CN118831079A - Medicine for treating drug-resistant tuberculosis and composition thereof - Google Patents

Abstract

The present invention relates to the field of biomedicine technology, and specifically discloses the application of Lefamulin in the treatment of drug-resistant tuberculosis. The present invention finds that Lefamulin can effectively inhibit standard strains and clinical isolates of Mycobacterium tuberculosis, and even has the same inhibitory effect on multidrug-resistant Mycobacterium tuberculosis. In addition, the present invention finds that Lefamulin can enhance its inhibitory effect when combined with other common clinical anti-tuberculosis drugs. Therefore, Lefamulin can be used as a candidate drug in the development of drugs against Mycobacterium tuberculosis infection, which is of great significance for the prevention and treatment of tuberculosis, especially drug-resistant tuberculosis.

Description

A medicine for treating drug-resistant tuberculosis and its composition

Technical Field

The invention relates to the technical field of biomedicine, and in particular to application of a semi-synthetic pleuromutilin drug in the treatment of drug-resistant tuberculosis.

Background Art

Tuberculosis is caused by infection with Mycobacterium tuberculosis (M.tb), a major infectious disease that seriously threatens human health. The Global Tuberculosis Report published by the World Health Organization (WHO) pointed out that there are about 2 billion to 3 billion people infected with M.tb worldwide, about 10.6 million new cases of tuberculosis in 2022, and about 1.3 million people died from tuberculosis-related diseases (World Health Organization. Global tuberculosis report 2023. [R] Geneva: World Health Organization, 2023.). Although the incidence of tuberculosis in my country has declined in recent years, it is still one of the countries with a high incidence of tuberculosis, and the prevalence of drug-resistant tuberculosis has exacerbated the difficulty of tuberculosis control in my country. In 2022, there were about 410,000 new cases of multidrug-resistant-tuberculosis (MDR-TB) worldwide, but only 43% of patients with multidrug-resistant or rifampicin-resistant tuberculosis were diagnosed and included in standardized treatment, with a treatment success rate of 63%. my country is a country with a high burden of tuberculosis, with approximately 750,000 new tuberculosis patients in my country in 2022. At the same time, my country is also one of the countries with a high burden of MDR-TB, ranking fourth in the world, with approximately 70,000 new cases of drug-resistant tuberculosis in 2022 (Gao Jingtao, Liu Yuhong. Interpretation of the key points of the 2019 World Health Organization Global Tuberculosis Report [J], International Journal of Respiratory Diseases, 2020, 40(3):161-166.). Due to the use of effective antibiotics, the cure rate of sensitive tuberculosis can reach 85%, but the treatment options for MDR-TB are limited, the treatment cycle is long, and the cure rate is only 50%. Anti-tuberculosis drugs are divided into first-line drugs and second-line drugs. First-line drugs play a major role in the treatment of tuberculosis. However, if Mycobacterium tuberculosis becomes resistant, the cure rate of first-line drugs will decrease and cannot meet the treatment needs. Second-line drugs need to be used for treatment in a timely manner. However, for the treatment of multidrug-resistant and extensively drug-resistant tuberculosis, the efficacy of many second-line drugs is not ideal, and some patients even have no drugs available (Ma Zhiqiang, Chen Wei, Wang Xiaoyan, et al. Analysis of drug resistance characteristics of 208 patients with drug-resistant tuberculosis [J]. Chinese Journal of Laboratory Diagnosis, 2024, 28(03): 304-306.). Therefore, new anti-tuberculosis drugs are urgently needed in clinical practice or the anti-Mycobacterium tuberculosis activity of existing drugs is crucial for the treatment of tuberculosis, especially drug-resistant tuberculosis.

