Application of eupatilin and pharmaceutical composition

Abstract

The invention discloses application of eupatilin and a pharmaceutical composition, and relates to the technical field of biological medicines, and the medicine is used for treating diseases caused by mycobacterium tuberculosis or nontuberculous mycobacterium infection. According to the application disclosed by the invention, the research finds that the eupatilin has a human macrophage level anti-tuberculosis effect and has an effect of inhibiting intracellular survival of mycobacterium tuberculosis or nontuberculous mycobacterium at a macrophage level for the first time, which indicates that the eupatilin has an application value as a drug for treating multidrug-resistant tuberculosis; the compound can be used as a novel anti-tuberculosis or anti-nontuberculous mycobacterium medicine.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an application of isoeupretinoin and a pharmaceutical composition. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) that primarily affects the lungs but may also involve other organs.

[0003] At present, the treatment of tuberculosis mainly relies on anti-tuberculosis drugs. Anti-tuberculosis drugs mainly include isoniazid, rifampicin, pyrazinamide, etc. However, with the widespread use of antibiotics and the characteristics of tuberculosis treatment and transmission, multidrug-resistant tuberculosis patients have emerged. Multidrug-resistant tuberculosis refers to resistance to at least two of the most important anti-tuberculosis drugs such as isoniazid and rifampicin, that is, the current anti-tuberculosis drugs are less than 50% effective against multidrug-resistant tuberculosis, and to a certain extent, it can develop into a progressive and incurable disease. In addition to pulmonary tuberculosis, the infection rate of pulmonary nontuberculous mycobacteria (PNTM) has increased year by year, and its importance has been increasingly recognized by clinicians. However, the diagnosis and treatment of PNTM in clinical practice is still lacking in standardization. Among them, Mycobacterium abscessus is one of the main mycobacterium pathogens that cause non-tuberculosis pulmonary infections. It is a highly resistant opportunistic pathogen that is resistant to multiple antibiotics and is difficult to treat. Summary of the invention

[0004] The main purpose of the present invention is to provide an application and pharmaceutical composition of isoeilflavin, aiming to solve the problem of lack of drugs that can be used to treat multidrug-resistant tuberculosis in the prior art.

[0005] To achieve the above object, the present invention provides a use of isoeupretinoic acid in the preparation of inhibitors of Mycobacterium tuberculosis or non-tuberculous mycobacteria.

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

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

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

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

[0010] The present invention also provides an application of isoeupretinoic acid in preparing medicines for treating diseases caused by infection with Mycobacterium tuberculosis or non-tuberculous mycobacteria.

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

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

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

[0014] In the technical scheme of the present invention, the present invention studies and discovers for the first time that isoeupretinoic acid has an anti-tuberculosis effect at the human macrophage level, and that isoeupretinoic acid has the effect of inhibiting the intracellular survival of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level, which indicates that isoeupretinoic acid has application value as a drug for treating multidrug-resistant tuberculosis, and that isoeupretinoic acid can be used as a new type of anti-tuberculosis or anti-non-tuberculous mycobacterium 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 This is a result diagram showing the effect of different concentrations of isoeilflavin on THP-1 cell viability in Example 1 provided by the present invention;

[0017] Figure 2 This is a graph showing the inhibition of Mycobacterium tuberculosis H37Rv by isoeilflavin in Example 2 provided by the present invention at the THP-1 macrophage level;

[0018] Figure 3 This is a graph showing the inhibition results of isoeilflavin combined with isoniazid on Mycobacterium tuberculosis H37Rv in THP-1 macrophages in Example 3 provided by the present invention;

[0019] Figure 4 This is a graph showing the inhibition of Mycobacterium abscessus by isoeilflavin in Example 5 provided by the present invention at the THP-1 macrophage level.

[0020] 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

[0021] 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.

[0022] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis) that primarily affects the lungs but may also involve other organs.

