Application of cefmenoxime hydrochloride in preparation of medicine for treating diseases caused by mycobacterium tuberculosis infection and / or nontuberculous mycobacterium infection

Abstract

The invention discloses application of cefmenoxime hydrochloride in preparation of a medicine for treating diseases caused by mycobacterium tuberculosis infection and / or nontuberculous mycobacterium infection, and belongs to the technical field of biological medicine. The invention provides an application of cefmenoxime hydrochloride in preparation of a medicine for treating diseases caused by mycobacterium tuberculosis infection and / or nontuberculous mycobacterium infection, mycobacterium tuberculosis comprises at least one of H37Rv, H37Ra and BCG, and nontuberculous mycobacterium comprises mycobacterium abscessus. It is found that the medicine exerts the antibacterial effect by inhibiting synthesis of bacterial cell walls and influencing synthesis of bacterial protein, and the medicine can effectively inhibit tuberculosis caused by mycobacterium tuberculosis infection and pulmonary infection, skin and soft tissue infection, lymphadenitis and other diseases caused by nontuberculous mycobacterium infection, so that the medicine has a good application prospect. The cefmenoxime hydrochloride can be used as a novel high-efficiency and low-toxicity anti-tuberculosis drug.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application of cefmenoxime hydrochloride in the preparation of a medicine for treating diseases caused by mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by the Mycobacterium tuberculosis complex. Tuberculosis can affect multiple organs throughout the body, with pulmonary tuberculosis being the most common. Mycobacterium tuberculosis (MTB) is the main pathogen of human tuberculosis. Mycobacterium tuberculosis is sensitive to streptomycin, isoniazid, rifampicin, ethambutol, pyrazinamide, etc., but it is prone to drug resistance due to long-term and frequent use of drugs in clinical treatment, and the continuous evolution of MTB also promotes the emergence of drug-resistant strains. Under the pressure of drugs, the expression of some MTB genes will undergo adaptive changes, leading to a variety of inherent drug resistance mechanisms.

[0003] At present, corresponding drug-resistant mutants have been found for both first-line and second-line anti-tuberculosis drugs. In recent years, although some new drugs have been successfully developed and applied to clinical practice, such as bedaquiline (Bdq), delamanid (Dlm), PA-824 and SQ-109. Bdq is a diarylquinoline that can specifically inhibit ATP synthase. However, MTB has been found to have a rich intrinsic drug resistance mechanism to Bdq. Dlm and PA-824 have bactericidal effects on dormant and intracellular MTB. However, any abnormality of the enzyme encoded by the glucose-6-phosphate dehydrogenase gene will lead to an increase in the resistance level of Dlm and PA-824 in MTB. In addition, the large side effects limit the widespread application of some new drugs. Therefore, it is urgent to develop a new anti-tuberculosis drug with high efficiency and low toxicity. Summary of the invention

[0004] The main purpose of the present invention is to provide a kind of cefmenoxime hydrochloride. The use of cefmenoxime hydrochloride in the preparation of a drug for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection aims to provide a new type of anti-tuberculosis drug with high efficiency and low toxicity for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection.

[0005] To achieve the above object, the present invention provides a use of cefmenoxime hydrochloride in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacteria infection, wherein the Mycobacterium tuberculosis includes at least one of H37Rv, H37Ra and BCG, and the non-tuberculous mycobacteria includes Mycobacterium abscessus.

[0006] In one embodiment, the disease comprises tuberculosis.

[0007] In one embodiment, the drug inhibits mycobacteria by inhibiting bacterial cell wall synthesis and / or affecting bacterial protein synthesis.

[0008] In one embodiment, the medicament further comprises isoniazid.

[0009] In one embodiment, the antibacterial dosage of the drug ranges from 0.000488 to 64 μg / mL.

[0010] In one embodiment, the drug is used to inhibit infection by Mycobacterium tuberculosis, wherein the Mycobacterium tuberculosis includes H37Rv, H37Ra or BCG, and the drug is used to inhibit infection by non-tuberculous mycobacteria, wherein the non-tuberculous mycobacteria include Mycobacterium abscessus: The dosage of the drug for inhibiting the H37Rv strain is not less than 1 μg / mL; and / or, The dosage of the drug for inhibiting the H37Ra strain is not less than 0.25 μg / mL; and / or, The dosage of the drug for inhibiting the BCG strain is not less than 0.25 μg / mL; and / or, The inhibitory dosage of the drug for inhibiting Mycobacterium abscessus is not less than 1 μg / mL.