Lefamulin was developed by Nabriva Therapeutics and approved by the FDA in August 2019 for the treatment of community-acquired bacterial pneumonia in adults. In my country, Lefamulin, developed by the Shanghai Branch of Sumitomo Pharma (Suzhou) Co., Ltd., was also approved by the State Food and Drug Administration on November 17, 2023 for the treatment of community-acquired bacterial pneumonia in adults. Community-acquired pneumonia (CAP) is one of the most common infectious diseases in the world, with high morbidity and mortality. Its incidence increases with age, placing a heavy economic burden on patients and the medical insurance system. In the United States, 5% to 15% of CAP cases are caused by Streptococcus pneumoniae, and other pathogens include atypical pathogens such as Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, and Legionella pneumophila. Lefamulin is the first semi-synthetic pleuromutilin antibiotic that can be used systemically by patients. In preclinical and clinical trials, Lefamulin has shown inhibitory activity against common pathogens that cause CAP. Its antibacterial mechanism is different from other antibiotics that inhibit protein synthesis, and follows a unique mode of action that binds to prokaryotic ribosomes. It interacts with the 50S ribosomal large subunit, its parent nucleus binds to the A site, and the C14 side chain extends to the P site of the peptide transfer center ribosome, which can bind to the 23S rRNA of the 50S large subunit and inhibit the activity of peptidyl transferase, so it can interfere with the synthesis of bacterial proteins and cause bacterial death. Lefamulin only selectively inhibits the synthesis of prokaryotic cell proteins, but has no effect on eukaryotic cells and does not interact with mammalian ribosomes. Because its mechanism of action is different from other commonly used antibiotics in clinical practice, Lefamulin has a lower tendency to cause drug resistance. In addition, based on the data from the two Phase III clinical trials LEAP 1 and LEAP 2, Lefamulin has a good safety profile, all adverse reactions are mild or moderate, and can resolve on their own within 2 days after discontinuation of the drug (Luo Zhimin, Zhu Yingchun, Wang Hairong. Lefamulin, a pleuromutilin antibiotic for the treatment of community-acquired pneumonia [J]. Chinese Journal of Medicine, 2020, 22(06): 397-400.). At present, there are no reports on the inhibition of Mycobacterium tuberculosis by Lefamulin.

Summary of the invention

The present invention was completed based on the discovery that Lefamulin has the effect of inhibiting the activity of Mycobacterium tuberculosis.

In a first aspect, the present invention provides a pharmaceutical composition, comprising Lefamulin and another drug for preventing Mycobacterium tuberculosis infection.

The pharmaceutical composition has at least one of the following effects:

a) inhibiting the activity of Mycobacterium tuberculosis;

b) Anti-Mycobacterium tuberculosis infection;

c) Prevention and/or treatment of diseases caused by Mycobacterium tuberculosis.

Furthermore, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

Preferably, the Mycobacterium tuberculosis is pan-sensitive and drug-resistant Mycobacterium tuberculosis.

Furthermore, the other anti-Mycobacterium tuberculosis infection drug includes one or more of antibiotics and other drugs that can help inhibit or kill Mycobacterium tuberculosis or provide resistance to patients.

Furthermore, the antibiotics include one or more of rifampicin, bedaquiline, linezolid, delamanid, levofloxacin, moxifloxacin, clofazimine, pyrazinamide, ethambutol, streptomycin, isoniazid and/or ethionamide; the other drugs include one or more of vitamins, amino acids, proteins and/or minerals.

Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

Furthermore, the carrier material includes but is not limited to one or more of water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.) and/or enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

Preferably, the carrier material is a water-soluble carrier material.

Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and/or lyophilized powder injections.

Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and/or various microparticle delivery systems.

Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and/or lubricants.

Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and/or aluminum silicate.

Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and/or polyvinyl pyrrolidone.

Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and/or ethyl cellulose.

Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and/or hydrogenated oil.

Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and/or sodium lauryl sulfate.

Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and/or polyethylene glycol.

Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and/or disintegrants.

Furthermore, the diluent and absorbent include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and/or talc.

Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and/or batter.

Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and/or ethyl cellulose.

Furthermore, the suppository may be made of a wide variety of carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and/or semi-synthetic glycerides.

Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and/or a suspension.

Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and/or polyoxyethylene sorbitan fatty acid esters.

Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and/or pH regulators.

Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

Furthermore, the product can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

In a second aspect, the present invention provides a use of Lefamulin in preparing a product for inhibiting Mycobacterium tuberculosis.

