor, often used in combination with other antiretroviral drugs as part of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus (HIV) infection. Currently, no studies have found that elvitegravir can be used to inhibit either Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0025] The structural formula of ertiravir is: .

[0026] This invention discloses for the first time that erteiravir inhibits the intracellular survival of Mycobacterium tuberculosis H37Rv and Mycobacterium abscessus at the macrophage level, exhibiting anti-tuberculosis activity at the macrophage level. It can be used to prepare inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria, serving as a novel anti-tuberculosis drug or anti-non-tuberculous mycobacteria drug. This invention also discloses that erteiravir's anti-tuberculosis effect at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits the intracellular survival of Mycobacterium tuberculosis by targeting and regulating the host's immune response. Therefore, erteiravir also has the potential as an immunomodulator, achieving anti-tuberculosis effects by regulating the body's immune response to Mycobacterium tuberculosis infection, unaffected by Mycobacterium tuberculosis drug resistance, thus showing good therapeutic effects against multidrug-resistant tuberculosis.

[0027] It should be noted that since erteiravir can also be used as an HIV integrase inhibitor to treat human immunodeficiency virus (HIV) infection, it is evident that erteiravir also has important application value in the clinical treatment of HIV co-infection with MTB.

[0028] In embodiments of the present invention, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv. The technical solution of the present invention uses erteiravir as an immunomodulator to inhibit the cell viability of Mycobacterium tuberculosis H37Rv by regulating the host's immune response; therefore, erteiravir has the effect of inhibiting the intracellular survival of Mycobacterium tuberculosis at the macrophage level.

[0029] In embodiments of the present invention, the nontuberculous mycobacteria include Mycobacterium abscessus. Etileravir has the effect of inhibiting the intracellular survival of Mycobacterium abscessus at the macrophage level, and therefore ertileravir has the effect of inhibiting the intracellular survival of nontuberculous mycobacteria at the macrophage level, and has the potential to be used as a drug for treating nontuberculous mycobacterial infections.

[0030] In embodiments of the present invention, the inhibitor is used to modulate the immune response of macrophages. Etelavir in the inhibitor can effectively inhibit the intracellular survival rate of Mycobacterium tuberculosis at the macrophage level by targeting and regulating the host immune response.

[0031] In embodiments of the present invention, the concentration range of erteiravir in the inhibitor is 0–20 μM. Controlling the concentration range of erteiravir below 20 μM can reduce potential side effects on macrophages, thereby reducing adverse effects on macrophage growth and development.

[0032] The present invention also proposes the use of erteiravir in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacterial infections.

[0033] Etileravir inhibits the cell viability of Mycobacterium tuberculosis by targeting and modulating the host's immune response, thereby killing the bacteria and controlling tuberculosis. Therefore, ertileravir can be used to treat diseases caused by Mycobacterium tuberculosis. Because ertileravir also effectively inhibits the intracellular survival of non-tuberculous mycobacteria at the macrophage level, it can also be used to treat diseases caused by non-tuberculous mycobacteria. Etileravir exhibits low cytotoxicity and good safety, providing a new drug option for the clinical treatment of diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria infections.

[0034] It should be noted that when preparing drugs for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria, erteiravir can be prepared alone as a drug for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria, or it can be used in combination with other drugs for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0035] In embodiments of the present invention, the disease includes tuberculosis. Mycobacterium tuberculosis is the pathogen causing tuberculosis. Among them, Mycobacterium tuberculosis H37Rv is a strain widely used globally in biomedical research, possessing intact virulence in animal models of pulmonary tuberculosis. The inhibitory activity of drugs against H37Rv is generally considered to have potential in treating tuberculosis. Macrophages are sentinel cells that detect invading microorganisms, possessing strong phagocytic capabilities and being the primary target cells for Mycobacterium tuberculosis infection. When Mycobacterium tuberculosis infects macrophages, the macrophages can phagocytose the Mycobacterium tuberculosis, generating an immune response. Eteiravir achieves the goal of controlling tuberculosis by targeting and modulating the host's immune response to inhibit the intracellular survival of Mycobacterium tuberculosis H37Rv at the macrophage level.

