Application of quarfloxin, a fluoroquinolone derivative, in the preparation of anti-tuberculosis drugs

Abstract

The present application relates to the field of medicine, especially relates to the application of quarfloxin in the preparation of anti-tuberculosis drugs. The present application shows that quarfloxin can inhibit the growth of clinical strains and drug-resistant strains of Mycobacterium tuberculosis, and obvious bacteriostatic effect appears in the clinical strains and drug-resistant strains of Mycobacterium tuberculosis, so the application of quarfloxin in the preparation of anti-tuberculosis drugs is provided.

Description

[0001] This application claims priority to Chinese Patent Application No. 2024104765311, filed on April 19, 2024, entitled "Application of quarfloxin, a fluoroquinolone derivative, in the preparation of anti-tuberculosis drugs", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of pharmaceutical technology, and in particular to the application of fluoroquinolone derivative quarfloxin in the preparation of anti-tuberculosis drugs. Background Technology

[0003] Tuberculosis (TB) is a serious infectious disease of global public health concern caused by Mycobacterium tuberculosis (Mtb). While Mtb can infect various organs of the human body, it is most commonly found in the lungs because it thrives in environments with ample blood supply and high oxygen levels. Extrapulmonary tuberculosis occurs when Mtb invades sites other than the lungs.

[0004] Tuberculosis can be divided into latent and active forms. Latent tuberculosis infection refers to the presence of Mycobacterium tuberculosis in the patient's body, but the body's immune system prevents it from developing into active tuberculosis. This means that symptoms do not appear immediately after infection, and the infection cannot be transmitted to others, but it can progress to active tuberculosis. Active tuberculosis refers to the presence of Mycobacterium tuberculosis in the body, causing symptoms and being highly contagious.

[0005] Currently, there are few first-line drugs for tuberculosis treatment, and the treatment cost for drug-resistant tuberculosis is even higher. Although the widespread use of anti-tuberculosis drugs such as rifampin, isoniazid, and pyrazinamide has reduced the development of tuberculosis, the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) in recent years has brought new challenges to tuberculosis control.

[0006] Recently approved anti-tuberculosis drugs such as bedaquiline, pretopanil, and delamanil have shown new potential in the treatment of drug-resistant tuberculosis, but they face challenges such as high medical costs, low accessibility, and insufficient validation, resulting in a smaller population benefiting from them. Furthermore, clinical resistance to these newly discovered drugs is gradually emerging. Therefore, there remains an urgent need to develop new drugs or reuse currently approved medications to prevent and control tuberculosis. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide the application of quarfloxin, a fluoroquinolone derivative, in the preparation of anti-tuberculosis drugs.

[0008] This invention provides the application of quarfloxin in the preparation of anti-tuberculosis drugs.

[0009] Quarfloxin is a fluoroquinolone derivative with antitumor activity, and its structural formula is as follows:

[0010]

[0011] The experiments of this invention show that quarfloxin has inhibitory activity against Mycobacterium tuberculosis, and has a significant inhibitory effect on both non-drug-resistant and drug-resistant Mycobacterium tuberculosis.

[0012] In this embodiment of the invention, the anti-tuberculosis treatment includes: inhibiting the activity of Mycobacterium tuberculosis and / or killing Mycobacterium tuberculosis.

[0013] In this invention, the inhibition of Mycobacterium tuberculosis by quarfloxin includes in vitro inhibition and / or subsequent in vivo inhibition. Therefore, this invention provides quarfloxin for the preparation of an in vivo anti-Mycobacterium tuberculosis drug. Alternatively, quarfloxin can be used to prepare an in vitro anti-Mycobacterium tuberculosis drug.

[0014] In this embodiment of the invention, the minimum inhibitory concentration of quarfloxin for inhibiting the activity of Mycobacterium tuberculosis is 6.25~25 μg / mL.

[0015] In this embodiment of the invention, the Mycobacterium tuberculosis is a drug-resistant strain or a non-drug-resistant strain.

