Use of isororientin and pharmaceutical compositions

Abstract

The application discloses application of isoschaftoside and a pharmaceutical composition, relates to the technical field of biological medicine, and the medicine is used for treating diseases caused by mycobacterium tuberculosis or non-tuberculous mycobacterial infection. The application discloses that isoschaftoside has human macrophage level anti-tuberculosis effect for the first time, and the isoschaftoside has the effect of inhibiting intracellular survival of mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level, which indicates that the isoschaftoside has the application value as a medicine for treating multidrug-resistant tuberculosis, and the isoschaftoside can be used as a novel anti-tuberculosis or anti-non-tuberculous mycobacterial medicine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of isozygoflavin and a pharmaceutical composition. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), which primarily affects the lungs but can also affect other organs.

[0003] Currently, tuberculosis treatment primarily relies on anti-tuberculosis drugs, including isoniazid, rifampin, and pyrazinamide. However, the widespread use of antibiotics and the characteristics of tuberculosis treatment and transmission have led to the emergence of multidrug-resistant tuberculosis (MDR-TB). MDR-TB refers to resistance to at least two of the most important anti-tuberculosis drugs, such as isoniazid and rifampin. This means that current anti-tuberculosis drugs are less than 50% effective against MDR-TB, and it can, to some extent, develop into a progressive and incurable disease. Besides pulmonary tuberculosis, the infection rate of nontuberculous mycobacteria (PNTM) in the lungs is increasing year by year, and their importance is increasingly recognized clinically. However, the diagnosis and treatment of PNTM in clinical practice are currently lacking in standardization. Among them, *Mycobacterium abscessatum* is one of the main mycobacterial pathogens causing nontuberculous pulmonary infections. It is a highly drug-resistant opportunistic pathogen, resistant to multiple antibiotics, and difficult to treat. Summary of the Invention

[0004] The main objective of this invention is to propose an application and pharmaceutical composition of isozyraxanthin, aiming to solve the problem of the lack of drugs available in the prior art for the treatment of multidrug-resistant tuberculosis.

[0005] To achieve the above objectives, this invention proposes the application of iso-zellin in the preparation of inhibitors for Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0006] In one embodiment, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv.

[0007] In one embodiment, the nontuberculous mycobacteria include Mycobacterium abscessus.

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

[0009] In one embodiment, the isozelin in the inhibitor has an inhibitory concentration of ≥25 μM against Mycobacterium tuberculosis at the cellular level.

[0010] The present invention also provides the use of iso-euphorbia flavonoids in the preparation of a drug for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacterial infections.

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

[0012] The present invention also provides a pharmaceutical composition comprising isozylan and isoniazid.

[0013] In one embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers, and / or diluents.

[0014] In the technical solution of this invention, it was discovered for the first time that isozygoside has anti-tuberculosis effects at the human macrophage level and inhibits the intracellular survival of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level. This indicates that isozygoside has application value as a drug for treating multidrug-resistant tuberculosis and can be used as a novel anti-tuberculosis or anti-non-tuberculous mycobacteria drug. Attached Figure Description

[0015] 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.

[0016] Figure 1 The graph shows the effect of different concentrations of isopropionol on the viability of THP-1 cells in Example 1 of this invention.

[0017] Figure 2 The graph shows the inhibition results of iso-zellin on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level in Example 2 of the present invention.

[0018] Figure 3 The figure shows the inhibition results of isozygoflavin combined with isoniazid on Mycobacterium tuberculosis H37Rv in THP-1 macrophages in Example 3 of the present invention;

[0019] Figure 4 The figure shows the inhibition results of isozygoflavin on Mycobacterium abscessus at the THP-1 macrophage level in Example 5 of this invention.

[0020] 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

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, 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 cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis), which primarily affects the lungs but can also affect other organs.

[0023] Currently, tuberculosis treatment primarily relies on anti-tuberculosis drugs. These drugs mainly include isoniazid, rifampin, and pyrazinamide. However, with the widespread use of antibiotics and the characteristics of tuberculosis treatment and transmission, multidrug-resistant tuberculosis (MDR-TB) has emerged. MDR-TB refers to resistance to at least two of the most important anti-tuberculosis drugs, such as isoniazid and rifampin. This means that current anti-tuberculosis drugs are less than 50% effective against MDR-TB, and to some extent, it can develop into a progressive, incurable disease.

