Application of TBA-7371 in prevention and treatment of nontuberculous mycobacterium infection

Abstract

According to the invention, systematic MIC determination is carried out on fast-growing and slow-growing nontuberculous mycobacteria, and it is found that TBA-7371 has stable low MIC in nontuberculous mycobacteria of different pedigree, so that it is technically ensured that TBA-7371 can reach an effective inhibitory concentration in vivo more easily and has broad-spectrum applicability. The invention further explores the combined medication effect of the TBA-7371 and a plurality of first-line anti-mycobacterium tuberculosis drugs, and finds that when the TBA-7371 is combined with other anti-tuberculosis drugs, the required concentrations of the other anti-tuberculosis drugs can be reduced.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically relates to the application of a drug. Background Technology

[0002] Non-tuberculous mycobacteria (NTM) refers to all mycobacteria except for the Mycobacterium tuberculosis complex and Mycobacterium leprae. NTMs are ubiquitous in nature and are opportunistic pathogens. The morbidity and mortality rates of lung infections they cause are rising globally, posing a serious threat to human health.

[0003] Nontuberculous mycobacteria (NTM) include fast-growing and slow-growing mycobacteria. Among them, *Mycobacterium abscessum*, one of the fast-growing mycobacteria, is highly pathogenic and exhibits extremely strong drug resistance, making it a multidrug-resistant bacterium. It typically causes inflammatory lung infections as well as severe skin, joint, soft tissue, surgical site, and disseminated infections. Current treatment mainly relies on long-term combination therapy with multiple drugs such as clarithromycin, azithromycin, rifampin, ethambutol, and aminoglycosides. However, factors such as the low permeability of the bacterium's outer membrane lipid barrier, drug-modified inactivating enzymes, efflux pump systems, and target mutations contribute to its high resistance rate to most clinically used antibiotics and anti-tuberculosis drugs, with antibiotic treatment achieving a cure rate of only 30-50%. Therefore, evaluating the efficacy of novel anti-tuberculosis drugs against nontuberculous mycobacteria will provide new options for treating nontuberculous mycobacterial infections, which is of great significance for improving patient treatment outcomes and prognosis.

[0004] Deformylphosphorylated β-d-ribose oxidase (DprE1) is a novel drug target in Mycobacterium tuberculosis. TBA-7371 is a non-covalent inhibitor of Mycobacterium tuberculosis DprE1 with an MIC of 0.64 μg / ml, and is currently in a phase 2 dose-escalation clinical trial. Robertson et al. evaluated the antibacterial effects of three DprE1 inhibitors in clinical trials, TBA-7371, PBTZ169, and OPC-167832, as single agents in a C3HeB / FeJ mouse model. They found that all three DprE1 inhibitors had significant therapeutic effects in C3HeB / FeJ mice (Comparative Analysis of Pharmacodynamics in the C3HeB / FeJ Mouse Tuberculosis Model for DprE1 Inhibitors TBA-7371, PBTZ169, and OPC-167832[J]. Antimicrob Agents Chemother. 2021 Oct 18;65(11):e0058321.). Li et al. used a BALB / c tuberculosis mouse model to evaluate the potential of a novel regimen combining bedaquiline or the more potent diacylquinoline TBAJ-587 with GSK2556286 and the DprE1 inhibitor TBA-7371 in the treatment of Mycobacterium tuberculosis infection (Bactericidal and sterilizing activity of novel regimens combining bedaquiline or TBAJ-587 with GSK2556286 and TBA-7371 in a mouse model of tuberculosis[J]. Antimicrob Agents Chemother. 2024;68(4):e0156223.).

[0005] Currently, domestic and international research on the DprE1 inhibitor TBA-7371 mainly focuses on its inhibition of Mycobacterium tuberculosis, and no research has been reported on its use in treating NTM infection. Summary of the Invention

[0006] The present invention discovered that TBA-7371 has the activity of inhibiting nontuberculous mycobacteria, and based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides the use of TBA-7371 in the preparation of a drug for preventing and treating nontuberculous mycobacterial infections, wherein the chemical structural formula of TBA-7371 is: .

[0008] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and treating nontuberculous mycobacterial infections.

[0009] Furthermore, the carrier is selected from water-soluble carriers, poorly soluble carriers, or enteric carriers.