[0011] In one embodiment, the medicament further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

[0012] In the technical scheme of the present invention, the present invention discovers for the first time that cefmenoxime hydrochloride has an anti-tuberculosis effect, and uses cefmenoxime hydrochloride in the preparation of a drug for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection. The present invention discovers that the drug exerts an antibacterial effect by inhibiting the synthesis of bacterial cell walls and affecting the synthesis of bacterial proteins. The drug can be used to inhibit diseases caused by Mycobacterium tuberculosis (MTB) infection and / or non-tuberculous mycobacterium (NTM) infection, that is, it can effectively inhibit tuberculosis caused by MTB infection and lung infection, skin and soft tissue infection, lymphadenitis and other diseases caused by NTM infection. Therefore, cefmenoxime hydrochloride can be used as a new type of anti-tuberculosis drug with high efficiency and low toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

[0014] Figure 1 This is a graph showing the inhibitory activity of cefmenoxime hydrochloride at different concentrations provided in an embodiment of the present invention against Mycobacterium tuberculosis strain H37Rv; Figure 2 This is a schematic diagram of the cytotoxicity of cefmenoxime hydrochloride to liver and kidney cells provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of the inhibition of Mycobacterium tuberculosis H37Ra by the combined use of cefmenoxime hydrochloride and isoniazid; Figure 4 This is a schematic diagram of the inhibition of Mycobacterium tuberculosis H37Ra protein synthesis after the use of cefmenoxime hydrochloride; Figure 5 This is a schematic diagram showing the inhibition of cell wall synthesis of Mycobacterium tuberculosis H37Ra after the use of ceftriaxone hydrochloride.

[0015] 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

[0016] 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.

[0017] At present, for both first-line and second-line anti-tuberculosis drugs, corresponding drug-resistant mutants have been found. In recent years, although some new drugs have been successfully developed and applied to clinical practice, such as bedaquiline (Bdq), delamanid (Dlm), PA-824 and SQ-109. Bdq is a diarylquinoline that can specifically inhibit ATP synthase. However, MTB has been found to have a rich intrinsic drug resistance mechanism to Bdq. Dlm and PA-824 have bactericidal effects on dormant and intracellular MTB. However, any abnormality of the enzyme encoded by the glucose 6-phosphate dehydrogenase gene will lead to an increase in the resistance level of Dlm and PA-824 in MTB. In addition, the large side effects limit the widespread application of some new drugs. Therefore, it is urgent to develop a new anti-tuberculosis drug with high efficiency and low toxicity.

[0018] In view of this, the present invention proposes an application of cefmenoxime hydrochloride in the preparation of a medicament, wherein the medicament is used for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection.

[0019] It should be noted that the specific chemical formula of cefmenoxime hydrochloride is as follows:

[0020] MTB (Mycobacterium tuberculosis) and NTM (Non-tuberculous mycobacteria) infections can cause a series of different diseases. They belong to tuberculosis and non-tuberculous mycobacterial infections respectively. That is, ceftriaxone hydrochloride is effective against a variety of mycobacteria, including MTB that causes tuberculosis and a variety of NTM. There are many types of NTM, including Mycobacterium avium, etc. They usually do not cause tuberculosis, but may cause other lung infections or immune system diseases.

[0021] In the technical scheme of the present invention, the present invention first discovered the use of cefmenoxime hydrochloride in the preparation of a drug for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection, wherein the Mycobacterium tuberculosis includes at least one of H37Rv, H37Ra and BCG, and the non-tuberculous mycobacteria include Mycobacterium abscessus. The present invention discovered that the drug exerts an antibacterial effect by inhibiting bacterial cell wall synthesis and affecting bacterial protein synthesis, and can effectively inhibit tuberculosis caused by Mycobacterium tuberculosis infection and lung infection, skin and soft tissue infection, lymphadenitis and other diseases caused by non-tuberculous mycobacterium infection. Therefore, cefmenoxime hydrochloride can be used as a new type of anti-tuberculosis drug with high efficiency and low toxicity.