[0023] At present, the treatment of tuberculosis mainly relies on anti-tuberculosis drugs. Anti-tuberculosis drugs mainly include isoniazid, rifampicin, pyrazinamide, etc. However, with the widespread use of antibiotics and the characteristics of tuberculosis treatment and transmission, multidrug-resistant tuberculosis patients have emerged. Multidrug-resistant tuberculosis refers to resistance to at least two of the most important anti-tuberculosis drugs such as isoniazid and rifampicin, which means that the current anti-tuberculosis drugs are less than 50% effective against multidrug-resistant tuberculosis, and to a certain extent, it can develop into a progressive, incurable disease.

[0024] In addition to pulmonary tuberculosis, the infection rate of pulmonary nontuberculous mycobacteria (PNTM) has increased year by year, and its importance has been increasingly recognized by clinicians. 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 nontuberculous pulmonary infections. It is a highly resistant opportunistic pathogen that is resistant to multiple antibiotics and is difficult to treat.

[0025] Therefore, there is still a lack of drugs available to treat MDR-TB.

[0026] The main reasons for latent infection, chronic protracted course of tuberculosis and poor effect of BCG vaccination are low immune function and inability to effectively kill tuberculosis bacteria. Regulating the immune status of the body is the most promising measure for treating tuberculosis.

[0027] In view of this, the present invention provides a use of isoeupretinoic acid in the preparation of an inhibitor of Mycobacterium tuberculosis or Mycobacterium abscessus.

[0028] In the technical scheme of the present invention, the present invention studies and discovers for the first time that isoeupretinoic acid has an anti-tuberculosis effect at the human macrophage level, and that isoeupretinoic acid has the effect of inhibiting the intracellular survival of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level, which indicates that isoeupretinoic acid has application value as a drug for treating multidrug-resistant tuberculosis, and that isoeupretinoic acid can be used as a new type of anti-tuberculosis drug or anti-non-tuberculous mycobacterium drug.

[0029] It should be noted that Eupatilin is an important flavonoid active ingredient in the Chinese medicine Artemisia argyi. Although Eupatilin is a bacteriostatic agent, in the present invention, Eupatilin kills Mycobacterium tuberculosis or non-tuberculous mycobacteria by regulating the immune response of the body to Mycobacterium tuberculosis infection. That is, Eupatilin of the present invention is an immunomodulator for optimizing the bactericidal activity of immune cells and controlling tissue damage caused by inflammatory reactions. Its therapeutic target is an immune response mediated by immune cells such as macrophages, so it is not affected by the drug resistance of Mycobacterium tuberculosis, thereby having a good therapeutic effect on multidrug-resistant tuberculosis.

[0030] The structural formula of isoeilanoxanthin is shown below:

[0031]

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

[0033] In some embodiments of the present invention, the non-tuberculous mycobacteria include Mycobacterium abscessus. The isoeuphenin in the inhibitor has the effect of inhibiting the intracellular survival of non-tuberculous mycobacteria at the macrophage level, and thus can be used as a drug for treating non-tuberculous mycobacterium infection.

[0034] In some embodiments of the present invention, the inhibitor is used to regulate the immune response of macrophages. The isoeuphenin in the inhibitor can effectively inhibit the intracellular survival rate of Mycobacterium tuberculosis at the macrophage level.

[0035] In some embodiments of the present invention, the inhibitory concentration of isoeupretinoic acid in the inhibitor on Mycobacterium tuberculosis at the cellular level is ≥25 μM. That is, the concentration of isoeupretinoic acid in the inhibitor can be 25 μM, 50 μM or 100 μM. When the concentration of isoeupretinoic acid is within the above range, it can be ensured that it plays the role of an inhibitor, and the survival rate of Mycobacterium tuberculosis in macrophages can be inhibited without affecting the survival rate of macrophages.

[0036] The present invention also provides an application of isoeupretinoic acid in the preparation of a drug for treating diseases caused by infection with Mycobacterium tuberculosis or non-tuberculous mycobacteria. Since the isoeupretinoic acid can well inhibit the intracellular survival rate of Mycobacterium tuberculosis or non-tuberculous mycobacteria at the macrophage level, the isoeupretinoic acid 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 mainly causes tuberculosis, isoeilflavin can be used to treat tuberculosis caused by Mycobacterium tuberculosis.