Furthermore, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

Preferably, the Mycobacterium tuberculosis is pan-sensitive and drug-resistant Mycobacterium tuberculosis.

Furthermore, the product for inhibiting Mycobacterium tuberculosis includes one or more of drugs and/or antibacterial agents.

In a third aspect, the present invention provides a use of Lefamulin in the preparation of a medicament for preventing and/or treating diseases caused by Mycobacterium tuberculosis infection.

Lefamulin works in the following ways:

1) Lefamulin inhibits the activity of Mycobacterium tuberculosis;

2) Lefamulin kills Mycobacterium tuberculosis.

Furthermore, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

Preferably, the Mycobacterium tuberculosis is pan-sensitive and drug-resistant Mycobacterium tuberculosis.

Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug for preventing and/or treating diseases caused by Mycobacterium tuberculosis infection.

Furthermore, the carrier material includes but is not limited to one or more of water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, organic acid, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.) and/or enteric carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).

Preferably, the carrier material is a water-soluble carrier material.

Furthermore, the drug for preventing and/or treating diseases caused by Mycobacterium tuberculosis infection can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, lozenges, suppositories and/or lyophilized powder injections.

Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and/or various microparticle delivery systems.

Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and/or lubricants.

Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and/or aluminum silicate.

Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and/or polyvinyl pyrrolidone.

Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and/or ethyl cellulose.

Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and/or hydrogenated oil.

Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and/or sodium lauryl sulfate.

Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and/or polyethylene glycol.

Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and/or disintegrants.

Furthermore, the diluent and absorbent include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and/or talc.

Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and/or batter.

Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and/or ethyl cellulose.

Furthermore, the suppository may be made of a wide variety of carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and/or semi-synthetic glycerides.

Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and/or a suspension.

Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and/or polyoxyethylene sorbitan fatty acid esters.

Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and/or pH regulators.

Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

Furthermore, the drug for preventing and/or treating diseases caused by Mycobacterium tuberculosis infection can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

Beneficial Effects

1. The present invention found that Lefamulin can effectively inhibit the standard strain of Mycobacterium tuberculosis, and the MIC of Lefamulin to the standard strain of Mycobacterium tuberculosis H37Rv is 1 μg/mL.

2. The present invention found that Lefamulin can effectively inhibit clinical isolates of Mycobacterium tuberculosis, with MIC ₅₀ of 0.5 μg/mL and MIC ₉₀ of 4 μg/mL for clinical isolates of Mycobacterium tuberculosis (non-MDR-TB). Even for multidrug-resistant Mycobacterium tuberculosis, Lefamulin can still effectively inhibit its growth, with MIC ₅₀ of 1 μg/mL and MIC ₉₀ of 4 μg/mL.

3. The present invention found that Lefamulin exhibited a synergistic effect when combined with the new anti-tuberculosis drug Bedaquiline, and exhibited an additive effect when combined with other common clinical anti-tuberculosis drugs.

4. The present invention found that Lefamulin has a good antibacterial effect on intracellular Mycobacterium tuberculosis alone, and its inhibitory effect can be enhanced when used in combination with anti-tuberculosis drugs; after treatment with Lefamulin and Bedaquiline, the bacterial load was reduced by 41.67%±12.42% and 76.67%±6.83% respectively compared with the control group; when the two were treated in combination, the bacterial load of Lefamulin+Bedaquiline was reduced by 83.33%±8.84% compared with the control group.

5. The present invention finds that Lefamulin can be used as a candidate drug for the development of drugs against Mycobacterium tuberculosis infection, which is of great significance for the prevention and treatment of tuberculosis, especially drug-resistant tuberculosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the distribution of MIC values of Lefamulin against 26 clinical isolates of Mycobacterium tuberculosis (non-MDR).

FIG2 shows the distribution of MIC values of Lefamulin against 25 clinical isolates of Mycobacterium tuberculosis (MDR).

Figure 3 is the evaluation of the antibacterial activity of Lefamulin combined with Bedaquiline against Mycobacterium tuberculosis in macrophages.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

Explanation of terms

Multidrug resistant (MDR-TB): Mycobacterium tuberculosis is resistant to at least two first-line anti-tuberculosis drugs, including isoniazid and rifampicin.