[0010] Furthermore, the dosage form of the drug for preventing and treating nontuberculous mycobacterial infections is selected from one of the following: tablets, capsules, aerosols, pills, powders, solutions, and granules.

[0011] Furthermore, the drug for preventing and treating nontuberculous mycobacterial infections is introduced into the body through physical or chemical means, such as muscle, intradermal, subcutaneous, venous, or mucosal tissues; or it is introduced into the body after being mixed or encapsulated with other substances.

[0012] Furthermore, the nontuberculous mycobacteria include fast-growing mycobacteria and slow-growing mycobacteria.

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising TBA-7371 and another drug against nontuberculous mycobacterial infection, as well as a pharmaceutically acceptable carrier or excipient.

[0014] Furthermore, the chemical structural formula of TBA-7371 is as follows: .

[0015] Furthermore, the other anti-tuberculosis drug is selected from one or more of the following: amikacin, bedaquiline, capreomycin, azithromycin, contazoline, linezolid, cycloserine, delamani, depazoline, ethambutol, ethionamide, isoniazid, kanamycin, levofloxacin, mycoxinone, moxifloxacin, ofloxacin, tigecycline, para-aminosalicylic acid, propylthiouracil, isoniazid, pyrazinamide, clofazimine, cefoxitin, fusidic acid, rifabutin, and / or rifampin.

[0016] Preferably, the other anti-tuberculosis drug is selected from one or more of rifampin, isoniazid, linezolid, azithromycin, amikacin, ethambutol, and levofloxacin.

[0017] Furthermore, the nontuberculous mycobacteria include fast-growing mycobacteria and slow-growing mycobacteria.

[0018] Thirdly, the present invention provides the use of the pharmaceutical composition described in the second aspect in the preparation of a medicament for preventing and treating nontuberculous mycobacterial infections.

[0019] Furthermore, the nontuberculous mycobacteria include fast-growing mycobacteria and slow-growing mycobacteria.

[0020] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and treating nontuberculous mycobacterial infections.

[0021] Furthermore, the carrier is selected from water-soluble carriers, poorly soluble carriers, or enteric carriers.

[0022] Furthermore, the dosage form of the drug for preventing and treating nontuberculous mycobacterial infections is selected from one of the following: tablets, capsules, aerosols, pills, powders, solutions, and granules.

[0023] Furthermore, the drug for preventing and treating nontuberculous mycobacterial infections is introduced into the body through physical or chemical means, such as muscle, intradermal, subcutaneous, venous, or mucosal tissues; or it is introduced into the body after being mixed or encapsulated with other substances.

[0024] Beneficial effects This invention systematically determined the MIC (microinhibitory MIC) of TBA-7371 in both fast-growing and slow-growing nontuberculous mycobacteria, finding that TBA-7371 exhibits consistently low MICs across different lineages of nontuberculous mycobacteria. This technically ensures that it is more easily achieved at effective inhibitory concentrations in vivo and possesses broad-spectrum applicability. Furthermore, this invention explored the combined efficacy of TBA-7371 with various first-line anti-tuberculosis drugs, finding that TBA-7371, when used in combination with other anti-tuberculosis drugs, can reduce the required concentrations of those drugs.

[0025] This invention optimizes multi-drug combination therapy, providing quantitative basis for the dosage of highly toxic drugs while ensuring efficacy, and offering a new treatment option for the prevention and treatment of nontuberculous mycobacterial infections. Attached Figure Description

[0026] Figure 1 The distribution of MICs for avian clinical strains and intracellular clinical strains is shown in Figure 1. A represents the MIC distribution of avian clinical strains, and B represents the MIC distribution of intracellular clinical strains.

[0027] Figure 2 The inhibitory effect of TBA-7371 on intracellular mycobacteria.

[0028] Figure 3 The in vitro bactericidal effect of TBA-7371 was demonstrated based on the THP-1 macrophage model.

[0029] Figure 4 To evaluate the combined use of TBA-7371 with commonly used clinical antibacterial drugs. Detailed Implementation

[0030] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0031] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0032] Example 1: TBA-7371 Drug Sensitivity Activity Test strain culture The Mycobacterium tuberculosis reference strain used in this invention is H37Rv (ATCC 27294), and the clinical isolates involved are from the Beijing Chest Hospital Biobank.