[0022] In some embodiments, the disease includes tuberculosis. Mycobacterium tuberculosis mainly causes tuberculosis (TB). According to the site of infection, tuberculosis can be divided into pulmonary tuberculosis and extrapulmonary tuberculosis. In some embodiments, ceftriaxone hydrochloride can be used to treat tuberculosis caused by Mycobacterium tuberculosis.

[0023] It should be noted that the mycobacteria include Mycobacterium tuberculosis and non-tuberculous mycobacteria.

[0024] In some embodiments, the medicament inhibits mycobacteria by inhibiting bacterial cell wall synthesis and / or affecting bacterial protein synthesis.

[0025] Further, the Mycobacterium tuberculosis includes H37Rv, H37Ra or BCG; and / or, the non-tuberculous mycobacteria includes Mycobacterium abscessus.

[0026] It is understandable that Mycobacterium tuberculosis is the main pathogen that causes tuberculosis. H37Rv is an important tool for studying tuberculosis and its treatment. The activity against H37Rv shows its potential in treating tuberculosis. H37Ra is a weak strain of H37Rv, which has lost most of its pathogenicity after cultivation. Because of its high safety, it is often used in research. BCG (Bacillus Calmette–Guérin): This is a weakened Mycobacterium tuberculosis used to make vaccines to prevent tuberculosis.

[0027] It should be noted that the cell wall structure of Mycobacterium tuberculosis is complex, and traditional drugs may encounter problems such as drug resistance when treating tuberculosis. However, ceftriaxone hydrochloride can improve the anti-tuberculosis effect of the drug by inhibiting Mycobacterium tuberculosis.

[0028] Tuberculosis is a highly contagious chronic disease. The use of ceftriaxone hydrochloride can prevent bacterial infection in the early stages and help control bacterial reproduction and slow down the progression of the disease during treatment, thereby achieving the effect of treating and preventing tuberculosis.

[0029] In some embodiments, the drug also includes isoniazid. The combined use of cefmenoxime hydrochloride and isoniazid can produce a synergistic anti-tuberculosis effect, improve the inhibitory effect on Mycobacterium tuberculosis, shorten the treatment time, and reduce the development of drug resistance.

[0030] In the present invention, isoniazid and cefmenoxime hydrochloride are used in combination, which has a better inhibitory effect on Mycobacterium tuberculosis.

[0031] In some embodiments, the dosage range of the drug is 0.000488-64 μg / mL, that is, the concentration range of the drug used in the MIC experiment is 0.000488-64 μg / mL.

[0032] In some embodiments, the drug is used to inhibit infection by Mycobacterium tuberculosis, wherein the Mycobacterium tuberculosis includes H37Rv, H37Ra or BCG, and the drug is used to inhibit infection by non-tuberculous mycobacteria, wherein the non-tuberculous mycobacteria include Mycobacterium abscessus: The inhibitory dosage of the drug for inhibiting the H37Rv strain is not less than 1 μg / mL; and / or, The inhibitory dosage of the drug for inhibiting the H37Ra strain is not less than 0.25 μg / mL; and / or, The inhibitory dosage of the drug for inhibiting the BCG strain is not less than 0.25 μg / mL.

[0033] Specifically, the MIC value of the drug against the H37Rv strain of Mycobacterium tuberculosis is 1 μg / mL; The MIC value of the drug to Mycobacterium tuberculosis H37Ra strain is 0.25 μg / mL, and the minimum bactericidal concentration MBC is 1 μg / mL. The MIC value of the drug to Mycobacterium tuberculosis BCG strain is less than 0.25 μg / mL. The MIC of the drug to Mycobacterium abscessus standard strain ATCC19977 is 1 μg / mL, and the MBC is 4 μg / mL.

[0034] It should be noted that MIC (Minimum Inhibitory Concentration) refers to the lowest concentration at which a drug can inhibit bacterial growth. For example, the MIC value of cefmenoxime hydrochloride against Mycobacterium tuberculosis H37Ra strain is 0.25 μg / mL, which means that when the concentration of the compound cefmenoxime hydrochloride reaches or exceeds 0.25 μg / mL, it can effectively prevent the growth of H37Ra strain.