First-line anti-tuberculosis drugs: isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin.

Community acquired bacterial pneumonia (CABP) refers to infectious lung parenchymal inflammation contracted outside the hospital, including pneumonia caused by pathogens with a clear incubation period after admission to the hospital. The main pathogen is Streptococcus pneumoniae, in addition, it also includes Haemophilus influenzae, Moraxella catarrhalis and Staphylococcus aureus.

Pleuromutilin: a natural product with antibacterial activity discovered in the 1950s. It is a class of diterpenoid compounds with a tricyclic skeleton produced by submerged culture of the higher fungi basidiomycetes Pleurotus mutilus and Pleurotus passeckerianus.

Material Description:

Mycobacterium tuberculosis standard strain H37Rv: purchased from ATCC, number ATCC27294.

Lefamulin: purchased from MedchemExpress, item number HY-16908A. The molecular formula is C ₃₀ H ₄₉ NO ₇ S, the molecular weight is 567.78, the CAS number is 1350636-82-6, and its structural formula is as follows:

Example 1 Detection of the antibacterial activity of Lefamulin against standard strains of Mycobacterium tuberculosis

1. Preparation of Lefamulin solution: Dissolve Lefamulin in dimethyl sulfoxide (DMSO) to prepare 8 mg/ml Lefamulin stock solution, filter and sterilize, and store. Use 7H9 culture medium to prepare Lefamulin stock solution into a 64 μg/mL Lefamuline solution. Add 64 μg/mL Lefamuline solution to the first column of the 96-well plate, mix well, and then dilute from the first column to the tenth column in sequence, aspirate 100 μl and discard; add 7H9 culture medium to the 11th and 12th columns as control wells. Set up 2 replicate wells for each concentration and repeat the experiment 3 times.

2. Preparation of bacterial solution: Culture Mycobacterium tuberculosis (ATCC27294) in neutral Roche medium to the logarithmic phase, scrape the strain in the logarithmic phase, place the colony in a grinding bottle and grind it evenly, dilute the bacterial suspension with 7H9 medium and measure the turbidity to 1 McFarland, then add it to 7H9 medium at a ratio of 1:20 and mix well to obtain Mycobacterium tuberculosis suspension.

3. Add an equal amount of Mycobacterium tuberculosis suspension to each well in columns 1-11, respectively, to make the final volume in each well consistent, and the final concentration of the bacterial solution is 4×10 ⁵ CFU/mL. At this time, the final drug concentrations in the wells in columns 1-10 are 32μg/mL, 16μg/mL, 8μg/mL, 4μg/mL, 2μg/mL, 1μg/mL, 0.5μg/mL, 0.25μg/mL, 0.125μg/mL, and 0.0625μg/mL, respectively. Set up positive control wells (column 11, bacterial culture medium without drug) and negative control wells (column 12, culture medium without bacterial solution and drug), set up 2 replicate wells for each concentration, and repeat the experiment 3 times.

4. Place the 96-well plate in an incubator and culture for 7 days.

5. Add the premixed colorimetric solution of Alamar blue and 5% Tween-80 to the microplate, continue culturing and observe the color change of the 96-well plate.

6. Judgment criteria: Blue wells indicate sterile growth, pink wells indicate bacterial growth, and the lowest drug concentration that prevents the color from changing from blue to pink is recorded as the minimum inhibitory concentration (MIC) that can inhibit the growth of Mycobacterium tuberculosis (ATCC27294).

The results showed that the MIC of Lefamulin against Mycobacterium tuberculosis H37Rv (ATCC27294) was 1 μg/mL.

Example 2 Detection of antibacterial activity of Lefamulin against clinically isolated Mycobacterium tuberculosis strains

According to the method in Example 1, the antibacterial activity of Lefamulin against 26 clinically isolated Mycobacterium tuberculosis strains (non-MDR-TB) was detected.

