Application of Lefamulin in intracellular mycobacterium infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a semi-synthetic pleuromutilin medicine in resisting intracellular mycobacterium infection. The Lefamulin disclosed by the invention has relatively good bacteriostatic activity on an intracellular mycobacterium standard strain and a clinical isolated strain, the MIC of the Lefamulin on the intracellular mycobacterium standard strain can reach 0.25 mu g / mL, and the best MIC of the Lefamulin on the clinical isolated strain of the intracellular mycobacterium can reach 0.03125 mu g / mL. According to the invention, the application field of the Lefamulin is widened, and a new method is provided for treatment of intracellular mycobacterium infection.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of a semi-synthetic pleuromutilin drug in resisting intracellular mycobacterium infection. Background Art

[0002] Non-tuberculous mycobacteria (NTM) are also called environmental mycobacteria or mycobacteria other than Mycobacterium tuberculosis complex (MTB) and Mycobacterium leprae (M.leprae). They can be found everywhere in the environment and are conditionally pathogenic bacteria. Most of them are parasites, and only a small number can cause disease (Cai Huijun, Tang Zhenghao. Current status of infection and research progress on diagnostic methods of non-tuberculous mycobacteria [J]. Chinese Journal of Clinical Medicine, 2023, 30(05): 900-907.). According to the growth rate, they can be divided into fast growing mycobacteria (RGM) and slow growing mycobacteria (SGM). Fast growing mycobacteria include Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium chelonae and Mycobacterium smegmatis, and slow growing mycobacteria include Mycobacterium kansasii, Mycobacterium avium, Mycobacterium intracellulare, etc. Studies have shown that Mycobacterium intracellulare, Mycobacterium paraintracellulare and Mycobacterium chimera are closely related, while they are distantly related to Mycobacterium avium, Mycobacterium Columbia and other slow growing mycobacteria (Mycobacterium gordonii, Mycobacterium paragordonii, Mycobacterium stenosis and Mycobacterium stenosis) (Huang Liping, He Huaqiang, Zou Wei, et al. Clinical characteristics of non-tuberculous mycobacterial pulmonary disease and its species identification [J]. Chinese Journal of Hospital Infection, 2023, 33(5): 641-646.). Currently, about 80 disease-causing NTMs have been found in the environment and animal hosts. Although most NTMs are non-pathogenic and have low clinical infection rates, as many as 60 pathogenic NTMs have been repeatedly isolated in clinical samples. Due to changes in population structure, advances in radiological diagnosis of lung abnormalities and mycobacterial identification technology, and the control and reduction of tuberculosis (TB), the incidence and disease burden of nontuberculous mycobacterial infections have increased significantly worldwide. Studies have found that among suspected patients with multidrug-resistant tuberculosis who have not responded to first-line anti-tuberculosis drug treatment for 2-3 months, about 30% are actually infected with NTM (SHAHRAKIA H, HEIDARIEH P, BOSTANABAD SZ, et al. "Multidrug-resistant tuberculosis" may be nontuberculous mycobacteria[J]. Eur J Intern Med, 2015, 26(4): 279-84.).

[0003] Mycobacterium intracellulare is the most common NTM with the highest isolation rate in my country, accounting for 35.69% of NTM clinical isolates. It mainly causes lung infection, but can also cause lymph node, skin and soft tissue infection, and even disseminated infection, especially in immunocompromised patients (DALEY CL, IACCARINO JM, LANGE C, et al. Treatment of non-tuberculous mycobacterial pulmonary disease: an official ATS / ERS / ESCMID / lDSA clinical practice guideline [J]. Clin Infect Dis, 2020, 71 (4): 905-913.). Mycobacterium intracellulare has a strong ability to survive in cells, can resist the phagocytosis and digestion of host macrophages, survive by inhibiting the maturation and acidification of lysosomes, and form a persistent infection in the host. In addition, the growth rate of intracellular mycobacterium is slow, which makes it difficult to diagnose its infection quickly, the treatment cycle is long and the recurrence rate is high. Infection with intracellular mycobacterium is on the rise worldwide, especially in patients with chronic lung disease or immunocompromised patients, and has become a clinically important public health problem. At the same time, intracellular mycobacterium is highly resistant to drugs, especially to first-line anti-tuberculosis drugs such as rifampicin and ethambutol (RYU YJ, KOH WJ, DALEY CL. Diagnosis and Treatment of Nontuberculous Mycobacterial Lung Disease: Clinicians' Perspectives [J]. Tuberc Respir Dis (Seoul), 2016, 79 (2): 74-84.). The current treatment regimen recommends multi-drug combination therapy based on macrolide drugs, but its overall cure rate is low, only 50% to 70%, reflecting the inherent resistance of intracellular mycobacterium to antibiotics and the challenges of treatment management.