[0016] In some specific embodiments, the non-drug-resistant strain is Mycobacterium tuberculosis H37Rv, and the drug-resistant strain of Mycobacterium tuberculosis is MDR-TB 34789, MDR-TB 34832, MDR-TB 34796, MDR-TB 34816 or MDR-TB 34786.

[0017] In this embodiment of the invention, the tuberculosis is human tuberculosis.

[0018] In some embodiments, the tuberculosis is human pulmonary tuberculosis.

[0019] In some specific embodiments, the tuberculosis is non-drug-resistant human pulmonary tuberculosis or drug-resistant human pulmonary tuberculosis.

[0020] Furthermore, experiments of this invention have shown that quarfloxin has a synergistic antibacterial effect with rifampin (RIF). Therefore, this invention provides compositions containing quarfloxin and other anti-tuberculosis drugs, and provides their use in the preparation of anti-tuberculosis medicaments.

[0021] A composition for the prevention and treatment of tuberculosis, comprising quarfloxin and an anti-tuberculosis drug, said anti-tuberculosis drug being rifampin, isoniazid, bedaquiline, or linezolid.

[0022] In some embodiments, the tuberculosis prevention and control composition comprises quarfloxin and rifampin.

[0023] Preferably, the mass ratio of quarfloxin to rifampin is (0.1~0.3):(0.01~0.05).

[0024] Preferably, the mass ratio of quarfloxin to rifampin is 0.2:0.03.

[0025] The present invention also provides a medicament for the prevention and treatment of tuberculosis, comprising pharmaceutically acceptable excipients and quarfloxin, or the medicament comprising pharmaceutically acceptable excipients and the composition as described above.

[0026] The drug described in this invention is in solid dosage form, liquid dosage form, or gaseous dosage form.

[0027] In some embodiments,

[0028] The solid dosage form is a tablet, pill, granule, powder, capsule, or micro-pellet;

[0029] The liquid dosage form is an injection, syrup, mixture, emulsion, suspension, or drop;

[0030] The gaseous formulation is an aerosol and / or a spray.

[0031] The present invention also provides a method for preventing and treating tuberculosis, comprising administering the drug described herein.

[0032] The drug described in this invention can be used preventively or as a treatment, and this invention does not limit its use. The timing of drug administration is also not limited to before contact with a tuberculosis patient, after contact with a tuberculosis patient, during the incubation period of tuberculosis, or during the active period of tuberculosis.

[0033] This invention demonstrates that Quarfloxin can inhibit the growth of both clinical and drug-resistant strains of Mycobacterium tuberculosis, exhibiting significant antibacterial effects in both strains. Therefore, it provides the application of the fluoroquinolone derivative Quarfloxin in the preparation of anti-tuberculosis drugs. Attached Figure Description

[0034] Figure 1 The killing time curve of quarfloxin against Mtb H37Rv is shown, where drug concentration is represented by different symbols;

[0035] Figure 2 The results of the intracellular killing assay (intracellular bactericidal assay) are shown. Detailed Implementation

[0036] This invention provides the application of the fluoroquinolone derivative quarfloxin in the preparation of anti-tuberculosis drugs. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0037] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0038] Example

[0039] 1. Experimental materials:

[0040] strain Reference strains and clinical isolates for pharmacodynamic experiments in the treatment of tuberculosis. Among them, the Mycobacterium tuberculosis reference strain H37Rv (ATCC27294) was collected from American-type cultures and is preserved at Beijing Chest Hospital, Capital Medical University. The five clinical multidrug-resistant tuberculosis (MDR-TB) isolates were all strains preserved at the National Tuberculosis Reference Laboratory.

[0041] culture medium Löwenstein-Jensen (LJ) medium.

