Application of chlorinated salicylanilide and medicine

Abstract

The invention discloses application of chlorinated salicylanilide and a medicine, and relates to the technical field of biology. It is found for the first time that chlorinated salicylanilide not only can well inhibit staphylococcus, streptococcus pneumoniae, klebsiella pneumoniae and other pathogenic bacteria, but also can well inhibit mycobacterium tuberculosis and nontuberculous mycobacterium, so that chlorinated salicylanilide can be used for preparing an inhibitor for pathogenic bacteria. Particularly, the compound can be used for preparing an inhibitor of mycobacterium tuberculosis and / or nontuberculous mycobacterium, and can also be used for preparing a medicine for treating diseases caused by pathogenic bacterium infection.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application and medicine of chlorinated salicylanilide. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by the Mycobacterium tuberculosis complex. Mycobacterium tuberculosis (MTB) is the main pathogen of human tuberculosis. Other mycobacteria that may infect humans include Mycobacterium leprae, Mycobacterium avium, Mycobacterium marinum, Mycobacterium kansasii, etc. MTB can infect tissues and organs of the body, among which pulmonary tuberculosis is the most common. BCG is the only vaccine to prevent tuberculosis, but the immune protection it induces only has a certain effect on infants and young children, and the protective effect on adults is not good. At present, the main means to curb tuberculosis is still anti-tuberculosis drug treatment.

[0003] Anti-tuberculosis drugs can be divided into two categories, namely first-line anti-tuberculosis drugs and second-line anti-tuberculosis drugs. First-line anti-tuberculosis drugs include rifampicin, isoniazid, pyrazinamide, etc., which have good efficacy and few side effects and are widely used. Second-line anti-tuberculosis drugs include rifapentine, cycloserine, linezolid, etc. Generally speaking, patients with first-time tuberculosis infection or mild cases should be treated with first-line drugs first. For patients who are ineffective / relapsed with first-line anti-tuberculosis drugs and patients with drug-resistant tuberculosis, second-line anti-tuberculosis drugs can be used for treatment, but second-line drugs not only have poor efficacy, but also have great toxic side effects.

[0004] Under the pressure of drug selection, MTB continues to evolve and develop drug resistance through mechanisms such as single nucleotide polymorphisms, chromosomal gene rearrangements encoding drug targets, enzymes in bacteria, reducing drug accumulation in bacteria or inactivating them. In addition, the various mechanisms of drug resistance also provide time and opportunities for the accumulation of mutations that lead to drug resistance. At present, corresponding drug-resistant mutants have been found for both first-line and second-line anti-tuberculosis drugs. With the increase in the number of patients with drug-resistant tuberculosis, research and development of new anti-tuberculosis drugs is imminent. Summary of the invention

[0005] The main purpose of the invention is to provide an application and medicine of chlorinated salicylanilide, aiming to provide a new anti-tuberculosis medicine.

[0006] To achieve the above object, the present invention provides a use of chlorinated salicylanilide in the preparation of inhibitors of pathogenic bacteria.

[0007] In one embodiment, the pathogenic bacteria include at least one of Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis and non-tuberculous mycobacteria.

[0008] In one embodiment, the Staphylococcus aureus comprises Staphylococcus epidermidis and / or Staphylococcus aureus; and / or,

[0009] The Mycobacterium tuberculosis includes at least one of Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis H37Ra and Mycobacterium tuberculosis BCG; and / or,

[0010] The nontuberculous mycobacteria include Mycobacterium marinum and / or Mycobacterium kansasii.

[0011] In one embodiment, the pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Ra, the minimum inhibitory concentration of chlorinated salicylanilide in the inhibitor to the Mycobacterium tuberculosis H37Ra is 4.0 μg / mL, and the minimum bactericidal concentration of chlorinated salicylanilide in the inhibitor to the Mycobacterium tuberculosis H37Ra is 32 μg / mL; and / or,

[0012] The pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv, and the minimum inhibitory concentration of chlorinated salicylanilide in the inhibitor to the Mycobacterium tuberculosis H37Rv is 4.0 μg / mL; and / or,

[0013] The pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis BCG, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Mycobacterium tuberculosis BCG is 4.0 μg / mL.