As shown in Table 1, the MIC range of Lefamulin against 26 clinical isolates of Mycobacterium tuberculosis was 0.0625 μg/mL-4 μg/mL, MIC ₅₀ = 0.5 μg/mL, MIC ₉₀ = 4 μg/mL, proving that Lefamulin has good in vitro antibacterial activity against clinically isolated Mycobacterium tuberculosis strains (non-MDR-TB). The statistical results of MIC concentration distribution are shown in Figure 1.

Table 1

According to the method in Example 1, the antibacterial activity of Lefamulin against 25 clinically isolated Mycobacterium tuberculosis strains (MDR-TB) was detected.

As shown in Table 2, the MIC range of Lefamulin against 25 clinical isolates of Mycobacterium tuberculosis was 0.25 μg/mL-4 μg/mL, MIC ₅₀ =1 μg/mL, MIC ₉₀ =4 μg/mL, proving that Lefamulin has good in vitro antibacterial activity against clinically isolated Mycobacterium tuberculosis strains (MDR-TB). The statistical results of MIC concentration distribution are shown in Figure 2.

Table 2

Example 3 Evaluation of the antibacterial activity of lefamulin combined with common clinical anti-tuberculosis drugs against standard strains of Mycobacterium tuberculosis

3.1 Preparation of drugs

Dissolve Lefamulin in DMSO to prepare 8 mg/ml Lefamulin stock solution, filter and sterilize, and store. The other anti-tuberculosis drugs: Rifampicin (RFP), Isoniazid (INH), Bedaquiline (BDQ), Linezolid (LZD), Clofazimine (CFZ), Delamanid (DLM) are the same as above, and the concentration is 8 mg/ml for use.

3.2 Preparation of drug plates

3.2.1 Preparation of Rifampicin Drug Plate

In columns 2-10 and B-G of a 96-well plate, add 50 μl of 7H9 medium (containing 10% OADC) to each well; add 100 μl of 16×MIC concentration of rifampicin solution to row H of columns 2-10; aspirate 50 μl from the wells of row H to row G, dilute to row B in sequence, aspirate 50 μl and discard; finally, add 50 μl of 7H9 medium to rows B-H of column 2 to complete the configuration of the rifampicin drug plate. The configuration of the remaining drug plates is the same as above.

3.2.2 Lefamulin drug plate preparation

Determine the volume of Lefamulin required according to the number of drug plates prepared in 1.1.1; prepare 20 ml of 16×MIC concentration of Lefamulin; prepare 7 test tubes, add 10 ml of 7H9 culture medium to each tube, and mark the serial number; perform serial dilution from the 16×MIC concentration of Lefamulin solution, so that the final drug concentration of each tube is 16μg/mL, 8μg/mL, 4μg/mL, 2μg/mL, 1μg/mL, 0.5μg/mL, 0.25μg/mL, 0.125μg/mL, marked as 16×, 8×, 4×, 2×, 1×, 1\2×, 1\4×, 1\8× respectively; draw 50μl Add 16× Lefamulin to the 10th column of the 96-well plate, 8× Lefamulin to the 9th column, and so on, until it is added to the 3rd column; add 100μl of 7H9 culture medium to wells A-D in the 1st column; add 200μl of 7H9 culture medium to wells E-H in the 1st column as a negative control; finally, add 50μl of 7H9 culture medium to columns 3-10 in row A to complete the drug preparation of the 96-well plate.

3.3 Preparation of bacterial culture

Mycobacterium tuberculosis (ATCC27294) was cultured in neutral Roche medium to the logarithmic phase, the strain in the logarithmic phase was scraped, the colony was placed in a grinding bottle and ground evenly, the bacterial suspension was diluted with 7H9 medium and the turbidity was measured to 1 McFarland, and then added to the 7H9 medium at a ratio of 1:20 and mixed to obtain a Mycobacterium tuberculosis suspension for use.

3.4 Adding bacterial solution

Add diluted bacterial solution to columns 3-10, rows B-H of column 2, and rows A-D of column 1 of the 96-well plate; among them, rows A-D of column 1 serve as positive controls.