[0024] Besides tuberculosis, the infection rate of nontuberculous mycobacteria (PNTM) in the lungs is increasing year by year, and their importance is becoming increasingly recognized in clinical practice. However, the diagnosis and treatment of PNTM in clinical practice are still lacking in standardization. Among them, Mycobacterium abscessus is one of the main mycobacterial pathogens causing nontuberculous lung infections. It is a highly drug-resistant opportunistic pathogen that is resistant to multiple antibiotics, making it difficult to treat.

[0025] Therefore, there is still a lack of drugs available for the treatment of multidrug-resistant tuberculosis.

[0026] A weakened immune system, which cannot effectively eradicate Mycobacterium tuberculosis, is the main reason for latent infection, the chronic and protracted course of tuberculosis, and the poor efficacy of BCG vaccination. Regulating the body's immune status is the most promising approach to treating tuberculosis.

[0027] In view of this, the present invention provides the application of isozygoflavin in the preparation of inhibitors of Mycobacterium tuberculosis or Mycobacterium abscessus.

[0028] In the technical solution of this invention, it was discovered for the first time that isozygoside has anti-tuberculosis effects at the human macrophage level and inhibits the intracellular survival of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level. This indicates that isozygoside has application value as a drug for treating multidrug-resistant tuberculosis and can be used as a novel anti-tuberculosis drug or anti-non-tuberculous mycobacteria drug.

[0029] It should be noted that eupatilin is an important flavonoid active ingredient found in the traditional Chinese medicine Artemisia argyi. Although eupatilin is an antibacterial agent, in this invention, it works by modulating the body's immune response to Mycobacterium tuberculosis infection to kill Mycobacterium tuberculosis or non-tuberculous mycobacteria. That is, the eupatilin of this invention is an immunomodulator used to optimize the bactericidal activity of immune cells and control tissue damage caused by inflammatory responses. Its therapeutic target is the immune response mediated by immune cells such as macrophages, and therefore it is not affected by the drug resistance of Mycobacterium tuberculosis, thus exhibiting good therapeutic effects against multidrug-resistant tuberculosis.

[0030] The structural formula of iso-euphorbia flavonoids is shown below:

[0031]

[0032] In some embodiments of the present invention, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv. The isozepine in the inhibitor has a macrophage-level inhibitory effect on the intracellular survival of Mycobacterium tuberculosis, thus tuberculosis can be treated by inhibiting Mycobacterium tuberculosis. The tuberculosis is preferably multidrug-resistant tuberculosis.

[0033] In some embodiments of the present invention, the nontuberculous mycobacteria include Mycobacterium abscessus. The iso-zellin in the inhibitor has the effect of inhibiting the intracellular survival of nontuberculous mycobacteria at the macrophage level, and therefore can be used as a drug for treating nontuberculous mycobacterial infections.

[0034] In some embodiments of the present invention, the inhibitor is used to modulate the immune response of macrophages. The iso-euphorbia fischeri in the inhibitor effectively inhibits the intracellular survival rate of Mycobacterium tuberculosis at the macrophage level.

[0035] In some embodiments of the present invention, the inhibitory concentration of isopropanol in the inhibitor against Mycobacterium tuberculosis at the cellular level is ≥25 μM. That is, the concentration of isopropanol in the inhibitor can be 25 μM, 50 μM, or 100 μM. When the concentration of isopropanol is within the above range, it can ensure that it exerts its inhibitory effect and inhibit the survival rate of Mycobacterium tuberculosis in macrophages without affecting the survival rate of macrophages.

[0036] This invention also provides the use of iso-euphorbia flavonoids in the preparation of medicaments for treating diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria. Because iso-euphorbia flavonoids can effectively inhibit the intracellular survival rate of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level, iso-euphorbia flavonoids can be used to treat diseases caused by Mycobacterium tuberculosis or non-tuberculous mycobacteria.

[0037] In some embodiments of the present invention, the disease includes tuberculosis. Since Mycobacterium tuberculosis is the primary cause of tuberculosis, isosorbide dinitrate can be used to treat tuberculosis caused by Mycobacterium tuberculosis.