[0038] The present invention also provides a pharmaceutical composition, which comprises isoeupretinoic acid and isoniazid. That is, isoeupretinoic acid and isoniazid are used in combination to produce a synergistic anti-tuberculosis effect and improve the inhibitory effect on Mycobacterium tuberculosis.

[0039] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier and / or diluent. The selection of the above 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.

[0040] 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.

[0041] Experimental Materials

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

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

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

[0045] Example 1 Effect of isoeilflavin 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. 5×10 cells were cultured in each well of a 96-well plate. 4 The cells were plated with 100 ng / mL of PMA (Phorbol 12-myristate 13-acetate) and stimulated overnight to differentiate them into macrophages. The culture medium was replaced with complete medium after 24 hours.

[0048] 2. Determination of Cell Viability

[0049] After the above-mentioned induced differentiated macrophages were cultured for 24 hours, isozygote was added, and the concentrations of isozygote were 5μM, 10μM, 30μM, 50μM, 80μM, 100μM, and 150μM, respectively. Three replicates were made for each concentration, which were recorded as experimental wells; dimethyl sulfoxide (DMSO) was used as a control well; the culture medium without cells was a blank well. Each group of cell wells was cultured for 48 hours at 37°C and 5% CO2, and the cell activity was detected using a CCK-8 kit: 10μL CCK-8 was added to each well, and after culturing in an incubator for 2 hours, the absorbance at OD450nm was measured using an enzyme marker.

[0050] The cell viability was calculated according to the following formula:

[0051] Cell survival rate = [(As-Ab) / (Ac-Ab)] × 100%,

[0052] Wherein, As: absorbance of experimental wells (culture medium containing cells, CCK-8, different concentrations of isoetzol); Ac: absorbance of control wells (culture medium containing cells, CCK-8, DMSO); Ab: absorbance of blank wells (culture medium without cells, containing CCK-8).

[0053] After calculating the cell survival rate of different concentrations of isoeilflavin, a bar graph was drawn using GraphPad software. The results are shown in Figure 1 shown.

[0054] Figure 1 The results show the effect of different concentrations of isoeilflavin on THP-1 cell viability. Figure 1 It can be seen that compared with the control wells, 5μM, 10μM, 30μM, 50μM, 80μM, 100μM, and 150μM concentrations of isoeupretinoin treatment had no effect on cell survival rate, and 150μM concentration of isoeupretinoin treatment significantly reduced cell survival rate. Calculation and analysis found that the IC50 (half-maximal inhibitory concentration, Inhibitory Concentration 50) concentration of isoeupretinoin on THP-1 cells was 336.7μM.

[0055] Example 2 Inhibitory effect of isoeilanine on Mycobacterium tuberculosis in macrophages

[0056] 1. Infection with Mycobacterium tuberculosis

[0057] THP-1 cells were plated at 5 × 10 per well in a 24-well plate. 5 The number of cells was induced to become macrophages according to the method in step 1 of Example 1. 25 μM, 50 μM, and 100 μM isoelastin were added for pretreatment 1 h before infection, and the solvent DMSO was used as a control. The cells were infected with Mycobacterium tuberculosis H37Rv at an MOI (multiplicity of infection) of 10. After 4 h of infection, the cells were washed three times with phosphate buffered saline (PBS), and then RPMI1640 complete medium was added to continue culturing in an incubator at 37°C and 5% CO2. During this process, the corresponding concentration of isoelastin was added.

[0058] 2. Count colony-forming units (CFU) on plates

[0059] After 4h and 72h of infection, THP-1 cells were lysed with 0.025% SDS (sodium dodecyl sulfate) and then 2 , 10 3 The lysed cells were plated with the dilution multiple and cultured in a 37°C bacterial incubator for about three weeks. The CFU were counted. The counting results were as follows: Figure 2 shown.

[0060] Figure 2 The inhibition of isoeupatilin on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level. DMSO is the control group and Eupatilin is isoeupatilin. Figure 2 It can be seen that compared with the DMSO control group, the survival rate of Mycobacterium tuberculosis H37Rv was significantly decreased after adding 25μM, 50μM, and 100μM isoelasiatin, and it had a dose-dependent inhibitory effect.