[0014] In one embodiment, the pathogenic bacteria include non-tuberculous mycobacteria, the non-tuberculous mycobacteria include Mycobacterium marinum, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor against Mycobacterium marinum is 8 μg / mL; and / or,

[0015] The pathogenic bacteria include non-tuberculous mycobacteria, and the non-tuberculous mycobacteria include Mycobacterium kansasii. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Mycobacterium kansasii is 8 μg / mL.

[0016] In one embodiment, the pathogenic bacteria include Staphylococcus, the Staphylococcus includes Staphylococcus epidermidis, the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Staphylococcus epidermidis is 0.5 μg / mL, and the minimum bactericidal concentration of salicylanilide chloride in the inhibitor to the Staphylococcus epidermidis is 2 μg / mL; and / or,

[0017] The pathogenic bacteria include Staphylococcus, and the Staphylococcus includes Staphylococcus aureus. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Staphylococcus aureus is 0.125 μg / mL.

[0018] In one embodiment, the pathogenic bacteria include Streptococcus pneumoniae, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor against the Streptococcus pneumoniae is 0.5 μg / mL.

[0019] In one embodiment, the pathogenic bacteria include Klebsiella pneumoniae, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor against Klebsiella pneumoniae is 0.5 μg / mL.

[0020] The present invention also provides a medicine, which comprises salicylanilide chloride and an anti-tuberculosis drug, wherein the anti-tuberculosis drug comprises rifampicin and / or isoniazid.

[0021] In the technical solution of the present invention, it is found for the first time that chlorinated salicylanilide can not only effectively inhibit pathogenic bacteria such as Staphylococcus aureus, Streptococcus pneumoniae and Klebsiella pneumoniae, but also effectively inhibit Mycobacterium tuberculosis and non-tuberculous mycobacteria. Therefore, chlorinated salicylanilide can be used to prepare inhibitors of pathogenic bacteria, especially inhibitors of Mycobacterium tuberculosis and / or non-tuberculous mycobacteria, and can also be used to prepare drugs for treating diseases caused by pathogenic bacteria infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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.

[0023] Figure 1 This is a graph showing the MIC test results of salicylanilide chloride on H37Ra in Example 1 provided by the present invention;

[0024] Figure 2 This is a graph showing the MIC test results of salicylanilide chloride on H37Rv in Example 1 provided by the present invention;

[0025] Figure 3 This is a graph showing the MIC test results of salicylanilide chloride against Mycobacterium kansasii in Example 1 provided by the present invention;

[0026] Figure 4 This is a graph showing the MIC test results of salicylanilide chloride on Mycobacterium marinum in Example 1 provided by the present invention;

[0027] Figure 5 This is a graph showing the toxicity results of salicylanilide chloride on 293T cells in Example 2 provided by the present invention;

[0028] Figure 6 This is a graph showing the toxicity of salicylanilide chloride to HEPG2 cells in Example 2 provided by the present invention.

[0029] 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

[0030] 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.

[0031] Tuberculosis (TB) is a chronic infectious disease caused by the Mycobacterium tuberculosis complex. Mycobacterium tuberculosis (MTB) is the main pathogen of human tuberculosis. Other mycobacteria that may infect humans include Mycobacterium leprae, Mycobacterium avium, Mycobacterium marinum, Mycobacterium kansasii, etc. MTB can infect tissues and organs of the body, among which pulmonary tuberculosis is the most common. BCG is the only vaccine to prevent tuberculosis, but the immune protection it induces only has a certain effect on infants and young children, and the protective effect on adults is not good. At present, the main means to curb tuberculosis is still anti-tuberculosis drug treatment.

[0032] Anti-tuberculosis drugs can be divided into two categories, namely first-line anti-tuberculosis drugs and second-line anti-tuberculosis drugs. First-line anti-tuberculosis drugs include rifampicin, isoniazid, pyrazinamide, etc., which have good efficacy and few side effects and are widely used. Second-line anti-tuberculosis drugs include rifapentine, cycloserine, linezolid, etc. Generally speaking, patients with first-time tuberculosis infection or mild cases should be treated with first-line drugs first. For patients who are ineffective / relapsed with first-line anti-tuberculosis drugs and patients with drug-resistant tuberculosis, second-line anti-tuberculosis drugs can be used for treatment, but second-line drugs not only have poor efficacy, but also have great toxic side effects.