[0033] All strains were cultured at 37°C on Müller-Hinton broth MH broth (BD Difco, USA) supplemented with 0.05% Tween-80 and 10% oleic acid-albumin-glucose-catalase (OADC) growth supplement (BD Difco, USA); and on solid Müller-Hinton broth MH medium (BD Difco, USA) supplemented with 10% OADC growth supplement (BD Difco, USA). All strains were stored at -80°C and cultured at 37°C on solid Löwenstein-Jensen (LJ) medium (Zhuhai, China).

[0034] Drug susceptibility assay TBA-7371 used in this invention was purchased from Med ChemExpress (USA). Other antibiotics, including rifampin, isoniazid, azithromycin, levofloxacin, amikacin, ethambutol, and linezolid, were purchased from Solarbio (China). Broth microdilution was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. MIC of the strain was tested using Müller-Hinton broth MH medium.

[0035] The final concentrations of TBA-7371 were from 0.0625 μg / mL to 64 μg / mL (the drug was serially diluted twofold starting from 64 μg / mL down to 0.0625 μg / mL (64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL) in 96-well microplates. Bacteria were scraped from LJ medium, homogenized in a grinding flask, adjusted to McFarland turbidity 1 with sterile saline, and then diluted 20-fold before inoculation. 100 µL of diluted inoculum was added to each well, and the microplates were incubated at 37°C for 7 days. Subsequently, 30 µL of AlamarBlue solution was added to each well, and the plates were incubated at 37°C for another 24 hours, observing the color changes.

[0036] The minimum inhibitory concentration (MIC) was defined as the lowest drug concentration at which the inhibitory indicator changed from blue to pink. Rifampicin (RIF) was used as a positive control. The results are shown in Tables 1 and 2. Table 1 lists the MICs of TBA-7371 against slow-growing nontuberculous mycobacteria and some reference strains of the Mycobacterium tuberculosis complex. TBA-7371 showed strong in vitro antibacterial activity against most slow-growing mycobacteria, with MICs mainly concentrated at 0.25–1 μg / mL. The MICs of some strains increased to 2–4 μg / mL, such as… M. celatum, M. africanum, M. shimoidei and M. chelonae .also, M. Kansas The MIC is 8 μg / mL. M. nonchromogenicum The MIC was the highest, at 16 μg / mL. It is worth noting that... M. tuberculosis The H37Ra had the lowest MIC (0.0625 μg / mL). The overall MIC range was 0.0625–16 μg / mL.

[0037] Table 1. MIC of TBA-7371 against slow-growing nontuberculous mycobacteria The results of the minimum inhibitory concentration (MIC) test of TBA-7371 against fast-growing nontuberculous mycobacteria showed that the in vitro antibacterial activity of TBA-7371 against different fast-growing mycobacteria varied significantly, with MICs ranging from 0.125 to 16 μg / mL. Mycobacterium neoaurum It has the lowest MIC (0.125 μg / mL). M. thermoresistant and M. Duval It also exhibited a low MIC (0.25 μg / mL); the MICs of most strains were concentrated in the range of 1–2 μg / mL. M. abscessand M. flavescens The MICs were the highest (all 16 μg / mL), indicating relatively low sensitivity to the drug.

[0038] Further analysis was conducted on the MIC distribution of the two types of clinical isolates. The results showed that the MIC frequency distribution of clinical Mycobacterium avium isolates ranged from 1 to 16 μg / mL, with 4 μg / mL being the most prevalent value (27 isolates), followed by 2 μg / mL (10 isolates) and 8 μg / mL (8 isolates). A few strains were distributed at 16 μg / mL (5 isolates) and 1 μg / mL (2 isolates). The MIC frequency distribution of clinical Mycobacterium endocytosis isolates ranged from 0.5 to 4 μg / mL, with the majority of strains concentrated at 1 μg / mL (45 isolates) and 2 μg / mL (21 isolates), and only a few distributed at 0.5 μg / mL and 4 μg / mL (2 isolates each). Overall, the MIC of TBA-7371 against clinical Mycobacterium endocytosis isolates was generally low, while the MIC distribution against clinical Mycobacterium avium isolates was higher and had a wider range.

