Application of urolithin A in drug preparation and drug

Abstract

This invention discloses the application of urolithin A in the preparation of a drug and the drug itself. In the application of urolithin A in the preparation of a drug, the drug is used to inhibit Mycobacterium tuberculosis. The anti-tuberculosis effect of urolithin A 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 application of urolithin A in the preparation of a drug for inhibiting Mycobacterium tuberculosis is disclosed for the first time in this invention.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of urolithin A in the preparation of drugs and the drugs themselves. Background Technology

[0002] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has consistently ranked among the leading causes of infectious disease morbidity and mortality worldwide for several years. With the widespread use of antibiotics and the characteristics of Mtb's treatment and transmission, the number of patients with multidrug-resistant TB is increasing daily. However, the efficacy of treatment for drug-resistant TB is less than 50%, and to some extent, it can develop into a progressive, incurable disease. Other immunocompromising infections and / or complications require more complex prevention and treatment methods. Summary of the Invention

[0003] The main objective of this invention is to propose the application of urolithin A in the preparation of drugs and the drugs themselves, which aim to effectively inhibit Mycobacterium tuberculosis.

[0004] To achieve the above objectives, in a first aspect, the present invention proposes the application of urolithin A in the preparation of a drug for inhibiting Mycobacterium tuberculosis.

[0005] Optionally, the Mycobacterium tuberculosis includes H37Rv and H37Ra.

[0006] Secondly, the present invention proposes the application of urolithin A in the preparation of a drug for the prevention and / or treatment of a disease, or for reducing the risk of a disease, wherein the disease is tuberculosis.

[0007] Thirdly, the present invention provides a drug for inhibiting Mycobacterium tuberculosis, the drug comprising urolithiasis A.

[0008] Optionally, the drug also includes isoniazid.

[0009] Fourthly, the present invention provides a medicament for the prevention and / or treatment of tuberculosis, the medicament comprising urolithiasis A.

[0010] Optionally, the drug also includes isoniazid.

[0011] Optionally, the drug may further comprise pharmaceutically acceptable excipients, carriers, and / or diluents.

[0012] In the technical solution of this invention, it is discovered for the first time that urolithin A has anti-tuberculosis effects at the human macrophage level. In the application of urolithin A in the preparation of drugs, the drugs can effectively inhibit Mycobacterium tuberculosis. Furthermore, urolithin A can be used to prepare drugs for the prevention and / or treatment of tuberculosis. Attached Figure Description

[0013] 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.

[0014] Figure 1 The dose-response curve of the human macrophage cell line THP-1 to urolithin A provided in Example 1 of the present invention;

[0015] Figure 2 The graph shows the inhibitory effect of urolithin A on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level provided in Example 2 of the present invention.

[0016] Figure 3 This is a graph showing the inhibitory effect of urolithin A on Mycobacterium tuberculosis H37Ra at the THP-1 macrophage level, as provided in Example 3 of the present invention.

[0017] Figure 4 The graph shows the inhibition of Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level by urolithin A, isoniazid, and the combination of urolithin A and isoniazid.

[0018] 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

[0019] 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.

[0020] Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has consistently ranked among the leading causes of infectious disease morbidity and mortality worldwide for several years. With the widespread use of antibiotics and the characteristics of Mtb's treatment and transmission, the number of patients with multidrug-resistant TB is increasing daily. However, the efficacy of treatment for drug-resistant TB is less than 50%, and to some extent, it can develop into a progressive, incurable disease. Other immunocompromising infections and / or complications require more complex prevention and treatment methods.

[0021] In view of this, the present invention proposes a first aspect, namely, the application of urolithin A in the preparation of a drug for inhibiting Mycobacterium tuberculosis.