[0033] Under the pressure of drug selection, MTB continues to evolve and develop drug resistance through mechanisms such as single nucleotide polymorphisms, chromosomal gene rearrangements encoding drug targets, enzymes in bacteria, reducing drug accumulation in bacteria or inactivating them. In addition, the various mechanisms of drug resistance also provide time and opportunities for the accumulation of mutations that lead to drug resistance. At present, corresponding drug-resistant mutants have been found for both first-line and second-line anti-tuberculosis drugs. With the increase in the number of patients with drug-resistant tuberculosis, research and development of new anti-tuberculosis drugs is imminent.

[0034] In addition, the incidence and prevalence of non-tuberculous mycobacteria (NTM) diseases are increasing, even exceeding the incidence and prevalence of tuberculosis. However, most NTM are resistant to commonly used anti-mycobacterial drugs.

[0035] In view of this, the present invention provides a use of chlorinated salicylanilide in preparing an inhibitor of pathogenic bacteria.

[0036] In the technical solution of the present invention, it is found for the first time that chlorinated salicylanilide can not only effectively inhibit pathogenic bacteria such as Staphylococcus aureus, Streptococcus pneumoniae and Klebsiella pneumoniae, but also effectively inhibit Mycobacterium tuberculosis and non-tuberculous mycobacteria. Therefore, chlorinated salicylanilide can be used to prepare inhibitors of pathogenic bacteria, especially inhibitors of Mycobacterium tuberculosis and / or non-tuberculous mycobacteria, and can also be used to prepare drugs for treating diseases caused by pathogenic bacteria infection.

[0037] The specific chemical formula of the chlorinated salicylanilide (Niclosamide) is as follows:

[0038]

[0039] In some embodiments of the present invention, the pathogenic bacteria include at least one of Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis and non-tuberculous mycobacteria. The pathogenic bacteria can cause various infections in different clinical environments, such as Staphylococcus aureus can form biofilms on the surface of medical devices to increase infection complexity and antibiotic resistance, Klebsiella pneumoniae is prone to cause lung infection, Mycobacterium tuberculosis is prone to cause pulmonary tuberculosis, and the clinical manifestations and imaging features of non-tuberculous mycobacterium infection are often similar to tuberculosis. In some embodiments of the present invention, the Staphylococcus aureus includes Staphylococcus epidermidis and / or Staphylococcus aureus. In some embodiments of the present invention, the Mycobacterium tuberculosis includes at least one of Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis H37Ra and Mycobacterium tuberculosis BCG. The above-mentioned Mycobacterium tuberculosis is an important member of the Mycobacterium tuberculosis complex. In some embodiments of the present invention, the non-tuberculous mycobacteria include Mycobacterium marinum and / or Mycobacterium kansasii.

[0040] In some embodiments of the present invention, the pathogenic bacteria include Mycobacterium tuberculosis, and the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Ra. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor for the Mycobacterium tuberculosis H37Ra is 4.0 μg / mL, and the minimum bactericidal concentration of salicylanilide chloride in the inhibitor for the Mycobacterium tuberculosis H37Ra is 32 μg / mL. H37Ra is a derivative strain of H37Rv and has partial pathogenicity. The minimum inhibitory concentration is also known as MIC. The minimum inhibitory concentration of 4.0 μg / mL indicates that salicylanilide chloride can effectively inhibit the growth of H37Ra at a concentration of 4.0 μg / mL. The low MIC value means that salicylanilide chloride has a strong inhibitory effect on H37Ra. The minimum bactericidal concentration is MBC (Minimum Bactericidal Concentration). The minimum bactericidal concentration refers to the lowest drug concentration that can kill more than 99.9% of the initial test bacterial concentration within a certain period of time (usually 24 hours). Unlike the minimum inhibitory concentration (MIC), MBC requires not only the inhibition of bacterial growth, but also a significant reduction in the number of bacteria. MBC of 32μg / mL indicates that salicylanilide chloride can effectively kill Mycobacterium tuberculosis H37Ra at a concentration of 32μg / mL.