[0004] Due to the various drug resistance mechanisms caused by NTM cell wall effects, biotransformation, induced resistance, efflux pumps, acquired target gene mutations, etc., it has a high resistance to a variety of antimicrobial drugs (Saxena S, Spain HP, Forn-Cuní G. Drug Resistance in Nontuberculous Mycobacteria: Mechanisms and Models. Biology (Basel). 2021 Jan 29; 10 (2): 96.). However, studies have found that different strains have different resistance rates to the same antimicrobial drug. Meilin et al. conducted drug sensitivity tests on 237 strains of NTM and found that for ethambutol, the resistance rate of Mycobacterium abscessus was 97.0%, while that of Mycobacterium kansasii was only 11.8%; for cefoxitin, the resistance rate of Mycobacterium kansasii was as high as 100.0%, while Mycobacterium abscessus had no resistance; for gatifloxacin, the resistance rate of Mycobacterium intracellulare was 52.5%, while Mycobacterium avium had no resistance to gatifloxacin (Meilin, Liu Shengsheng, Lin Wenhong, et al. Analysis of distribution, clinical characteristics and drug resistance of non-tuberculous mycobacterium pulmonary disease in Anhui Chest Hospital [J]. Chinese Journal of Antibiotics, 2023, 48(11): 1288-1294.). Lin Xuefeng et al. conducted a drug resistance analysis of 308 strains and 19 types of non-tuberculous mycobacteria (NTM) in Yueqing area and found that Mycobacterium intracellulare had good sensitivity to clarithromycin, with a sensitivity rate of 95.6%, a resistance rate of less than 3.0%, and a MIC of 1. 50 The sensitivity of Mycobacterium avium to clarithromycin was poor, only 30.8%, and the MIC 50 16 μg / mL (Lin Xuefeng, Chen Jing, Jiang Danying, et al. Study on the identification, distribution and drug resistance of 308 strains of non-tuberculous mycobacteria in Yueqing area [J]. Zhejiang Medicine, 2024, 46(3): 317-322.). Therefore, choosing an appropriate antimicrobial treatment regimen is crucial for NTM treatment, which is beneficial to improve the efficacy and reduce excessive exposure to antimicrobial drugs.