[0039] Table 2. MIC of TBA-7371 against rapidly growing nontuberculous mycobacteria Example 2: Evaluation of the bactericidal activity of TBA-7371 Bacterial-drug mixtures at concentrations of 1×MIC, 2×MIC, 4×MIC, 8×MIC, 16×MIC, and 32×MIC as described in the MIC test plate were taken, and 100 µL of culture medium from each well was serially diluted and inoculated onto MH agar plates supplemented with 10% OADC. After inoculation, the plates were incubated upside down at 37°C for 3–4 weeks, and the colony units (CFUs) formed were counted. The CFUs associated with the detection of 99.9% bacterial kill or a reduction of 3 log10 surviving bacteria were determined; this was considered the minimum bactericidal concentration (MBC).

[0040] Changes in the survival rate of intracellular mycobacteria in infected cells after drug treatment. Compared with the untreated control group (0), the bacterial load in the 0.5×MIC group decreased only slightly; when the concentration reached 1×MIC, the bacterial load decreased significantly, and further decreased at 4×MIC, suggesting that the drug has a significant concentration-dependent bactericidal / cleaning effect on intracellular mycobacteria.

[0041] Example 3: In vitro bactericidal assay based on the THP-1 macrophage model Seed 500 µL of THP-1 cells into each well of a 24-well plate, resulting in a final concentration of 1×10⁻⁶ cells. 6Cells / mL. Cells were induced to differentiate into macrophages with 200 ng / mL PMA for 48 hours. THP-1 cells infected with intracellular mycobacteria (ATTC: 13950) were cultured in RPMI 1640 medium containing 10% fetal bovine serum at a multiplicity of infection (MOI) of 1:1. After infection at 37°C and 5% CO2 for 4 hours, cells were washed three times with pre-warmed 1×PBS to remove extracellular bacteria. DMSO medium was used as a negative control. To quantify CFU counts, cells were cultured on 7H10 agar medium supplemented with OADC.

[0042] TBA-7371 was co-administered at concentrations of 4 µg / mL, 8 µg / mL, and 16 µg / mL, with DMSO medium serving as a negative control. Cells were lysed with 0.01% Triton X-100 on days 1, 3, and 5, followed by serial dilutions with 1×PBS. To quantify CFU counts, cells were cultured on MH agar medium supplemented with OADC.

[0043] The inhibitory and eliminative effects of the drug on intracellular mycobacteria were evaluated using a cell infection model. Results showed that at 0 h, the bacterial loads in all groups were similar, with no significant differences (ns), indicating a consistent initial infection load. By 24 h, compared to the control group (NC), the bacterial load in the TBA-7371-treated groups significantly decreased in a dose-dependent manner: 4 μg / mL reduced CFU, and 8 μg / mL and 16 μg / mL further reduced it; there was no significant difference between 8 μg / mL and 16 μg / mL (ns). By 72 h, the drug effect was further enhanced; the 4, 8, and 16 μg / mL groups all significantly reduced the bacterial load compared to the control group (NC), showing a dose-dependent decreasing trend, with the 16 μg / mL group exhibiting the lowest bacterial load. Overall, this suggests that TBA-7371 has a time-dependent, dose-dependent eliminative effect on intracellular mycobacteria.

[0044] Example 4: Checkerboard Combined Drug Experiment This invention evaluates the synergistic activity of TBA-7371 in combination with first-line anti-tuberculosis drugs using an intracellular mycobacterium (ATTC: 13950) checkerboard test. The antibacterial activity of TBA-7371 in combination with rifampin (RIF), isoniazid (INH), linezolid (LZD), azithromycin (AZI), amikacin (AMK), ethambutol (EMB), and levofloxacin (LFX) was tested.