[0042] 2. Experimental Methods: The isolates were placed on LJ medium, and the presence of Mycobacterium tuberculosis complex was detected using MPT64 antigen. The specific experiments included the following:

[0043] 2.1) Minimum Inhibitory Concentration (MIC) Test: Quarfloxin (manufactured by MedChemExpress, USA) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 or 10 mg / mL. Broth microdilution was performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. MICs for Mycobacterium tuberculosis and multidrug-resistant tuberculosis isolates were detected using Middlebrook 7H9 broth (Becton, Dickinson) containing 10% oleic acid-albumin-glucose-catalase (OADC). Inoculi were prepared using fresh cultures from LJ medium. The drug concentration range was 0.049–100 μg / mL. Specifically, Mycobacterium tuberculosis and multidrug-resistant tuberculosis isolates were scraped from LJ medium, homogenized, and adjusted to a turbidity of 1 McFarland standard. The suspension was then diluted and inoculated into 96-well microplates at a density of 10 μL per well. 5 Colony forming units (CFU) were reached to achieve the final bacterial load. After incubation at 37°C for 7 days, 30 μL of resazurin (0.02%, wt / vol) was added to each well, and incubated at 37°C for 24 h. A change from blue to pink or purple indicates bacterial growth. The MIC is defined as the lowest antibiotic concentration that prevents the color from changing from blue to pink.

[0044] 2.2) Time-kill curve:

[0045] 2.2.1 Take 100 μL of the original bacterial culture from 7H9 Middlebrook liquid medium, add OADC growth supplement (BDBioscience) and 0.05% Tween 80 surfactant, and dilute to a concentration of 5 × 10⁻⁶. 5 A bacterial suspension of CFU / mL was used as the test bacterial solution.

[0046] 2.2.2 Prepare test sample solutions with working concentrations of 1×, 2×, 4×, 8×, and 16×MIC. Then, pipette the above sample solutions and add the test bacterial culture.

[0047] 2.2.3 Add 30 μL of the above-mentioned drug-containing bacterial solution to a 96-well plate, and at the same time add 30 μL of the test bacterial solution to the well plate as a blank control group, and then incubate in a 37°C incubator.

[0048] 2.2.4 Samples were taken at incubation times of 0, 1, 2, 3, and 6 days, and each sample was subsequently diluted 10⁻⁶. −1 Up to 10 −610 μL was spread onto a 7H9 Middlebrook agar plate and incubated at 37°C. After 3 weeks, colony counts were performed, and the colony count was expressed as CFU / mL.

[0049] 2.2.5 Data processing: Plot a time-sterilization curve with Lg CFU / mL as the ordinate and time as the abscissa.

[0050] 2.3) Intracellular killing assay (intracellular bactericidal assay):

[0051] 2.2.1 THP-1 cells were cultured at a rate of 5 × 10⁻⁶. 5 Cells were seeded at a dose of 1 cell / well in 24-well plates and induced to differentiate into macrophages at 48 hours with phorbol myristate acetate (PMA, 100 ng / mL).

[0052] 2.2.2 Cells were infected with Mtb H37Rv (ATCC27294) at a fold increase in infection (MOI) of 5:1. After infection at 37°C and 5% CO2 for 4 h, the cells were gently washed three times with pre-warmed 1x PBS to remove extracellular bacteria.

[0053] 2.2.3 In the intracellular killing assay, RPMI complete culture medium containing quarfloxin was used at concentrations of 1×MIC, 2×MIC, and 4×MIC. DMSO-containing medium served as a negative control, and RPMI 1640 medium containing isoniazid (INH, 3 μg / mL) served as a positive control. Macrophages were extensively washed with PBS at 24 and 72 hours post-infection and lysed with 0.1% Triton X-100. The number of cfu (cfu units) was determined by serial dilution of the lysate on 7H10 agar plates. Bacterial viability was calculated using the formula: Viability = (Cfu of quinacrine / doxorubicin-treated bacteria or INH of DMSO-treated bacteria / Cfu) × 100%.

[0054] 2.4) Combined drug antibacterial experiment:

[0055] The checkerboard method was used to evaluate the effects of drug combinations. The test compound (Aldoxorubicin or Quarfloxin) was added to a 96-well plate and serially diluted 2-fold, from 8x MIC to 1 / 32x MIC, with a volume of 50 μl per well. Next, 50 μl of four anti-tuberculosis drugs with different targets (RIF, INH, LZD, and BDQ) was added to each well, from 8x MIC to 1 / 8x MIC. Logarithmic-phase Mycobacterium tuberculosis was diluted, and 100 μl of bacterial culture was added to each well, similar to the MIC assay, resulting in a total volume of 200 μl per well. The final bacterial load per well was 10-1. 5CFU. Positive and negative control wells were also included. Incubate at 37°C for 7 days, then add resazurin and incubate again for 24 hours before viewing the results. Calculate FICs using the following formula:

[0056] ∑FIC = [MIC of compound X in combination with Y] / [MIC of X alone] + [MIC of compound Y in combination with X] / [MIC of Y alone].