3.5 Cultivation and determination of results

The 96-well plate was placed in an incubator for 7-9 days; Resazurin (purchased from SIGMA, catalog number: R7017-5G) colorimetric solution was added to the microplate, and after continued culturing, the color change of the 96-well plate was observed and the result was read.

3.6 Result judgment criteria

The blue wells represent sterile growth, the pink wells represent bacterial growth, the lowest drug concentration that prevents the color from changing from blue to pink is recorded as the minimum inhibitory concentration (MIC) that can inhibit the growth of Mycobacterium tuberculosis (ATCC27294); the lowest concentration of antibiotics that inhibit growth in the combination (CIC); FICI is determined as the sum of the fractional inhibitory concentrations (FIC) of the individual antibiotics, and the formula is as follows:

FIC＝CIC/MIC

FICI＝CICA/MICA+CICB+MICB

The combined effect of antibiotics was considered synergistic, additive, unrelated, or antagonistic when the FICI value was ≤0.5, 0.5-1.0, 1.0-4.0, or >4.0, respectively.

3.7 Results

As shown in Table 3, Lefamulin exhibited synergistic effects with the new anti-tuberculosis drug bedaquiline, and additive effects with other tested anti-tuberculosis drugs.

Table 3. In vitro interactions between Lefamulin and core anti-tuberculosis drugs

Antibiotic Name FICI Combined effect Lefamulin/Rifampicin RIF 0.625 Additive effect Lefamulin/Isoniazid INH 0.75 Additive effect Lefamulin/bedaquiline BDQ 0.5 Synergy Lefamulin/Linezolid LZD 0.75 Additive effect Lefamulin/clofazimine CFZ 0.625 Additive effect Lefamulin/Delamanid DLM 0.625 Additive effect

Example 4. Evaluation of the antibacterial activity of lefamulin combined with bedaquiline against tuberculosis in macrophages 4.1 Cell preparation

1. Cultivate cells: Prepare 1640 complete medium (0.1% double antibody + 10% fetal bovine serum (FBS)) and culture THP-1 cells in a cell culture flask.

2. Collect, count and dilute cells: THP-1 cells are suspension cells. Collect and centrifuge the cultured cells and discard the supernatant. Add complete cell culture medium containing double antibodies to resuspend the cells and count the cells. Dilute the cells to 1×10 ⁶ /mL according to the counting results.

3. Add phorbol ester (PMA): Prepare PMA, a macrophage differentiation stimulator, and add it to the cell suspension to a final concentration of 100 ng/mL.

4. Plate: Prepare a culture plate and plate the above cells into the culture plate. Make the number of cells in each well 5×10 ⁵ and culture statically to allow the monocytes to differentiate into macrophages.

5. Wash away PMA: After culture, macrophages adhere to the wall and grow. Discard the supernatant, wash with PBS buffer, add complete cell culture medium without double antibody, and continue incubating in the cell culture incubator.

4.2 Bacterial preparation

1. Centrifuge the collected bacterial suspension and discard the supernatant. Resuspend it in complete cell culture medium without double antibodies, measure the OD ₆₀₀ value, and adjust the bacterial OD ₆₀₀ value to 0.6, corresponding to a bacterial concentration of about 1×10 ⁸ cells/mL.

2. Infect cells at MOI = 1:1. The bacterial suspension with OD ₆₀₀ = 0.6 needs to be diluted 100 times by adding complete cell culture medium before being used to infect cells.

4.3 Bacterial infection of cells

1. Discard the supernatant of macrophages that have grown adherently, and discard the supernatant after washing with PBS solution.

2. Add the prepared bacterial suspension.

3. Incubate in a cell culture incubator, discard the supernatant, and wash with PBS to remove extracellular bacteria.

4.4 Target drug incubation

To the 24-well macrophage plates from which extracellular bacteria had been washed away, 1640 complete medium containing 2 μg/ml of Lefamulin, Bedaquiline, Bedaquiline+Lefamulin and the same volume of DMSO was added, and the incubation continued.

4.5 Colony count

1. Use 0.01% Triton (diluted with PBS) to lyse the cells at 0, 1, and 3 days of drug incubation, and the cells are completely lysed after incubation.