[0005] As early as the 1970s, pleuromutilin antibiotics were used as veterinary antibiotics. In recent years, research on them as human antibiotics has become more popular. In 2019, the FDA approved the first systemic pleuromutilin antibiotic Lefamulin (including intravenous and oral formulations) for clinical treatment of community-acquired pneumonia. The antibacterial mechanism of Lefamulin is mainly through acting on bacterial ribosomes and selectively inhibiting bacterial protein synthesis. Its chemical structure mainly includes a tricyclic mutilin core, a C-21 keto group and a C-14 side chain. The tricyclic mutilin core and the C-14 side chain bind to the A site and P site of the bacterial ribosome 50S subunit and the 23S rRNA peptidyl transferase center through hydrogen bonds, hydrophobic bonds, conformational changes and other pathways, blocking the correct positioning of tRNA and affecting peptidyl transfer, thereby inhibiting bacterial protein synthesis. The binding site of Lefamulin is in the highly conserved core of the ribosomal peptidyl transferase center, acting at an earlier stage of bacterial protein synthesis and having a stronger binding force with the target site. This unique mechanism of action makes the drug theoretically less likely to cause bacterial resistance, and is also less likely to develop cross-resistance with other antibacterial drugs that act on bacterial ribosomes (Eyal Z, Matzov D, Krupkin M, et al. A novel pleuromutilin antibacterial compound, its binding mode and selectivity mechanism. Sci Rep. 2016 Dec 13; 6: 39004.). In vitro antibacterial studies have shown that Lefamulin has strong in vitro activity against Gram-positive and Gram-negative bacteria (such as Neisseria gonorrhoeae, Haemophilus influenzae and Moraxella catarrhalis) and atypical bacteria, including vancomycin-resistant Enterococcus faecalis, ofloxacin-resistant Streptococcus pneumoniae, methicillin-resistant Staphylococcus aureus, multidrug-resistant (MDR) Streptococcus pneumoniae and macrolide-resistant Mycoplasma pneumoniae (Veve MP, Wagner JL. Lefamulin: Review of a Promising Novel Pleuromutilin Antibiotic. Pharmacotherapy. 2018 Sep; 38(9): 935-946.). The global antimicrobial surveillance data (SENTRY) from 2015 to 2016 showed that Lefamulin at a concentration of 1 μg / mL could inhibit 100% of Streptococcus pneumoniae isolates, 99.8% of Staphylococcus aureus isolates, 93.8% of Haemophilus influenzae isolates, and 100% of Moraxella catarrhalis isolates.Lefamulin's activity against S. pneumoniae was not affected by resistance to other antimicrobial classes, including β-lactams, fluoroquinolones, and macrolides, as well as multidrug-resistant (MDR) or extensively drug-resistant strains; MICs for all strains. 50 / 90 The values ​​were all 0.06 / 0.12 μg / mL, except for strains insensitive to levofloxacin (0.06 / 0.25 μg / mL) (Paukner S, Gelone SP, Arends SJR, et al. Antibacterial Activity of Lefamulin against Pathogens Most Commonly Causing Community-Acquired Bacterial Pneumonia: SENTRY Antimicrobial Surveillance Program (2015-2016). Antimicrob Agents Chemother. 2019 Mar 27; 63 (4): e02161-18.). In general, the antibacterial spectrum of Lefamulin is similar to that of macrolide drugs, but it has better antibacterial activity against drug-resistant bacteria. At present, there are no reports on the inhibition of intracellular mycobacteria by Lefamulin. Summary of the invention

[0006] The present invention was completed based on the discovery that Lefamulin has the effect of inhibiting the activity of intracellular mycobacterium.

[0007] In a first aspect, the present invention provides a pharmaceutical composition comprising Lefamulin and another drug for preventing intracellular mycobacterium infection.

[0008] The pharmaceutical composition has at least one of the following effects:

[0009] a) inhibiting the activity of intracellular mycobacterium;

[0010] b) Anti-intracellular Mycobacterium infection;

[0011] c) preventing and / or treating diseases caused by Mycobacterium intracellulare.

[0012] Furthermore, the intracellular mycobacterium includes a standard strain of intracellular mycobacterium, a clinical isolate of intracellular mycobacterium, or mycobacterium carried by a patient infected with intracellular mycobacterium.

[0013] Furthermore, the other anti-intracellular mycobacterium infection drug includes one or more of antibiotics and other drugs that can help inhibit or kill intracellular mycobacterium or provide resistance to patients.

[0014] Furthermore, the antibiotics include one or more of rifampicin, streptomycin, ethambutol, moxifloxacin, clarithromycin and / or amikacin; and the other drugs include one or more of vitamins, amino acids, proteins and / or minerals.

[0015] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0016] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0017] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories; the drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0018] Furthermore, the dosage form may be one or more of a conventional preparation, a sustained-release preparation and / or a controlled-release preparation.

[0019] Furthermore, the pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or encapsulated with other substances.

[0020] In a second aspect, the present invention provides the use of Lefamulin in preparing a product for inhibiting intracellular mycobacterium.

[0021] Furthermore, the intracellular mycobacterium includes a standard strain of intracellular mycobacterium, a clinical isolate of intracellular mycobacterium, or mycobacterium carried by a patient infected with intracellular mycobacterium.

[0022] Furthermore, the product for inhibiting intracellular mycobacterium is selected from one or more of experimental reagents and / or antibacterial agents.

[0023] Furthermore, the antibacterial agent may be a detergent, which inhibits / eliminates intracellular mycobacteria through Lefamulin and its pharmaceutically acceptable salts.