[0041] In some embodiments of the present invention, the pathogenic bacteria include Mycobacterium tuberculosis, and the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Mycobacterium tuberculosis H37Rv is 4.0 μg / mL. H37Rv is a typical reference strain of Mycobacterium tuberculosis and has complete pathogenicity. The MIC is 4.0 μg / mL, that is, salicylanilide chloride can effectively inhibit the growth of H37Rv at a concentration of 4.0 μg / mL, indicating that salicylanilide chloride can effectively inhibit H37Rv.

[0042] In some embodiments of the present invention, the pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis BCG, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Mycobacterium tuberculosis BCG is 4.0 μg / mL. BCG is a live attenuated vaccine strain of Mycobacterium tuberculosis. The MIC is 4.0 μg / mL, that is, salicylanilide chloride can effectively inhibit the growth of BCG at a concentration of 4.0 μg / mL, indicating that salicylanilide chloride can effectively inhibit BCG.

[0043] In some embodiments of the present invention, the pathogenic bacteria include non-tuberculous mycobacteria, the non-tuberculous mycobacteria include Mycobacterium marinum, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to Mycobacterium marinum is 8 μg / mL. That is, salicylanilide chloride can effectively inhibit the growth of Mycobacterium marinum at a concentration of 8 μg / mL, indicating that salicylanilide chloride can effectively inhibit Mycobacterium marinum.

[0044] NTM is ubiquitous in the environment and can cause human diseases, including lung infections, skin and soft tissue infections, etc. In some embodiments of the present invention, the pathogenic bacteria include non-tuberculous mycobacteria, and the non-tuberculous mycobacteria include Mycobacterium kansasii. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor against Mycobacterium kansasii is 8 μg / mL. That is, salicylanilide chloride can effectively inhibit the growth of Mycobacterium kansasii at a concentration of 8 μg / mL, indicating that salicylanilide chloride can effectively inhibit Mycobacterium kansasii.

[0045] In some embodiments of the present invention, the pathogenic bacteria include Staphylococcus, the Staphylococcus includes Staphylococcus epidermidis, the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Staphylococcus epidermidis is 0.5 μg / mL, and the minimum bactericidal concentration of salicylanilide chloride in the inhibitor to the Staphylococcus epidermidis is 2 μg / mL. The MBC of 0.5 μg / mL indicates that salicylanilide chloride can effectively kill Staphylococcus epidermidis at a concentration of 0.5 μg / mL.

[0046] In some embodiments of the present invention, the pathogenic bacteria include Staphylococcus, the Staphylococcus includes Staphylococcus aureus, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Staphylococcus aureus is 0.125 μg / mL.

[0047] The pathogenic bacteria include Streptococcus pneumoniae, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Streptococcus pneumoniae is 0.5 μg / mL. That is, salicylanilide chloride can effectively inhibit the growth of Streptococcus pneumoniae at a concentration of 0.5 μg / mL, indicating that salicylanilide chloride can effectively inhibit Streptococcus pneumoniae.

[0048] The pathogenic bacteria include Klebsiella pneumoniae, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Klebsiella pneumoniae is 0.5 μg / mL. That is, salicylanilide chloride can effectively inhibit the growth of Klebsiella pneumoniae at a concentration of 0.5 μg / mL, indicating that salicylanilide chloride can effectively inhibit Klebsiella pneumoniae.

[0049] The present invention also provides an antibacterial agent, which includes salicylanilide chloride and an anti-tuberculosis drug, wherein the anti-tuberculosis drug includes rifampicin and / or isoniazid. Since salicylanilide chloride has a strong inhibitory effect on the above-mentioned Mycobacterium tuberculosis, salicylanilide chloride can also be used in combination with an existing antibacterial agent (such as rifampicin, isoniazid, etc.) for inhibiting Mycobacterium tuberculosis infection to improve the antibacterial effect.