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

[0062] 1. Infection with Mycobacterium tuberculosis

[0063] The THP-1 cells of Example 1 were plated at 2.5×10 5The number of cells was induced to become macrophages according to the above method, and 50 μM isoniazid was added for pretreatment 1 hour before infection. The cells were infected with Mycobacterium tuberculosis H37Rv 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 isoniazid / isoniazid was maintained.

[0064] 2. Plate and count CFU

[0065] After 24, 48, and 72 hours of infection, cells were lysed with 0.025% SDS and cultured 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.

[0066] Figure 3 This is the inhibition of isoniazid combined with eupatilin on Mycobacterium tuberculosis H37Rv, where INH is isoniazid, Eupatilin is isoniazid, INH+Eupatilin is isoniazid combined with isoniazid, and DMSO is the control group. Figure 3 The results showed that isoniazid or isoelastin alone can effectively inhibit or kill Mycobacterium tuberculosis in macrophages, and isoniazid combined with isoelastin can significantly enhance its killing effect compared with either alone.

[0067] Example 4 Detection of Minimum Inhibitory Concentration (MIC) of Isozepin

[0068] Take a 96-well plate, add 100 μL ddH2O to each well at the outermost edge of the 96-well plate, add 98 μL 7H9-OADC full culture medium to each well from B2 to G2 in the 96-well plate, and add 50 μL 7H9-OADC full culture medium to each well. Add 2 μL of isocyanate to each well from B2 to D2, and add 2 μL INH (isoniazid) to each well from E2 to G2 as a positive control. From B2 to G2 to B10 to G10, two-fold dilutions are made, and B11 to G11 are negative control wells without drug treatment. Finally, add 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 10 to 14 days. After taking out, the bacterial inhibition of drugs with different concentrations was observed and the MIC value was read.

[0069] Table 1 shows the MIC of isoeilanine and INH against Mycobacterium tuberculosis H37Rv

[0070] drug MIC Isozepin >900μM INH 0.02 μg / mL

[0071] As shown in Table 1, the minimum inhibitory concentration of isoeilin against Mycobacterium tuberculosis in vitro is greater than 900 μM, which is much greater than the concentration used at the cellular level. It can be seen that the anti-tuberculosis effect of isoeilin at the macrophage level is not directly acting on bacteria as an antibiotic, but by targeting the host to regulate the host immune response to inhibit the intracellular survival of Mycobacterium tuberculosis. The minimum inhibitory concentration of INH against Mycobacterium tuberculosis is 0.02 μg / mL, which is used as a positive control to illustrate the reliability of the experimental data.

[0072] Example 5 Inhibitory effect of isoeilanine on Mycobacterium abscessus in macrophages

[0073] 1. Infection with Mycobacterium abscessus

[0074] The THP-1 cells of Example 1 were plated at 5×10 5 The number of cells was induced to become macrophages according to the above method, and 50 μM isoelastin 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, washed three times with PBS after 4 hours, and added with 1640 complete medium and continued to be cultured in an incubator at 37°C, 5% CO2, and the addition of isoelastin was maintained during this process.

[0075] 2. Plate and count CFU

[0076] After 4 and 72 hours of infection, cells were lysed with 0.025% SDS and 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 Eupatilin was used as isoeupatilin. The CFU of the two groups was calculated. The results showed that compared with the DMSO control group, the survival rate of Mycobacterium abscessus in THP1 macrophages was significantly decreased after the addition of isoeupatilin.

[0077] 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 isoeupretinoic acid in the preparation of an inhibitor of Mycobacterium tuberculosis or non-tuberculous mycobacteria.

2. The use according to claim 1, characterized in that The Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv.

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

4. The use according to claim 1, characterized in that The inhibitor is used to modulate the immune response of macrophages.

5. The use according to claim 1, characterized in that The inhibitory concentration of isoeupretinoic acid in the inhibitor on Mycobacterium tuberculosis at the cellular level is ≥25 μM.

6. Use of isoeupretinoic acid in the preparation of medicines for treating diseases caused by infection with Mycobacterium tuberculosis or non-tuberculous mycobacteria.

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

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises isoelastin and isoniazid.

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

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