[0024] In a third aspect, the present invention provides the use of Lefamulin in the preparation of a drug for preventing and / or treating diseases caused by Mycobacterium intracellulare infection;

[0025] Lefamulin works in the following ways:

[0026] 1) Lefamulin inhibits intracellular mycobacterial activity;

[0027] 2) Lefamulin kills intracellular mycobacteria.

[0028] Furthermore, the intracellular mycobacterium includes a standard strain of intracellular mycobacterium, a clinical isolate of intracellular mycobacterium, or mycobacterium carried by a patient infected with intracellular mycobacterium.

[0029] Furthermore, one or more pharmaceutically acceptable carriers may be added to the drug.

[0030] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0031] Furthermore, the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories; the drugs in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0032] Furthermore, the dosage form may be one or more of a conventional preparation, a sustained-release preparation and / or a controlled-release preparation.

[0033] Furthermore, the drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or introduced into the body after being mixed or encapsulated with other substances.

[0034] Beneficial Effects

[0035] Lefamulin in the present invention has good antibacterial activity against standard strains and clinical isolates of intracellular mycobacterium, and the MIC of the standard strain of intracellular mycobacterium can reach 0.25 μg / mL, and the MIC of the clinical isolate of intracellular mycobacterium can reach 0.03125 μg / mL at best.

[0036] The invention broadens the application field of Lefamulin and provides a new method for treating intracellular mycobacterium infection. DETAILED DESCRIPTION

[0037] The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0038] Explanation of terms

[0039] 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.

[0040] Mycobacterium intracellulare standard strain: ATCC 13950.

[0041] Mycobacterium chimera standard strain: DSM 44612.

[0042] Lefamulin: purchased from MedchemExpress, product number: HY-16908A, molecular formula: C 28 H 45 NO5S, CAS No. 1350636-82-6. The structural formula is as follows:

[0043]

[0044] Example 1. Detection of the antibacterial activity of Lefamulin against standard strains of Mycobacterium intracellulare and Mycobacterium chimera

[0045] 1. Prepare standard strain suspension: Inoculate the standard strain of intracellular mycobacterium (ATCC 13950) and chimera mycobacterium (DSM 44612) in neutral Roche medium, culture in a 37°C incubator for 3 to 4 weeks, and scrape the strains that have grown to the logarithmic phase from the neutral Roche medium. After grinding the bacteria, adjust the concentration of the bacterial suspension to 1 McFarland by turbidimetry, and use Mueller Hinton (MH) medium containing 5% OADC to dilute it at a ratio of 1:20 for later use.

[0046] 2. Drug dilution: Add 100 μL of MH medium (containing 5% OADC) to each well of a 96-well plate. After completion, add 100 μL of a 128 μg / mL drug solution (prepared with DMSO) to the first column, mix well, and add 100 μL from the first column to the second column, and so on, performing a two-fold gradient dilution column by column until the 11th column, and then take 100 μL and discard it. No drug is added to the 12th column, which serves as a positive control well. Set up 3 replicate wells for each concentration.

[0047] 3. Inoculation of bacterial suspension: Add 100 μL of the standard strain of intracellular mycobacterium suspension to each well, so that the final volume of each well reaches 200 μL. The final concentration of the bacterial solution is 4×10^5 CFU / mL. The specific final drug concentration in each well is shown in Table 1.

[0048] 4. Culture: Place the 96-well plate in a 37°C incubator for 7 days.

[0049] 5. Add color development reagent: After the culture is completed, add 20 μL of A Lamar blue and 50 μL of 5% Tween 80 solution to each well of the 96-well plate and incubate in a 37°C incubator for 24 hours to perform a color development reaction.

[0050] 6. Reading results: Take a 96-well plate, read the minimum inhibitory concentration (MIC) and calculate the inhibition rate.

[0051] Table 1

[0052]

[0053] Minimum inhibitory concentration (MIC) reading method: Minimum inhibitory concentration (MIC) is the drug concentration that can inhibit 90% of bacterial colony growth. The minimum drug concentration that inhibits >90% of the production of reduced Alamar blue by fluorescence detection (Ex / Em, 530nm / 600nm).

[0054] Inhibition rate % = 100% - (fluorescence value of the detection well - background fluorescence value) / (fluorescence value of the growth control well - background fluorescence value) × 100%.