[0036] In embodiments of the present invention, the drug further comprises at least one of pharmaceutically acceptable excipients, carriers, and diluents. The use of excipients can improve the physical properties or chemical stability of the drug; carriers and excipients can also regulate the drug release rate or targeting; diluents are commonly used in low-dose drugs to ensure precise drug content per unit volume and avoid dosage unevenness by increasing volume.

[0037] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the following embodiments are only used to explain the present invention and are not intended to limit the present invention.

[0038] Experimental materials THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0039] Mycobacterium tuberculosis H37Rv (ATCC 27294) was preserved by the Third People's Hospital of Shenzhen.

[0040] Mycobacterium abscessus (ATCC19977) was preserved by the Third People's Hospital of Shenzhen.

[0041] Example 1 This example illustrates the effect of different concentrations of erteiravir on the survival rate of THP-1 macrophages, including the following steps: (1) Differentiation of THP-1 macrophages: THP-1 cells were cultured in 1640 medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2. 5 × 10⁶ cells were cultured in each well of a 96-well plate. 4 The cells were seeded into plates and stimulated overnight with PMA (Phorbol 12-myristate 13-acetate) at a final concentration of 100 ng / mL to differentiate into macrophages. After 24 h, the medium was replaced with complete medium. (2) Determination of cell viability: After culturing the macrophages induced in step (1) for another 24 h, erteiravir was added. The concentrations of erteiravir were 1 μM, 2.5 μM, 5 μM, 10 μM and 20 μM, and each concentration was repeated three times. DMSO was used as the control well and cell-free medium was used as the blank well. The cells were cultured at 37℃ and 5% CO2 for another 48 h. Cell viability was detected using the CCK-8 kit. 10 μL of CCK-8 was added to each well and the cells were cultured in an incubator for 2 h. The absorbance at OD 450nm was measured using an ELISA reader.

[0042] Cell viability is calculated using the following formula: Cell viability = [(As-Ab) / (Ac-Ab)] × 100%; As: Absorbance of experimental wells (containing cell culture medium, CCK-8, and different concentrations of erteiravir); Ac: Absorbance of control wells (containing cell culture medium, CCK-8, DMSO); Ab: Absorbance of blank wells (cell-free culture medium, CCK-8).

[0043] After calculating the cell viability at different concentrations of erteiravir, a bar chart was generated using GraphPad software. The test results are shown below. Figure 1 As shown.

[0044] Figure 1 The study showed the effect of different concentrations of erteiravir on the viability of THP-1 macrophages. The results indicated that there was no significant difference in cell viability after treatment with 1 μM, 2.5 μM, 5 μM, 10 μM and 20 μM erteiravir compared with the control well, suggesting that erteiravir has low toxicity to THP-1 cells and good safety profile.

[0045] Example 2 This embodiment describes the effect of erteiravir on the survival of Mycobacterium tuberculosis in macrophages, including the following steps: (1) Infection with Mycobacterium tuberculosis: THP-1 cells were cultured in 24-well plates at a density of 5 × 10⁶ cells per well. 5 The number of cells was induced to become macrophages according to the method in step (1) of Example 1. 10 μM erteiravir was added for pretreatment 1 h before infection. An equal volume of DMSO was added to the control group. After 1 h of pretreatment, Mycobacterium tuberculosis H37Rv was used to infect the cells at an MOI (Multiplicity of Infection) of 10. 4 h later, the cells were washed 3 times with PBS (phosphate buffer saline) and cultured in 1640 complete medium in a 37°C, 5% CO2 incubator. During this process, 10 μM erteiravir was added to the experimental group and an equal volume of DMSO was added to the control group. (2) Colony-Forming Units (CFUs): Cells were lysed with 0.025% SDS at 4 h and 72 h after infection, and counted at 10 CFUs. 2 10 3 The dilution factor was plated and incubated in a bacterial incubator at 37 °C for about three weeks before CFU were counted.