[0050] The present invention also provides a medicine, which includes salicylanilide chloride and an anti-tuberculosis drug, wherein the anti-tuberculosis drug includes rifampicin and / or isoniazid. Since salicylanilide chloride has a strong inhibitory effect on the above-mentioned non-tuberculous mycobacteria and mycobacterium tuberculosis, it can inhibit tuberculosis caused by infection with the above-mentioned non-tuberculous mycobacteria or mycobacterium tuberculosis. At the same time, compared with existing anti-tuberculosis drugs, salicylanilide chloride has lower liver toxicity and kidney toxicity. Therefore, salicylanilide chloride can be used as a new type of anti-tuberculosis drug with high efficiency and low toxicity.

[0051] 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.

[0052] Experimental Materials

[0053] 1. Strains

[0054] The strains and sources used in the experimental verification process of the present invention are as follows:

[0055] MTB H37Ra (ATCC 25177), MTB H37Rv (ATCC 27294), BCG (ATCC 35733), Mycobacterium marinum (ATCC 927), and Mycobacterium kansasii (ATCC 12478) were deposited by the Shenzhen National Clinical Research Center for Infectious Diseases. Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis (ATCC 12228), Klebsiella pneumoniae (ATCC 700603), and Streptococcus pneumoniae (ATCC 49619) were deposited at the Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences.

[0056] 2. Main reagents

[0057] Middle Brook OADC Enrichment, Middle Brook 7H11 Agar, and Middle Brook 7H9 Broth were purchased from Becton, Dickinson and Company, USA;

[0058] MH broth and MH agar were purchased from Beijing Solebow Technology Co., Ltd.;

[0059] Phosphate buffered saline (PBS) was purchased from Gibco (Thermo Fisher SCIENTIFIC);

[0060] Dimethyl sulfoxide (DMSO), isoniazid (INH), rifampicin (RIF), ethambutol, and salicylanilide chloride were purchased from Sigma Aldrich;

[0061] Resazurin and glycerol were purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0062] CCK-8 kit was purchased from Amresco, fetal bovine serum (FBS) was purchased from Guangzhou Eukaryotic Biotechnology Co., Ltd., and DMEM cell culture medium was purchased from Beyotime Biotechnology Co., Ltd.;

[0063] 293T cells and HEPG2 cells were purchased from Bio-Tech.

[0064] 3. Preparation of culture medium and main solutions

[0065] Middle Brook 7H9 medium: 4.7 g 7H9 powder, 2 mL glycerol (0.05% Tween-80 can be added as needed), dilute to 900 mL with distilled water, sterilize with high pressure steam at 121°C for 15 min. After cooling to about 45°C, add 100 mL OADC supplement solution and store at 4°C.

[0066] Middle Brook 7H11 medium: 21g 7H11 powder, 5mL glycerol, dilute to 900mL with distilled water, sterilize at 121℃ high temperature and high pressure steam for 15min. When the temperature cools to about 45℃, add 100mL OADC supplement solution, mix well, pour into 90mm plate at 18-20mL / dish, dry and store at room temperature.

[0067] MH broth medium: 24g powder, add distilled water to 1L, sterilize with high pressure steam at 121℃ for 15min, and store at 4℃.

[0068] MH agar medium: 36.5g powder, add distilled water to 1L, sterilize at 121℃ with high pressure steam for 15min. When the temperature cools to about 45℃, pour 18-20mL / dish into a 90mm plate, dry and store at room temperature.

[0069] Weigh 1 g of resazurin (sodium salt) powder and dissolve it in 100 mL of ddH2O to obtain a 1% resazurin stock solution, which is then diluted with ddH2O to a 0.05% resazurin solution.

[0070] DMEM cell culture medium supplemented with 10% FBS was used to culture 293T cells and HEPG2 cells.

[0071] Example 1

[0072] 1. Strain activation

[0073] Mycobacterium tuberculosis H37Rv (ATCC 27294), BCG (ATCC 35733), Mycobacterium tuberculosis H37Ra (ATCC25177), Mycobacterium marinum (ATCC 927), and Mycobacterium kansasii (ATCC 12478) were all carried out in a biosafety level 3 laboratory (BSL-3) in strict accordance with the relevant experimental technical standard operating procedures. The specific operations are as follows:

[0074] (1) Thaw the bacterial strain stored at -80°C in the laboratory, use an inoculation loop to dip the bacterial strain and streak it in Middle Brook 7H11 medium, and invert it in a 5% CO2 incubator at 37°C for about 4 weeks.