[0038] The present invention also provides a pharmaceutical composition comprising isoflavone and isoniazid. That is, the combined use of isoflavone and isoniazid produces a synergistic anti-tuberculosis effect, enhancing the inhibitory effect against Mycobacterium tuberculosis.

[0039] In some embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients, carriers, and / or diluents. The choice of carrier is related to the route of administration and can be selected according to actual needs, while the diluents or excipients are those conventionally used in the pharmaceutical field.

[0040] 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.

[0041] Experimental materials

[0042] Mycobacterium tuberculosis H37Rv (ATCC 27294) was preserved by the Third People's Hospital of Shenzhen.

[0043] Mycobacterium abscessus (ATCC19977) was preserved by the Third People's Hospital of Shenzhen.

[0044] THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0045] Example 1: Effect of iso-euphorbia flavonoids on macrophage survival rate

[0046] 1. Differentiation of THP-1 macrophages

[0047] THP-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in a cell culture incubator at 37°C and 5% CO2. Cells were cultured in 96-well plates at a density of 5 × 10⁶ cells per well. 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 hours, the medium was replaced with complete medium.

[0048] 2. Determination of cell viability

[0049] After culturing the induced macrophages for another 24 hours, isoflavone was added at concentrations of 5 μM, 10 μM, 30 μM, 50 μM, 80 μM, 100 μM, and 150 μM, with each concentration performed in triplicate. These were recorded as experimental wells. Dimethyl sulfoxide (DMSO) was used as a control well, and cell-free culture medium served as a blank well. The cells in each well were cultured at 37°C and 5% CO2 for another 48 hours. Cell viability was assessed using a CCK-8 assay kit: 10 μL of CCK-8 was added to each well, and after incubation for 2 hours, the absorbance at OD450 nm was measured using a microplate reader.

[0050] Cell viability is calculated using the following formula:

[0051] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0052] Wherein, As: absorbance of experimental wells (containing cell culture medium, CCK-8, and different concentrations of isopropanol); Ac: absorbance of control wells (containing cell culture medium, CCK-8, and DMSO); Ab: absorbance of blank wells (containing cell-free culture medium and CCK-8).

[0053] After calculating the cell viability at different concentrations of isoflavone, a bar chart was plotted using GraphPad software. The results are as follows: Figure 1 As shown.

[0054] Figure 1 The results show the effect of different concentrations of isosorbide dinitrate on the viability of THP-1 cells. Figure 1 It was found that, compared with the control wells, treatment with isocyanin at concentrations of 5 μM, 10 μM, 30 μM, 50 μM, 80 μM, 100 μM, and 150 μM had no effect on cell viability, while treatment with 150 μM of isocyanin significantly reduced cell viability. Calculations revealed that the IC50 (half-maximal inhibitory concentration) of isocyanin for THP-1 cells was 336.7 μM.

[0055] Example 2: Inhibitory effect of iso-euphorbia flavonoids on Mycobacterium tuberculosis in macrophages

[0056] 1. Mycobacterium tuberculosis infection

[0057] 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. One hour before infection, the cells were pretreated with 25 μM, 50 μM, and 100 μM isosorbide, respectively, with DMSO as a control. Mycobacterium tuberculosis H37Rv was used to infect the cells at an MOI (Multiplicity of Infection) of 10. Four hours after infection, the cells were washed three times with phosphate-buffered saline (PBS) and then cultured in RPMI 1640 complete medium at 37°C and 5% CO2. During this process, the corresponding concentration of isosorbide was continuously added.

[0058] 2. Plate count of colony-forming units (CFU)

[0059] 4 h and 72 h after THP-1 cell infection, the cells were lysed with 0.025% SDS (sodium dodecyl sulfate), and then subjected to 10... 2 10 3 The diluted cells were plated and incubated at 37°C for approximately three weeks. CFU were then counted, and the results are as follows: Figure 2 As shown.