[0035] MBC (Minimum Bactericidal Concentration): refers to the lowest concentration at which a drug can kill bacteria. For example, the MBC of cefmenoxime hydrochloride against Mycobacterium tuberculosis H37Ra strain is 1μg / mL, which means that cefmenoxime hydrochloride at a concentration of 1μg / mL can not only inhibit the growth of H37Ra bacteria, but also kill bacteria and reduce the number of colonies by 99.9%. The above two indicators can be used to measure the antibacterial activity of the drug.

[0036] In some embodiments, the medicament further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

[0037] 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.

[0038] 1. Experimental Materials 1. Strains The strains and sources used in the experimental verification process of the present invention are as follows: The Mycobacterium tuberculosis includes H37Rv (ATCC 27294), H37Ra (ATCC 25177) and BCG (ATCC35733), and the non-tuberculous mycobacteria include Mycobacterium abscessus (ATCC19977), which are preserved by the Shenzhen National Clinical Research Center for Infectious Diseases.

[0039] 2. Main Reagents The main reagents and sources used in the experimental verification process of the present invention are as follows: Middle Brook OADC Enrichment, Middle Brook 7H11 Agar, and Middle Brook 7H9Broth were purchased from Becton, Dickinson and Company, USA.

[0040] Phosphate buffered saline (PBS), fetal bovine serum (FBS), and DMEM medium were purchased from Gibco.

[0041] Resazurin (sodium salt), dimethyl sulfoxide (DMSO), and isoniazid (INH) were purchased from Sigma Aldrich, and isoniazid (INH) and ethambutol (EMB) were purchased from MedChemExpress (MCE).

[0042] CCK-8 kit was purchased from Biotechnology Co., Ltd.

[0043] Disposable bacterial culture dishes were purchased from Shanghai Bioengineering Co., Ltd. Four-partition 90 mm bacterial culture dishes and cell culture flasks T25 and T75 were purchased from Wuxi Nice Life Science Co., Ltd. (NEST).

[0044] 5 xSDS loading buffer and SDS-PAGE rapid gel preparation kit were purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0045] 3. Preparation of culture medium and main solutions 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 at 121°C with high-temperature and high-pressure steam for 10 min. When the temperature cools to about 45°C, add 100 mL OADC and store at 4°C.

[0046] 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 50-55℃, add 100mL OADC, mix well, pour into 90mm plates at 18-20mL / dish, dry and store at room temperature.

[0047] Preparation of complete culture medium: Add 50 ml FBS to 450 ml DMEM basal culture medium and mix well to make DMEM complete culture medium with 10% FBS.

[0048] Preparation of Resazurin staining solution: Weigh 1g of Resazurin (sodium salt) powder and dissolve it in 100mL ddH2O. Prepare 1% Resazurin stock solution for use. Dilute it 100 times to 0.05% when using it. Mix 0.05% Resazurin and 10% Tween-80 (after high pressure steam sterilization) in a volume ratio of 1:1. Add 30μl of the previously mixed staining solution to each 100μl bacterial solution well, incubate overnight and take pictures.

[0049] Preparation of INH and EMB solutions: Weigh 10 mg of isoniazid powder or EMB powder and dissolve it in 1 ml of cell-grade DMSO to prepare a 10 mg / ml stock solution. Aliquot 50 μl into each tube and store at -20°C. When using, dilute according to the required concentration and add to the corresponding solution.

[0050] 2. Embodiment 1 The strain activation of Mycobacterium tuberculosis strains H37Rv, H37Ra, Mycobacterium tuberculosis BCG and Mycobacterium abscessus standard strain NTM ATCC19977 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: (1) Thaw the bacterial strain stored at -80℃ in the laboratory, use an inoculation loop to dip the bacterial strain into Middlebrook 7H11 medium, and then incubate it upside down in a 5% CO2 incubator at 37℃ for about 4 weeks.

[0051] (2) Pick a single colony from Middlebrook 7H11 medium and inoculate it into Middlebrook 7H9 liquid medium. Incubate at 37°C for about 2 weeks until the strain reaches the logarithmic growth phase.