[0075] (2) A single colony on Middle Brook 7H11 medium was selected and inoculated into Middle Brook 7H9 liquid medium and cultured at 37°C for about 2 weeks until the strain reached the logarithmic growth phase.

[0076] (3) Collect bacterial suspension in the logarithmic growth phase, centrifuge at 4000 r / min for 10 min, discard the supernatant, add an appropriate amount of Middle Brook 7H9 medium to resuspend the precipitate, use a bacterial disperser to fully disperse the precipitate, let it stand for 5 to 10 min, collect the supernatant, divide it into cell cryopreservation tubes, and store it at -80°C.

[0077] (4) Randomly take one tube of bacterial frozen stock solution, take an appropriate amount of bacterial solution and dilute it in multiples with sterile PBS, use a disposable spreading stick to evenly spread it on a Middle Brook 7H11 medium plate, and culture it upside down at 37°C in a 5% CO2 incubator for about 4 weeks; when bacterial colonies can be clearly distinguished with the naked eye, calculate the colony forming unit (CFU).

[0078] 2. Strain dilution

[0079] The bacterial strains cultured to the logarithmic phase were measured using a bacterial dispersion instrument and their turbidity was diluted to 1 McFarland turbidity.

[0080] 3. Preparation and dilution of salicylanilide chloride compounds

[0081] The salicylanilide chloride compound is diluted to 10 mg / mL with DMSO (dimethyl sulfoxide) to obtain a salicylanilide chloride solution.

[0082] The specific operation process is to weigh 10 mg of salicylanilide chloride compound and dissolve it in 1 mL of cell-grade DMSO.

[0083] 4. In vitro MIC determination of salicylanilide chloride against Mycobacterium tuberculosis

[0084] (1) Anti-tuberculosis activity was determined using a sterile 96-well culture plate with a total volume of 100 μL in each well.

[0085] (2) Add 100 μL of 5×10 5 CFU / mLH37Rv, H37Ra and BCG strain culture fluid, and then add different volumes of salicylanilide chloride solution, so that the final concentration of salicylanilide chloride in the final system is 64.0μg / mL, 32.0μg / mL, 16.0μg / mL, 8.0μg / mL, 4.0μg / mL, 2.0μg / mL, 1.0μg / mL, 0.5μg / mL, 0.25μg / mL, 0.125μg / mL. At the same time, take a new 96-well plate, add 100μL of 5×10 5CFU / mL of culture fluid of H37Rv, H37Ra and BCG strains, and then adding different volumes of isoniazid (INH) solution as a positive control, so that the final concentration of isoniazid in the final system is the same as that of salicylanilide chloride, in the above 10 concentration gradient settings;

[0086] (3) Three growth positive control wells (equal volume of DMSO without salicylanilide chloride) and three growth negative control wells (equal volume of culture medium without any Mycobacterium tuberculosis) were set up in the culture plate for each strain;

[0087] (4) Cover the 96-well plate and seal it with sealing film, then place it in an incubator at 37°C;

[0088] (5) On the sixth day of incubation, add 0.05% resazurin solution to each well at 10% of the total volume of the solution, and continue to incubate at 37°C in an incubator for 4 h;

[0089] (6) Observe the positive growth control well and the negative growth control well. When a clear difference is observed between the two, observe the color of each test well. The concentration corresponding to the blue culture solution under the lowest concentration sample is interpreted as the minimum inhibitory concentration. Determine inhibition or resistance and record the results.

[0090] (7) Observe again on the 8th day to confirm and record the results.

[0091] The results showed that salicylanilide chloride had an effect on the MTB standard strain H37Ra ( Figure 1 , three rows for three parallel experimental groups), H37Rv( Figure 2 , three rows as three parallel experimental groups) and the minimum inhibitory concentration (MIC) of BCG were 4.0 μg / mL, 4.0 μg / mL and 4.0 μg / mL, respectively.

[0092] 5. In vitro MBC determination of salicylanilide chloride against Mycobacterium tuberculosis

[0093] (1) Anti-tuberculosis activity was determined using a sterile 96-well culture plate with a total volume of 100 μL in each well.