[0055] The background fluorescence value is the fluorescence value of the negative control, and the fluorescence value of the growth control well is the fluorescence value of the positive control.

[0056] The negative control was a culture medium without drugs and bacterial solution, and the positive control was a culture medium containing bacteria without drugs.

[0057] The results showed that the MIC of Lefamulin against the standard strain of intracellular Mycobacterium was 0.25 μg / mL, and the MIC against Mycobacterium chimaera was>32 μg / mL.

[0058] Example 2: Detection of the antibacterial activity of Lefamulin against clinically isolated Mycobacterium intracellulare and Mycobacterium chimera

[0059] Clinical isolates: 16SrRNA, hsp65, rpoB, and 16-23S rRNA intergenic region sequencing were used to identify the strains, and 27 clinical isolates of intracellular mycobacterium were selected for subsequent testing. Due to the low isolation rate of chimaera mycobacterium, all chimaera mycobacterium (a total of 5 strains) stored in this laboratory were used for subsequent testing.

[0060] Drug to be tested: Lefamulin

[0061] According to the method in Example 1, the antibacterial activity of the drug to be tested against 27 clinically isolated intracellular mycobacterium and 5 mycobacterium chimaera strains was detected.

[0062] The MICs of lefamulin against 5 strains of Mycobacterium chimera were as follows: MIC = 8 μg / mL for 1 strain, and MIC > 32 μg / mL for 4 strains. Therefore, lefamulin had low antibacterial activity against Mycobacterium chimera, suggesting that different strains have different resistance spectra to the same antimicrobial drug.

[0063] As shown in Table 2 and Table 3, the results indicate that Lefamulin has good antibacterial activity against intracellular mycobacteria, with the best MIC reaching 0.03125 μg / mL. It is expected to find new applications in the clinical treatment of intracellular mycobacterium infections.

[0064] Table 2 MIC of Lefamulin against 5 strains of intracellular mycobacterium

[0065]

[0066]

[0067] Table 3 Statistical results of MIC concentration distribution of Lefamulin against 5 strains of intracellular mycobacterium

[0068]

Claims

1. A pharmaceutical composition comprising Lefamulin and another drug for preventing intracellular mycobacterium infection, wherein the other drug for preventing intracellular mycobacterium infection comprises one or more of antibiotics and other drugs that can help inhibit or kill intracellular mycobacterium or provide resistance to patients; The pharmaceutical composition has at least one of the following effects: a) inhibiting the activity of intracellular mycobacterium; b) Anti-intracellular mycobacterium infection; c) preventing and / or treating diseases caused by Mycobacterium intracellulare.

2. The pharmaceutical composition according to claim 1, wherein the intracellular mycobacterium comprises a standard strain of intracellular mycobacterium, a clinical isolate of intracellular mycobacterium, or mycobacterium carried by a patient infected with intracellular mycobacterium.

3. Application of Lefamulin in the preparation of products for inhibiting intracellular mycobacteria.

4. The use according to claim 3, wherein the intracellular mycobacterium comprises a standard strain of intracellular mycobacterium, a clinical isolate of intracellular mycobacterium, or mycobacterium carried by a patient infected with intracellular mycobacterium.

5. The use according to claim 3, wherein the product for inhibiting intracellular mycobacterium is selected from one or more of experimental reagents and / or antibacterial agents.

6. The use according to claim 5, wherein the antibacterial agent can be a detergent, and the detergent inhibits / eliminates intracellular mycobacteria through Lefamulin and its pharmaceutically acceptable salts.

7. Use of Lefamulin in the preparation of drugs for preventing and / or treating diseases caused by Mycobacterium intracellulare infection; Lefamulin works in the following ways: 1) Lefamulin inhibits intracellular mycobacterial activity; 2) Lefamulin kills intracellular mycobacteria.

8. The use according to claim 7, wherein the intracellular mycobacterium comprises a standard strain of intracellular mycobacterium, a clinical isolate of intracellular mycobacterium, or mycobacterium intracellulare carried by a patient infected with intracellular mycobacterium.

9. The use as claimed in claim 7, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents and / or suppositories; the drugs in the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

10. The use as claimed in claim 7, wherein the drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or wrapped with other substances.

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