[0052] (3) Collect the bacterial suspension in the logarithmic growth phase, centrifuge at 4000 r / min for 10 min, discard the supernatant, add an appropriate amount of Middlebrook 7H9 medium to resuspend the precipitate, use a bacterial disperser to fully disperse it, let it stand for 5 to 10 min, collect the supernatant, divide it into cell cryopreservation tubes, and store it at -80℃.

[0053] (4) Randomly select 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 Middlebrook 7H11 medium plate, and culture it upside down in a 5% CO2 incubator at 37°C for about 4 weeks. When bacterial colonies can be clearly distinguished with the naked eye, calculate the colony forming unit (CFU).

[0054] 2. Strain dilution The bacterial strains cultured to the logarithmic phase were measured using a bacterial dispersion instrument and their turbidity was diluted to 1 McFarland turbidity.

[0055] 3. Preparation and dilution of cefmenoxime hydrochloride compounds The cefmenoxime hydrochloride compound was diluted to 10 mg / mL with DMSO (dimethyl sulfoxide). The specific operation process is as follows: Weigh 10 mg of cefmenoxime hydrochloride compound and dissolve it in 1 mL of cell-grade DMSO.

[0056] 4. In vitro activity determination of cefmenoxime hydrochloride against sensitive Mycobacterium tuberculosis The anti-tuberculosis activity assay was performed using a sterile 96-well culture plate with a total volume of 100 μL in each well.

[0057] like Figure 1 As shown, 100 μL of the bacterial solution in the logarithmic growth phase obtained above was added to each test well, and the culture solution of Mycobacterium tuberculosis H37Rv, H37Ra and BCG strains was added at a concentration of 0.001 at OD600nm. During the MIC experiment for H37Rv, cefmenoxime hydrochloride was diluted from the highest concentration of 64.0 μg / mL to 0.000488 μg / mL in the form of 3 replicates. At the same time, the positive control drugs (positive) INH and EMB and the test samples of different concentrations prepared with DMSO were added at appropriate concentrations. The final concentrations of the test samples in the determination system are as follows: Figure 1 Labeled as 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, 0.062 5μg / ml, 0.03125μg / ml, 0.015625μg / mL, 0.007813μg / mL, 0.003906μg / mL, 0.001953μg / mL, 0.000977μg / mL, 0.000488μg / mL; Three growth positive control wells (equal volume and concentration of H37Ra bacterial solution) and three growth negative control wells (equal volume of culture medium without any Mycobacterium tuberculosis) were set up in the culture plate; The 96-well plate was covered and sealed with sealing film, and then placed in an incubator at 37°C for incubation; On the 7th day, observe the positive growth control wells and the negative growth control wells. When a clear difference is observed between the two, observe the quantity and morphology of bacterial growth in each test well, determine inhibition or drug resistance, and record the results; Observe again on the 10th day to confirm and record the results.

[0058] The results showed that the MIC concentration of H37Rv was 1 μg / ml.

[0059] 1) Anti-tuberculosis activity assay was performed using a sterile 96-well culture plate with a total volume of 100 μL in each well; 2) Add 100 μL of Mycobacterium tuberculosis with an OD600nm of 0.001 in the logarithmic growth phase to each test well, add cefmenoxime hydrochloride in a doubling dilution from 32 times the MIC to 1 times the MIC, and culture at 37°C for 7-10 days; 3) Take all the liquid in each well (100 μL) and spread it on 7H11 plates. After 4 weeks of culture, count the number of colonies to determine the exact initial bacterial count. 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; 5) Observe the inhibition and killing of bacteria by drugs with different concentrations, read the MIC value and MBC value. The results show that the MIC of ceftriaxone hydrochloride against Mycobacterium tuberculosis H37Ra is 0.25 μg / mL, and the MBC is 1 μg / mL.