[0022] It should be noted that currently, it is believed that a weakened immune system, unable to 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. Therefore, in recent years, a new type of immunotherapy—host-directed therapy (HDT)—has been considered an effective measure for treating drug-resistant tuberculosis. HDT refers to the use of 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. The method involves using immunomodulators to optimize the bactericidal activity of immune cells and control tissue damage caused by inflammatory responses. Therefore, HDT is not affected by Mycobacterium tuberculosis resistance and will also have good efficacy in patients with concurrent Mycobacterium tuberculosis infection, such as those with AIDS, diabetes, or rheumatic diseases. However, the key to HDT lies in the selection of immune response targets and immunomodulators.

[0023] Urolithin A (UroA), produced by intestinal flora, is a natural metabolite of ellagitannins, a class of compounds found in pomegranates and some other fruits and nuts, possessing anti-inflammatory, anti-proliferative, and antioxidant properties. In this invention, urolithin A is found for the first time to exhibit anti-tuberculosis activity at the human macrophage level, specifically, it inhibits the intracellular survival of Mycobacterium tuberculosis at the macrophage level. The anti-tuberculosis effect of urolithin A 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 application of urolithin A in the preparation of drugs for inhibiting Mycobacterium tuberculosis is disclosed for the first time in this invention.

[0024] In some embodiments, the drug is used to inhibit Mycobacterium tuberculosis H37Rv and Mycobacterium tuberculosis H37Ra.

[0025] Secondly, the present invention proposes the application of urolithin A in the preparation of a drug for the prevention and / or treatment of a disease, or for reducing the risk of a disease, wherein the disease is tuberculosis.

[0026] In this invention, the term "prevention and / or treatment" includes not only the prevention and / or treatment of disease, but also generally includes reducing or inhibiting the number of pathogens associated with the disease, preventing the onset of the disease, slowing or reversing the progression of the disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and / or alleviating one or more symptoms associated with the disease, reducing the severity and / or duration of the disease and / or any symptoms associated with it and / or preventing a further increase in the severity of the disease and / or any symptoms associated with it, preventing, reducing or reversing any physiological damage caused by the disease, and any pharmacological effects that are generally beneficial to the patient being treated. The pharmaceuticals of this application do not need to achieve a complete cure or eradication of any symptoms or manifestations of the disease to be considered useful therapeutic agents. As recognized in the relevant art, pharmaceuticals used as therapeutic agents may reduce the severity of a given disease state, but do not need to eliminate every manifestation of the disease to be considered useful therapeutic agents. Similarly, preventatively applied treatments constituting feasible preventative agents do not need to completely and effectively prevent the onset of the disease. Simply reducing the impact of the disease on the subject (e.g., by reducing the number or severity of their symptoms, or by increasing the effectiveness of another treatment, or by producing another beneficial effect), or reducing the likelihood of the disease occurring or worsening, is sufficient.

[0027] Thirdly, the present invention provides a drug for inhibiting Mycobacterium tuberculosis, the drug comprising urolithiasis A.

[0028] Optionally, the drug also includes isoniazid.

[0029] Fourthly, the present invention provides a medicament for the prevention and / or treatment of tuberculosis, the medicament comprising urolithiasis A.

[0030] Optionally, the drug also includes isoniazid.

[0031] It should be noted that this invention also found that the combined use of urolithiasis A and isoniazid has a better effect on inhibiting Mycobacterium tuberculosis.

[0032] In some embodiments, the drug further comprises pharmaceutically acceptable excipients, carriers, and / or diluents.

[0033] 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.

[0034] Example 1:

[0035] 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 in a cell culture incubator with 5% CO2. Cells were cultured in 96-well plates at a density of 2.5 × 10⁶ cells per well. 4 The cells were seeded into plates, and PMA at a final concentration of 50 ng / ml was added to stimulate them overnight to differentiate into macrophages. After 24 hours, the medium was replaced with complete culture medium.