[0094] (2) Add 100 μL of 5×10 5 CFU / mL Mycobacterium tuberculosis H37Ra, add salicylanilide chloride at a concentration of 1 to 32 times the MIC, and culture at 37°C for 5 to 7 days. The exact initial bacterial count was determined by plating.

[0095] (3) Take all the liquid in each well (100 μL) and apply it to a 7H11 plate.

[0096] (4) Taking the initial colony count in the culture medium as a reference, the concentration of the compound that reduces the colony count by 99.9% is the minimum bactericidal concentration.

[0097] The results show that: Figure 2 As shown, the MBC of salicylanilide chloride against Mycobacterium tuberculosis H37Ra is 8×MIC=32 μg / mL.

[0098] 6. In vitro MIC determination of salicylanilide against nontuberculous mycobacteria

[0099] (1) Antimicrobial activity was determined using a sterile 96-well culture plate with a total volume of 100 μL in each well.

[0100] (2) Add 100 μL of 5×10 5 CFU / mL culture medium of Mycobacterium marinum (ATCC 927) and Mycobacterium kansasii (ATCC 12478) strains, and then add different volumes of salicylanilide chloride solution, so that the final concentration of salicylanilide chloride in the final system is 64.0μg / mL, 32.0μg / mL, 16.0μg / mL, 8.0μg / mL, 4.0μg / mL, 2.0μg / mL, 1.0μg / mL, 0.5μg / mL, 0.25μg / mL, 0.125μg / mL. At the same time, take a new 96-well plate, add 100μL of 5×10 5 CFU / mL culture fluid of Mycobacterium marinum (ATCC 927) and Mycobacterium kansasii (ATCC 12478) strains, and then different volumes of rifampicin (RIF) solution were added as a positive control, so that the final concentration of rifampicin in the final system was the same as that of salicylanilide chloride, in the above 10 concentration gradient settings;

[0101] (3) setting up three growth positive control wells (equal volume of DMSO without salicylanilide chloride) and three growth negative control wells (equal volume of culture medium without any non-tuberculosis bacilli) in the culture plate;

[0102] (4) Cover the 96-well plate and seal it with sealing film, then place it in an incubator at 37°C;

[0103] (5) On the third day of incubation, add 0.05% resazurin solution at 10% of the total volume and continue to incubate at 37°C in an incubator for 4 h;

[0104] (6) Observe the positive growth control wells and the negative growth control wells. When a clear difference is observed between the two, observe the color of each test well and interpret the concentration corresponding to the blue culture solution under the lowest concentration sample as the minimum inhibitory concentration. Determine inhibition or resistance and record the results.

[0105] The results showed that salicylanilide chloride had an effect on Mycobacterium kansasii ( Figure 3) and Mycobacterium marinum ( Figure 4 ) strains were 4 μg / mL and 4 μg / mL, respectively.

[0106] 7. In vitro MIC and MBC determination of salicylanilide against Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae and Klebsiella pneumoniae

[0107] MIC and MBC determinations were performed in a biosafety level 2 laboratory (BSL-2) using MH broth and MH agar media.

[0108] (1) The MIC determination method is similar to that in step 6 of “In vitro MIC determination of salicylanilide against nontuberculous mycobacteria” except that the strains are different, and the overnight culture is read using resazurin colorimetric reading;

[0109] (2) Except for the difference in strains, the MBC determination method is similar to the "In vitro MBC determination of salicylanilide against Mycobacterium tuberculosis" in step 5. The values ​​are read by plate count after the strains are treated with the compounds overnight.

[0110] The results showed that the MICs of salicylanilide chloride against Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae and Klebsiella pneumoniae were 0.5μg / mL, 0.125μg / mL, 0.5μg / mL and 0.5μg / mL, respectively, and the MBC of salicylanilide chloride against Staphylococcus epidermidis was 2μg / mL.