[0060] 5. Inhibitory activity of ceftriaxone hydrochloride against Mycobacterium abscessus 1) Anti-tuberculosis activity assay was performed using a sterile 96-well culture plate with a total volume of 100 μL in each well; 2) Add 100 μl of Mycobacterium abscessus with an OD600nm of 0.001 in the logarithmic growth phase to the test wells, and add an appropriate concentration of the positive control drug ofloxacin (OFX) or different concentrations of the test samples prepared with DMSO. The final concentrations of the test samples in the assay system are: 64.0 μg / mL, 32.0 μg / mL, 16.0 μg / mL, 8.0 μg / mL, 4.0 μg / mL, 2.0 μg / mL, 1.0 μg / mL, 0.5 μg / mL, and 0.25 μg / mL; 3) Set up 3 growth positive control wells (equal volume and concentration of H37Ra bacterial solution) and 3 growth negative control wells (equal volume of culture medium without any bacteria) in the culture plate; 4) Cover the 96-well plate and seal it with sealing film, then place it in an incubator at 37°C; 5) On the third day, add 30 μL of resazurin staining solution to each well and continue to incubate in the incubator at 37°C overnight; observe the positive growth control wells and negative growth control wells. When a clear difference is observed between the two, observe the number and morphology of bacterial growth in each test well, determine inhibition or resistance, and record the results; 6) The results showed that the minimum inhibitory concentration (MIC) of ceftriaxone hydrochloride against the standard strain of Mycobacterium abscessus ATCC19977 was 1 μg / mL.

[0061] 1) Anti-tuberculosis activity assay was performed using a sterile 96-well culture plate with a total volume of 100 μL in each well; 2) Add 100 μL of Mycobacterium abscessus ATCC19977 in the logarithmic growth phase with an OD600nm of 0.001 to each test well, add cefmenoxime hydrochloride in a serial dilution from 32 times the MIC to 1 times the MIC, and culture at 37°C for 7-10 days; 3) Take all the liquid in each well (100 μL) and spread it on 7H11 plates. After 4 weeks of culture, count the number of colonies to determine the exact initial bacterial count. 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; 5) The MBC of ceftriaxone hydrochloride against Mycobacterium abscessus ATCC19977 is 4 μg / mL, and the MIC is 1 μg / mL; Test results: The minimum bactericidal concentration (MBC) of cefmenoxime hydrochloride against Mycobacterium abscessus ATCC19977 was 4 μg / mL.

[0062] Example 2 Cytotoxicity of cefmenoxime hydrochloride on hepatic and renal cells 1) Take cells in logarithmic growth phase, digest and count them, and use 4×10 3 The cells were seeded in a 96-well cell culture plate and incubated in a 37°C, 5% CO2 cell culture incubator; 2) After culturing for 24 h, different concentrations of drugs were added after the cells adhered to the wall. 293T and hepG2 cells were diluted 3-fold from 1000 μg / ml for 9-10 times, with 3 parallel wells in each group, and the cells were treated for 72 h; 3) Add the detection solution according to the CCK-8 kit and obtain the corresponding absorbance on the microplate reader.

[0063] 4) Calculate the cell survival rate: Cell survival rate % = (OD of drug-treated cells-background OD) / (OD of control cells-background OD) × 100%.

[0064] 5) Calculate IC using curve fitting on GraphPad Prism 8.0.2 50 (The median toxic concentration in drug toxicity tests).

[0065] The results are as follows Figure 2 As shown, Figure 2 The hepG2 in A belongs to the liver cancer cell line, which shows the effect of different concentrations of cefmenoxime hydrochloride on the viability of hepG2 cells. The horizontal axis is "Log compound 2", which represents the logarithmic concentration of cefmenoxime hydrochloride; the vertical axis is "cell viability", which represents the percentage of cell viability. Figure 2 In A, with the increase of ceftriaxone hydrochloride concentration, the cell survival rate showed a downward trend, and IC 50 The value is 148.3 μg / mL: This means that at this concentration, cefmenoxime hydrochloride reduces the survival rate of hepG2 cells by 50%. The 293T in B belongs to the human embryonic kidney cell line, which shows the effect of different concentrations of cefmenoxime hydrochloride on the survival rate of 293T cells. Figure 2 IC of B 50 The value is 111.9 μg / mL: that is, at this concentration, cefmenoxime hydrochloride reduces the survival rate of 293T cells by 50%. In summary, according to the definition of safety selection index (SI), combined with the MIC of cefmenoxime hydrochloride to H37Ra, the SI index is greater than 200. This shows that the action concentration of cefmenoxime hydrochloride is very safe, which proves that cefmenoxime hydrochloride has low biological toxicity and its action concentration is very safe.