[0036] Cell viability determination: After culturing the induced macrophages for another 24 h, urolithin A was added at concentrations of 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM, 120 μM, and 200 μM, with three replicates for each concentration. DMSO was used as the control wells, and cell-free medium was used as the blank wells. The cells were cultured at 37°C and 5% CO2 for another 48 h. Cell viability was detected using the CCK-8 assay kit. 10 μl of CCK-8 was added to each well, and the cells were incubated for 2 h. The absorbance at OD450 nm was then measured using a microplate reader.

[0037] Cell viability is calculated using the following formula:

[0038] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0039] As: Experimental wells (containing cell culture medium, CCK-8, and different concentrations of urolithin A)

[0040] Ac: Control wells (containing cell culture medium, CCK-8, DMSO)

[0041] Ab: Blank wells (cell-free culture medium, CCK-8)

[0042] After calculating the cell viability of different concentrations of urolithin A, dose-response curves were plotted and IC50 was calculated using GraphPad software.

[0043] See Figure 1 , Figure 1 The dose-response curve of THP-1 macrophages to urolithin A shows that the IC50 concentration of urolithin A for THP-1 cells is 149.5 μM, which means that the concentration of urolithin A is 149.5 μM when the THP-1 cell viability is 50%.

[0044] Example 2:

[0045] Mycobacterium tuberculosis infection: THP-1 cells were used in 24-well plates at 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 30 μM, 60 μM, and 90 μM urolithin A, with DMSO as a control. 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. Urolithin A was added throughout the process.

[0046] CFU count by plating: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and counted at 10⁻⁶ ppm. 2 10 3 Dilute the sample and plate it. Incubate at 37°C for approximately three weeks to count CFU.

[0047] Figure 2 The study investigated the inhibitory effect of urolithin A on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level. Results showed that 30 μM, 60 μM, and 90 μM urolithin A effectively inhibited the growth of Mycobacterium tuberculosis H37Rv in macrophages, with inhibition rates of 40–47%, 54–60%, and 70–75%, respectively. This indicates that the survival rate of Mycobacterium tuberculosis H37Rv was significantly reduced after the addition of urolithin A compared to the DMSO control group.

[0048] Example 3

[0049] Mycobacterium tuberculosis infection: THP-1 cells were used in 24-well plates at 5 × 10⁶ cells per well. 5 The number of cells was induced to become macrophages using the method described above. One hour before infection, the cells were pretreated with 30 μM, 60 μM, and 90 μM urolithin A, with DMSO as a control. Mycobacterium tuberculosis H37Ra 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. Urolithin A was added throughout the process.

[0050] CFU count by plating: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and counted at 10⁻⁶ ppm. 2 10 3 Dilute the sample and plate it. Incubate at 37°C for approximately three weeks to count CFU.

[0051] Figure 3The study investigated the inhibitory effect of urolithin A on Mycobacterium tuberculosis H37Ra at the THP-1 macrophage level. Results showed that 30 μM, 60 μM, and 90 μM urolithin A effectively inhibited the growth of Mycobacterium tuberculosis H37Ra within macrophages, with inhibition rates of 39–45%, 50–57%, and 73–80%, respectively. This indicates that the survival rate of Mycobacterium tuberculosis H37Ra was significantly reduced after the addition of urolithin A compared to the DMSO control group.

[0052] Example 4

[0053] Mycobacterium tuberculosis infection: THP-1 cells were used in 24-well plates at 5 × 10⁶ cells per well. 5 The number of cells was induced to become macrophages using the method described above. Urolithin A was added for pretreatment 1 hour before infection, with DMSO as a control. 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 with 5% CO2. The experiment was then divided into four groups: a solvent control group, a urolithin A treatment group, an isoniazid treatment group, and a combined isoniazid and urolithin A treatment group.

[0054] CFU count by plating: 4 and 72 hours after infection, cells were lysed with 0.025% SDS and counted at 10⁻⁶ ppm. 2 10 3 Dilute the sample and plate it. Incubate at 37°C for approximately three weeks to count CFU.