[0060] Figure 2 This study demonstrates the inhibitory effect of eupatilin on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level. DMSO served as the control group, and eupatilin was used as the eupatilin. Figure 2 It can be seen that, compared with the DMSO control group, the survival rate of Mycobacterium tuberculosis H37Rv was significantly reduced after the addition of 25μM, 50μM and 100μM isozygotene, and the inhibitory effect was dose-dependent.

[0061] Example 3: Inhibitory effect of isozygotene combined with isoniazid on Mycobacterium tuberculosis in macrophages

[0062] 1. Mycobacterium tuberculosis infection

[0063] The THP-1 cells from Example 1 were used in 24-well plates at a density of 2.5 × 10⁶ cells per well. 5The number of cells was induced to become macrophages using the method described above. One hour before infection, the cells were pretreated with 50 μM isoniazid. Mycobacterium tuberculosis H37Rv was used to infect the cells at an MOI of 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, isoniazid / isoniacin was continuously added.

[0064] 2. Coating Count (CFU)

[0065] Cells were lysed with 0.025% SDS at 24, 48, and 72 hours after infection, and then divided into 10 groups. -2 10 -3 Dilute the sample and plate it. Incubate at 37°C for approximately three weeks to count CFU.

[0066] Figure 3 The study investigated the inhibitory effect of isoniazid combined with isoniazid on Mycobacterium tuberculosis H37Rv. In this study, INH represented isoniazid, Eupatilin represented isoniazid, INH+Eupatilin represented isoniazid combined with isoniazid, and DMSO represented the control group. Figure 3 The results showed that isoniazid or isosorbide dinitrate alone could effectively inhibit or kill Mycobacterium tuberculosis in macrophages, and the combination of isoniazid and isosorbide dinitrate significantly enhanced the killing effect compared with the use of either alone.

[0067] Example 4: Determination of the minimum inhibitory concentration (MIC) of isopropionitanol

[0068] 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 from B2 to G2, and 50 μL of 7H9-OADC complete medium to each of the remaining wells. Add 2 μL of isoniazid to each well from B2 to D2, and 2 μL of isoniazid to each well from E2 to G2 as positive controls. Double-dilute wells are used for B2 to G2 to B10 to G10. Wells B11 to G11 are negative controls without any drug treatment. Finally, add 2 × 10⁻⁶ ppm of ddH2O to each well (except the ddH2O wells). 5 A 50 μL solution of Mycobacterium tuberculosis H37Rv was sealed with sealing film and incubated at 37°C for 10 to 14 days. After incubation, the bacterial inhibition at different drug concentrations was observed, and the MIC values ​​were recorded.

[0069] Table 1 shows the MICs of isopropionate and INH against Mycobacterium tuberculosis H37Rv.

[0070] drug MIC Iso-euphorbia >900μM INH 0.02 μg / mL

[0071] As shown in Table 1, the minimum inhibitory concentration (MIC) of iso-euphorbia nitrate against Mycobacterium tuberculosis in vitro is greater than 900 μM, which is much higher than the concentration used at the cellular level. This indicates that the anti-tuberculosis effect of iso-euphorbia nitrate 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 serves as a positive control to demonstrate the reliability of the experimental data.

[0072] Example 5: Inhibitory effect of iso-zellin on Mycobacterium abscessus in macrophages

[0073] 1. Infection with Mycobacterium abscessus

[0074] THP-1 cells from Example 1 were used in a 24-well plate 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, 50 μM isozygotene was added for pretreatment, and DMSO was added as a control. Mycobacterium abscessus ATCC19977 was used to infect the cells at an MOI of 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. Isozygotene was added continuously throughout the process.

[0075] 2. Coating Count (CFU)

[0076] 4 and 72 hours after infection, cells were lysed with 0.025% SDS, and then divided into groups of 10. 2 10 3 Diluted plating, incubated at 37°C for approximately three weeks, CFU counted, and results calculated as follows. Figure 4 As shown, Figure 4 DMSO was used as the control group, and Eupatilin was used as the flavonoid. The CFU of the two groups were calculated. 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 flavonoid.

[0077] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. Use of isoalantolactone in the preparation of an inhibitor of non-tuberculous mycobacteria; said non-tuberculous mycobacteria being Mycobacterium abscessus.

2. Use according to claim 1, wherein The inhibitor is used to modulate the immune response of macrophages.

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