[0046] Figure 2 This figure shows the inhibitory effect of erteiravir on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level. DMSO represents the control group, and EVG represents erteiravir. Figure 2 It was found that, compared with the DMSO control group, the survival rate of Mycobacterium tuberculosis was not significantly changed after 4 hours of treatment with 10 μM erteiravir, but a significant decrease in the survival rate was observed after 72 hours of treatment. These results indicate that erteiravir can inhibit Mycobacterium tuberculosis infection.

[0047] Example 3 This example demonstrates the determination of the minimum inhibitory concentration (MIC) of erteiravir, including the following steps: Take a 96-well plate. Add 100 μL of ddH2O to each well on the outermost edge. Add 98 μL of 7H9-OADC complete medium to each well of B2-G2, and 50 μL of 7H9-OADC complete medium to each of the remaining wells. Add 2 μL of erteiravir to each well of B2-D2, and 2 μL of isoniazid to each well of E2-G2 as positive controls. Dilute B2-G2 to B10-G10 two-fold. B11-G11 are negative control wells without drug treatment. Finally, add 2 × 10⁻⁶ ppm of ddH2O to each well (except the ddH2O wells). 5 CFU / 50 μL of Mycobacterium tuberculosis H37Rv bacterial culture was sealed with sealing film and incubated in a 37℃ bacterial incubator for 10-14 days. After incubation, the bacterial inhibition of different drug concentrations was observed, and the MIC values ​​were read. The test results are shown in Table 1.

[0048] Table 1 shows the MIC test results of erteiravir and INH against Mycobacterium tuberculosis H37Rv.

[0049] As shown in Table 1, the minimum inhibitory concentration (MIC) of erteiravir against Mycobacterium tuberculosis in vitro is greater than 200 μM, which is much higher than the concentration used at the cellular level. This indicates that the anti-tuberculosis effect of erteiravir at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits the intracellular survival of Mycobacterium tuberculosis by targeting the host and regulating the host's immune response. The MIC of INH against Mycobacterium tuberculosis is 0.02 μg / mL, which is consistent with the concentration range reported in the literature, serving as a positive control to demonstrate the reliability of the experimental data.

[0050] Example 4 This embodiment describes the effect of erteiravir on the survival of Mycobacterium abscesses within macrophages, including the following steps: (1) Infection with Mycobacterium abscessus: THP-1 cells from Example 1 were cultured in 24-well plates at a density of 5 × 10⁶ cells per well. 5 The number of cells was induced to become macrophages using the method described above. One hour before infection, 10 μM erteiravir was added for pretreatment, and DMSO was added as a control. Mycobacterium abscessus ATCC19977 was used to infect cells at MOI=10. After 4 hours, the cells were washed three times with PBS and cultured in 1640 complete medium at 37°C in a 5% CO2 incubator. During this process, 10 μM erteiravir was continuously added.

[0051] (2) CFU count on plate: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and counted at 10... 2 10 3 Diluted plating, incubated at 37°C for approximately three weeks, CFU counted, and results calculated as follows. Figure 3 As shown.

[0052] Figure 3 The figure shows the inhibition results of erteiravir on Mycobacterium abscessus at the THP-1 macrophage level, with DMSO as the control group and EVG as erteiravir. Figure 3 The results showed that, compared with the DMSO control group, the survival rate of Mycobacterium abscessus in THP1 macrophages was significantly reduced after the addition of 10 μM erteiravir.

[0053] The above description is merely an exemplary embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural transformations made using the contents of the present invention specification and drawings under the technical concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. The use of erteiravir in the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria.

2. The application as described in claim 1, characterized in that, The tuberculosis mycobacteria include Mycobacterium tuberculosis H37Rv.

3. The application as described in claim 1, characterized in that, The nontuberculous mycobacteria include Mycobacterium abscessus.

4. The application as described in claim 1, characterized in that, The inhibitor is used to modulate the immune response of macrophages.

5. The use of erteiravir in the preparation of a medicament for treating diseases caused by infection with Mycobacterium tuberculosis or non-tuberculous mycobacteria.

6. The application as described in claim 5, characterized in that, The diseases mentioned include tuberculosis.

7. The application as described in claim 5, characterized in that, The drug also includes at least one of pharmaceutically acceptable excipients, carriers, and diluents.

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