[0111] Example 2 Cytotoxicity of salicylanilide chloride to liver and kidney cells

[0112] (1) Take 293T cells and HEPG2 cells in the logarithmic growth phase, digest them, count them, and dilute them to an appropriate concentration in DMEM medium containing 10% fetal bovine serum (FBS). 3 The cells were inoculated into 96-well cell culture plates and incubated in a 37°C, 5% CO2 cell culture incubator;

[0113] (2) Dilute the salicylanilide chloride compound to 1000 μg / mL with DMSO (dimethyl sulfoxide) to obtain a salicylanilide chloride solution, and dilute the drug in a 3-fold ratio, with a total of 9 to 10 concentration gradients. After the 293T cells in (1) are cultured for 24 hours and the cells are attached to the wall, they are added to the drugs with different dilution concentrations, with 3 parallel wells in each group, and the cells are treated with drugs for 72 hours;

[0114] (3) Add the detection solution according to the CCK-8 kit, obtain the corresponding absorbance on the microplate reader, and calculate the cell survival rate, cell survival rate (%) = (addition cell OD-background OD) / (control cell OD-background OD) × 100%.

[0115] (4) GraphPad Prism 8.0.2 was used to fit the curve and calculate the IC 50 (the half toxic concentration in drug toxicity tests), the results are as follows Figure 5 and Figure 6 shown.

[0116] The results show that: Figure 5 and Figure 6 It can be seen that the IC of chlorinated salicylanilide compounds on 293T cells 50 The value is 91.160 μg / mL, and the IC 50 The value is 0.2300 μg / mL, which indicates that the biological toxicity of the chlorinated salicylanilide compound is low.

[0117] 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 chlorinated salicylanilide in the preparation of inhibitors of pathogenic bacteria.

2. The use according to claim 1, wherein the pathogenic bacteria include at least one of Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Mycobacterium tuberculosis and non-tuberculous mycobacteria.

3. The use according to claim 2, characterized in that The staphylococci include Staphylococcus epidermidis and / or Staphylococcus aureus; and / or, The Mycobacterium tuberculosis includes at least one of Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis H37Ra and Mycobacterium tuberculosis BCG; and / or, The nontuberculous mycobacteria include Mycobacterium marinum and / or Mycobacterium kansasii.

4. The use according to claim 2, characterized in that The pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Ra, the minimum inhibitory concentration of chlorinated salicylanilide in the inhibitor to the Mycobacterium tuberculosis H37Ra is 4.0 μg / mL, and the minimum bactericidal concentration of chlorinated salicylanilide in the inhibitor to the Mycobacterium tuberculosis H37Ra is 32 μg / mL; and / or, The pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis H37Rv, and the minimum inhibitory concentration of chlorinated salicylanilide in the inhibitor to the Mycobacterium tuberculosis H37Rv is 4.0 μg / mL; and / or, The pathogenic bacteria include Mycobacterium tuberculosis, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis BCG, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Mycobacterium tuberculosis BCG is 4.0 μg / mL.

5. The use according to claim 2, characterized in that The pathogenic bacteria include non-tuberculous mycobacteria, the non-tuberculous mycobacteria include Mycobacterium marinum, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to Mycobacterium marinum is 8 μg / mL; and / or, The pathogenic bacteria include non-tuberculous mycobacteria, and the non-tuberculous mycobacteria include Mycobacterium kansasii. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Mycobacterium kansasii is 8 μg / mL.

6. The use according to claim 2, characterized in that The pathogenic bacteria include Staphylococcus, the Staphylococcus includes Staphylococcus epidermidis, the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Staphylococcus epidermidis is 0.5 μg / mL, and the minimum bactericidal concentration of salicylanilide chloride in the inhibitor to the Staphylococcus epidermidis is 2 μg / mL; and / or, The pathogenic bacteria include Staphylococcus, and the Staphylococcus includes Staphylococcus aureus. The minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Staphylococcus aureus is 0.125 μg / mL.

7. The use according to claim 2, characterized in that The pathogenic bacteria include Streptococcus pneumoniae, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Streptococcus pneumoniae is 0.5 μg / mL.

8. The use according to claim 2, characterized in that The pathogenic bacteria include Klebsiella pneumoniae, and the minimum inhibitory concentration of salicylanilide chloride in the inhibitor to the Klebsiella pneumoniae is 0.5 μg / mL.

9. A drug, characterized in that The drugs include salicylanilide chloride and anti-tuberculosis drugs, and the anti-tuberculosis drugs include rifampicin and / or isoniazid.

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