[0066] Example 3 Experimental study on the combined use of cefmenoxime hydrochloride and isoniazid H37Ra in logarithmic growth phase, take appropriate amount of bacterial solution 7H9 and dilute to OD 0.001, 100 μl bacterial solution per well, and use the chessboard method to make the final drug concentration Figure 3 As shown above, by Figure 3 It can be seen that: Figure 3 The horizontal part in the middle shows the dilution of isoniazid (INH) starting from 1 times the MIC, and the vertical part shows the dilution of cefmenoxime hydrochloride starting from 1 times the MIC. The OD was tested after 7-10 days of static culture in a 37°C incubator. 600After measuring the nm absorbance, 30 μl of resazurin (a 1:1 mixture of 0.05% resazurin and 10% TWEEN-80) was added to each well for color development. Among them, the wells with bacterial growth showed pink, and the wells without growth activity showed blue. The fractional inhibitory concentration index (FICI) was calculated according to the formula FIC = (MIC of drug A in combination) / (MIC of drug A alone) + (MIC of drug B in combination) / (MIC of drug B alone). The interaction between drugs was determined based on the FICI. When FICI ≤ 0.5, the mode of action of the two drugs was synergistic; when 0.5 < FICI ≤ 1, the mode of action of the two drugs was additive; when 1 < FICI ≤ 2, the mode of action of the two drugs was irrelevant; when FICI > 2, the mode of action of the two drugs was antagonistic.

[0067] The results showed that under the action of cefmenoxime hydrochloride, the MIC of isoniazid decreased from the original 0.125 μg / ml to 0.003906 μg / ml; at the same time, cefmenoxime hydrochloride also decreased from the original 0.25 μg / ml to 0.015625 μg / ml. The fractional inhibitory concentration index calculated from the above data was 0.093748. Therefore, the mode of action of the two drugs was synergistic, bringing hope for reducing drug dosage, shortening the treatment course, and reducing the medication cycle.

[0068] Example 4 Inhibitory effect of cefmenoxime hydrochloride on the total protein synthesis of H37Ra After 10-fold dilution of H37Ra in the logarithmic growth phase, from Figure 4It can be seen that the vertical Marker icon represents the molecular weight standard, which is used to determine the molecular weight of each band. The molecular weight unit is kDa (kilodaltons). From top to bottom, 170kDa, 130kDa, 100kDa, 70kDa, 55kDa, 45kDa, 35kDa, 25kDa, 15kDa and 10kDa are marked. These markers are references and are used to estimate the molecular weight of the bands. The horizontal direction is the samples to be tested, which are grouped according to different concentrations of ceftriaxone hydrochloride (2xMic, 1xMic, 0.5xMic, control). Among them, the control group is the bacterial control group without drug addition, 2 mL per group, placed at 37℃, 180 rpm shaking incubator for 7 days, 5000 × g for 10 min, centrifuged to collect the precipitate, and the corresponding volume of B-PER™ bacterial protein extraction reagent and 0.1 mm glass beads were added according to the weight of the precipitate. The mixture was centrifuged at 5000RPM for 1 min, 3-5 cycles, and cooled on ice for 5 min in between. Then, the supernatant was collected by centrifugation at 12000 × g, 4°C, for 5 min, and the corresponding volume of 5 xSDS loading buffer was added, and SDS-PAGE electrophoresis was performed after the metal bath was set at 95°C for 10 min.

[0069] The electrophoresis results are as follows Figure 4 As shown in the figure, the protein levels of H37Ra after the action of cefmenoxime hydrochloride in different concentration groups, the results show that the addition of cefmenoxime hydrochloride reduced most of the proteins in H37Ra, and this effect became more obvious with the increase of compound concentration, small molecular weight proteins were greatly inhibited at a concentration of 0.5xMic, and the reduction of proteins became more and more with the increase of cefmenoxime hydrochloride concentration.