2. Transfer the lysate stock solution to a 96-well plate, and make two replicate wells in each well of the 96-well plate.

3. Use a 96-well plate to perform gradient dilutions of the stock solution.

4. Take 10 μl of each gradient dilution of the bacterial solution and drop it on a solid agar plate containing 7H10 (containing 10% OADC).

5. Wait for the liquid on the plate to absorb and dry, then close the lid, seal it with sealing film, and place it upside down in an incubator for 3-4 weeks.

4.6 Interpretation of results

As shown in Table 4, after 3 days of Lefamulin treatment alone, the bacterial load was reduced by 5×10 ³ CFU compared with the DMSO control group. After 3 days of Bedaquiline treatment alone, the bacterial load was reduced by 9.2×10 ³ CFU compared with the DMSO group; after 3 days of Lefamulin + Bedaquiline combined treatment, the bacterial load was reduced by 1.02×10 ⁴ CFU compared with the DMSO control group.

As shown in Figure 3, after 3 days of Lefamulin treatment, the bacterial load was reduced by 41.67% ± 12.42% compared with the DMSO control group. After 3 days of Bedaquiline treatment, the bacterial load was reduced by 76.67% ± 6.83% compared with the DMSO control group; after 3 days of combined treatment with Lefamulin + Bedaquiline, the bacterial load was reduced by 83.33% ± 8.84% compared with the DMSO control group. Compared with single-drug treatment, Lefamulin combined with Bedaquiline effectively improved its bactericidal effect on intracellular Mycobacterium tuberculosis after 3 days of treatment.

Table 4 Intracellular survival of H37Rv infected THP-1 cells after treatment with different drugs

Claims (10)

1. A pharmaceutical composition comprising Lefamulin and another drug against a mycobacterium tuberculosis infection, the Lefamulin having the structural formula:

2. The pharmaceutical composition of claim 1, wherein the mycobacterium tuberculosis comprises mycobacterium tuberculosis carried by a mycobacterium tuberculosis standard strain, a mycobacterium tuberculosis clinical isolate, or a mycobacterium tuberculosis infected patient; preferably, the mycobacterium tuberculosis is a pan-sensitive and drug-resistant mycobacterium tuberculosis.

3. The pharmaceutical composition of any one of claims 1 or 2, wherein the other anti-mycobacterium tuberculosis infection agent comprises one or more of an antibiotic and other agents that can facilitate inhibition or killing of mycobacterium tuberculosis or provide patient resistance.

4. The pharmaceutical composition of any one of claims 1-3, one or more of rifampin, bedaquiline, linezolid, delamanib, levofloxacin, moxifloxacin, clofazimine, pyrazinamide, ethambutol, streptomycin, isoniazid, and/or ethionamide; the other drugs include one or more of vitamins, amino acids, proteins, and/or minerals.

5. The pharmaceutical composition of any one of claims 1-4, further comprising one or more pharmaceutically acceptable carriers.

6. The pharmaceutical composition of any one of claims 1-5, which can be formulated into a variety of dosage forms, including, but not limited to, one or more of tablets, capsules, drops, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories, and/or lyophilized powder for injection.

7. Use of Lefamulin in the preparation of a product for inhibiting mycobacterium tuberculosis.

8. The mycobacterium tuberculosis inhibiting product of claim 7, comprising one or more of a drug and/or a bacteriostatic agent.

9. Use of Lefamulin in the manufacture of a medicament for the prevention and/or treatment of a disease caused by a mycobacterium tuberculosis infection, wherein Lefamulin functions by:

1) Lefamulin inhibit mycobacterium tuberculosis activity;

2) Lefamulin kill Mycobacterium tuberculosis.

10. The use according to claim 9, wherein the mycobacterium tuberculosis comprises mycobacterium tuberculosis carried by a mycobacterium tuberculosis standard strain, a mycobacterium tuberculosis clinical isolate or a mycobacterium tuberculosis infected patient; preferably, the mycobacterium tuberculosis is a pan-sensitive and drug-resistant mycobacterium tuberculosis.