[0055] Figure 4 This study investigated the inhibitory effect of urolithiasis A combined with the first-line clinical drug isoniazid on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level. Results showed that urolithiasis A, isoniazid, and the combination of isoniazid and urolithiasis A all effectively inhibited the growth of Mycobacterium tuberculosis H37Rv within macrophages. Notably, the inhibitory effect of urolithiasis A combined with isoniazid on Mycobacterium tuberculosis H37Rv was significantly higher than that of isoniazid alone. Therefore, the combined use of urolithiasis A and isoniazid shows promise as a combination drug to enhance the therapeutic effect on tuberculosis.

[0056] Example 5:

[0057] Minimum inhibitory concentration (MIC) assay: 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 (B2-G2). Add 50 μL of 7H9-OADC complete medium to each of the remaining wells. Add 2 μL of urolithin A to each well (B2-D2) and 2 μL of isoniazid (INH) to each well (E2-G2) as positive controls. Dilute wells from B2-G2 to B10-G10 (two-fold dilution). Wells B11-G11 are negative controls without drug treatment. Finally, add 50 μL of Mycobacterium tuberculosis H37Rv bacterial suspension (2 × 10⁵ CFU / mL) to each well (except the ddH2O well). Seal the wells and incubate at 37°C for 10-14 days. After incubation, observe the bacterial inhibition at different drug concentrations and read the MIC values.

[0058] Table 1 shows the minimum inhibitory concentrations (MICs) of urolithin A and INH against Mycobacterium tuberculosis H37Rv. The results indicate that the minimum inhibitory concentration of urolithin A against Mycobacterium tuberculosis H37Rv in vitro is greater than 1.2 mM, significantly higher than the concentrations used at the cellular level (30 μM, 60 μM, and 90 μM). This suggests that the anti-tuberculosis effect of urolithin A at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits intracellular survival of Mycobacterium tuberculosis by targeting the host and regulating the host's immune response. The minimum inhibitory concentration of INH against Mycobacterium tuberculosis is 0.02 μg / mL, serving as a positive control to demonstrate the reliability of the experimental data.

[0059] Table 1

[0060] MIC Urolithin A >1.2mM Isoniazid (INH) 0.02 μg / mL

[0061] Example 5:

[0062] Minimum inhibitory concentration (MIC) assay: 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 (B2-G2). Add 50 μL of 7H9-OADC complete medium to each of the remaining wells. Add 2 μL of urolithin A to each well (B2-D2) and 2 μL of isoniazid (INH) to each well (E2-G2) as positive controls. Dilute wells from B2-G2 to B10-G10 (two-fold dilution). Wells B11-G11 are negative controls without drug treatment. Finally, add 50 μL of Mycobacterium tuberculosis H37Ra bacterial suspension containing 2 × 10⁵ CFU / well to each well (except the ddH2O well). Seal the wells and incubate at 37°C for 10-14 days. After incubation, observe the bacterial inhibition at different drug concentrations and read the MIC values.

[0063] Table 1 shows the minimum inhibitory concentrations (MICs) of urolithin A and INH against Mycobacterium tuberculosis H37Ra in vitro. The results indicate that the minimum inhibitory concentration of urolithin A against Mycobacterium tuberculosis H37Ra in vitro is greater than 1.2 mM, significantly higher than the concentrations used at the cellular level (30 μM, 60 μM, and 90 μM). This suggests that the anti-tuberculosis effect of urolithin A at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits intracellular survival of Mycobacterium tuberculosis by targeting the host and regulating the host's immune response. The minimum inhibitory concentration of INH against Mycobacterium tuberculosis was 0.02 μg / mL, serving as a positive control to demonstrate the reliability of the experimental data.

[0064] Table 2

[0065] MIC Urolithin A >1.2mM Isoniazid (INH) 0.02 μg / mL

[0066] 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. The application of urolithin A in the preparation of a drug, characterized in that, The drug is used to inhibit Mycobacterium tuberculosis.

2. The application according to claim 1, characterized in that, The tuberculosis mycobacteria include H37Rv and H37Ra.

Images