[0070] Example 5 Inhibitory effect of cefmenoxime hydrochloride on H37Ra cell wall synthesis After 10-fold dilution of H37Ra in the logarithmic growth phase, 0.5xMIC of ceftriaxone hydrochloride was added to each tube with 2 ml of bacterial solution, and a control group without drug addition was set up and cultured at 37°C, 180rpm for 5 days. After fixation with 2.5% glutaraldehyde, scanning electron microscopy was performed. like Figure 5As shown in the figure, the cell wall of Mycobacterium tuberculosis was observed after adding cefmenoxime hydrochloride at a concentration of 0.5 times the MIC concentration compared with the control group. The scanning electron microscopy results showed that after the action of cefmenoxime hydrochloride, the total amount of bacteria also decreased significantly. Under the action of 0.5xMIC concentration that did not completely inhibit growth, it can be observed that Mycobacterium tuberculosis easily agglomerated, the cell boundaries were unclear, and the cells became sticky and aggregated. Cracks, deformations, and broken threads appeared on the cell surface. This shows that cefmenoxime hydrochloride targets the cell wall of Mycobacterium tuberculosis to exert its antibacterial activity.

[0071] 3. Verification conclusion The MIC value of cefmenoxime hydrochloride compound against H37Rv is 1μg / mL. Further studies have shown that the MIC against Mycobacterium tuberculosis H37Ra is 0.25μg / mL, and the minimum bactericidal concentration (MBC) is 1μg / mL. The MIC against Mycobacterium tuberculosis BCG is less than 0.25μg / mL. The MIC against the standard strain of Mycobacterium abscessus ATCC19977 is 1μg / mL, and the MBC is 4μg / mL; and the mechanism studies have shown that cefmenoxime hydrochloride exerts antibacterial activity by inhibiting the synthesis of cell wall and protein of Mycobacterium tuberculosis. In view of the good activity of the compound, its cytotoxicity to liver and kidney cells was determined, and the results showed that the concentration of cefmenoxime hydrochloride is very safe.

[0072] Through experiments, the inhibitory and killing activity of cefmenoxime hydrochloride on MTB and NTM was verified, and the application of cefmenoxime hydrochloride in the treatment of infectious diseases caused by MTB, especially drug-resistant MTB and drug-resistant NTM infection was determined, thereby expanding the application of cefmenoxime hydrochloride as an anti-tuberculosis compound. Ultimately, the result was obtained that cefmenoxime hydrochloride is a new type of anti-tuberculosis drug with high efficiency and low toxicity.

[0073] 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. A use of cefmenoxime hydrochloride in the preparation of a medicament for treating diseases caused by Mycobacterium tuberculosis infection and / or non-tuberculous mycobacterium infection, characterized in that: The Mycobacterium tuberculosis includes at least one of H37Rv, H37Ra and BCG, and the non-tuberculous mycobacteria includes Mycobacterium abscessus.

2. The use according to claim 1, characterized in that The diseases include tuberculosis.

3. The use according to claim 1, characterized in that The drug inhibits mycobacteria by inhibiting bacterial cell wall synthesis and / or affecting bacterial protein synthesis.

4. The use according to claim 1, characterized in that The drugs also include isoniazid.

5. The use according to claim 1, characterized in that: The antibacterial dosage range of the drug is 0.000488~64μg / mL.

6. The use according to claim 1, characterized in that: The drug is used to inhibit infection by Mycobacterium tuberculosis, wherein the Mycobacterium tuberculosis includes H37Rv, H37Ra or BCG, and the drug is used to inhibit infection by non-tuberculous mycobacteria, wherein the non-tuberculous mycobacteria include Mycobacterium abscessus: The dosage of the drug for inhibiting the H37Rv strain is not less than 1 μg / mL; and / or, The dosage of the drug for inhibiting the H37Ra strain is not less than 0.25 μg / mL; and / or, The dosage of the drug for inhibiting the BCG strain is not less than 0.25 μg / mL; and / or, The inhibitory dosage of the drug for inhibiting Mycobacterium abscessus is not less than 1 μg / mL.

7. The use according to claim 1, characterized in that: The medicament further comprises a pharmaceutically acceptable excipient, carrier and / or diluent.

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