[0045] Add 50 µL of MH medium supplemented with 10% OADC to each well of a 96-well microplate. Then, add commonly used clinical antibiotics in the horizontal row and TBA-7371 in the vertical row. Specifically, add 100 µL of a commonly used clinical antibiotic at a concentration of 16×MIC in row H, then serially dilute the 16×MIC antibiotic by 2-fold, adding 100 µL to each well in column A according to the concentration from highest to lowest. Next, dilute TBA-7371 from 1 / 8×MIC to 16×MIC, then add 50 µL from column 10 in column 1 according to the concentration from highest to lowest to complete the drug plate for the combined experiment. The bacterial solution was prepared as described in Example 1. Add 100 µL of bacterial culture to each well of the 96-well plate, and each combination was statistically repeated three times. Incubate at 37°C for 7 days, then add an indicator and read the bacterial growth inhibition data based on the color change. The lowest dilution at which each drug combination exhibits bacterial inhibition is used to determine the fractional inhibitory concentration index (FICI); the FICI is determined as the sum of the fractional inhibitory concentrations (FICs) of the individual antibiotics, using the following formula: FICI = The sum of the FIC values ​​when a single antibiotic acts alone; FIC = CIC / MIC.

[0046] Wherein, CIC is the combined inhibitory concentration, representing the minimum concentration of antibiotics that inhibit growth in the combination; MIC is the minimum concentration of antibiotics that inhibit growth when used alone; the combined use of antibiotics is considered as follows: synergistic effect, ≤0.5; additive effect, 0.5–1.0; no difference, 1.0–4.0; or antagonistic effect, >4.0.

[0047] The interaction between TBA-7371 and various antimycobacterial drugs was evaluated using the checkerboard dilution method, and the fractional inhibitory concentration index (FICI) was used for determination. Results are as follows: Figure 4 As shown, the FICI values ​​for TBA-7371 in combination with amikacin (AMK), linezolid (LZN), and ethambutol (EMB) were 0.3125, 0.3125, and 0.375, respectively, all ≤0.5, suggesting a synergistic effect between these drugs and TBA-7371 in pairwise combinations. The FICI values ​​for TBA-7371 in combination with azithromycin (AZI), levofloxacin (LFX), rifampin (RIF), and isoniazid (INH) were 0.53125, 0.5625, 0.625, and 0.75, respectively, all within the 0.5–1.0 range, suggesting an additive effect. Overall, TBA-7371 exhibits synergistic effects with some drugs, while the remaining combinations are predominantly additive, with no antagonistic results observed.

Claims

1. The application of TBA-7371 in the preparation of drugs for the prevention and treatment of nontuberculous mycobacterial infections, wherein the chemical structural formula of TBA-7371 is: 。 2. In the application described in claim 1, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing nontuberculous mycobacterial infections.

3. As described in claim 1, the drug for preventing nontuberculous mycobacterial infection is introduced into the body, such as through muscles, intradermal tissue, subcutaneous tissue, veins, or mucous membranes, by physical or chemical means; or is introduced into the body after being mixed with or encapsulated by other substances.

4. The application as described in claim 1, wherein the nontuberculous mycobacteria include fast-growing mycobacteria and slow-growing mycobacteria.

5. A pharmaceutical composition comprising TBA-7371 and another drug against nontuberculous mycobacterial infections, and a pharmaceutically acceptable carrier or excipient, wherein the chemical structural formula of TBA-7371 is: 。 6. The pharmaceutical composition of claim 5, wherein the other anti-tuberculosis drug is selected from one or more of amikacin, bedaquiline, capreomycin, azithromycin, contazoline, linezolid, cycloserine, delamanide, depazoline, ethambutol, ethionamide, isoniazid, kanamycin, levofloxacin, mycoxinone, moxifloxacin, ofloxacin, tigecycline, para-aminosalicylic acid, propylthiouracil, isoniazid, pyrazinamide, clofazimine, cefoxitin, fusidic acid, rifabutin, and / or rifampin.

7. The pharmaceutical composition of claim 5, wherein the other anti-tuberculosis drug is selected from one or more of rifampin, isoniazid, linezolid, azithromycin, amikacin, ethambutol and levofloxacin.

8. The use of the pharmaceutical composition of claim 5 in the preparation of a medicament for preventing and treating nontuberculous mycobacterial infections.

9. The application as described in claim 8, wherein the nontuberculous mycobacteria include fast-growing mycobacteria and slow-growing mycobacteria.

10. The application as described in claim 8, wherein the drug for preventing nontuberculous mycobacterial infection is introduced into the body, such as into muscles, intradermal tissue, subcutaneous tissue, veins, or mucous membranes, by physical or chemical means; or is introduced into the body after being mixed with or encapsulated by other substances.

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