[0057] According to the definition of ∑FIC value: ≤0.5 is a synergistic effect; 0.5~0.75 is a partial synergistic effect; 0.75~1.0 is an additive effect; 1.0~4.0 is an irrelevant effect; and antagonistic effect is greater than 4.0.

[0058] 2.5) Security Verification

[0059] Quarfloxin has entered clinical trials related to anti-tumor drug development. We will conduct a literature search to find the results of existing clinical trials on quarfloxin to demonstrate its safety in drug development.

[0060] 3. Experimental Results:

[0061] 3.1 The results of the minimum inhibitory concentration (MIC) test are shown in Tables 1 and 2:

[0062] Table 1. MIC of Quarfloxin against Mycobacterium tuberculosis clinical isolate H37Rv

[0063]

[0064] Table 2. MIC of Quarfloxin against resistant strains of Mycobacterium tuberculosis isolated clinically.

[0065]

[0066] The MICs of the selected inhibitor Quarfloxin against the Mtb reference strain H37Rv are shown in Table 1. Quarfloxin exhibited high antibacterial activity against H37Rv, with an MIC of 12.5 μg / mL. Further antibacterial activity tests were conducted on Quarfloxin against MDR strains (i.e., resistant strains). The antibacterial activity of Quarfloxin against MDR strains ranged from 6.25 μg / mL to 25 μg / mL, demonstrating high antibacterial activity against resistant strains as well, and exhibiting higher inhibitory activity against Mycobacterium tuberculosis compared to fluoroquinolone derivatives.

[0067] 3.2 The results of the Time-kill curve test are as follows: Figure 1 .

[0068] Time-kill curve analysis further evaluated the anti-tuberculosis efficacy of quarfloxin. Specifically, quarfloxin exhibited a moderate growth and killing pattern against H37Rv, showing a clear concentration dependence. At all tested concentrations on day 2, compared to the growth control, the time-kill curves of quarfloxin showed a decrease in quarfloxin concentration of approximately 0.5 log10 CFU / mL at 1 times the MIC (12.5 μg / mL). Quarfloxin demonstrated strong antibacterial activity, especially at concentrations ≥16× MIC, and exhibited higher inhibitory activity against Mycobacterium tuberculosis compared to fluoroquinolone derivatives.

[0069] 3.3 Intracellular killing assay (intracellular bactericidal assay) results are as follows: Figure 2 .

[0070] like Figure 2 As shown, quarfloxin exhibited effective inhibition at a concentration of 4×MIC 24 hours after treatment. After 72 hours of treatment, quarfloxin showed an antibacterial effect comparable to that of INH at a concentration of 1×MIC. In summary, quarfloxin at a concentration of 1×MIC reduced Mtb by 1.92 log10 CFU·ml. -1 INH, on the other hand, reduced [the concentration] by 2.07 log10 CFU / ml. -1 .

[0071] 3.4 The results of the combined drug antibacterial experiment are shown in the table below:

[0072]

[0073] The above experiments were conducted on strain H37Rv, where RIF was rifampicin, INH was isoniazid, BDQ was bedaquiline, and LZD was linezolid.

[0074] 3.5 Security Test Results

[0075] As a potential anti-tumor drug (the first g-quadruple interaction drug to enter human clinical trials), a series of preclinical trials have shown that Quarfloxin induces selective apoptosis of tumor cells and inhibits tumor growth by inhibiting the synthesis of overexpressed ribosomal RNA in cancer cells [Anticancer Activity of CX-3543: A Direct Inhibitor of rRNA Biogenesis, Cancer Res (2009) 69 (19): 7653–7661.]. Meanwhile, to determine the dose-limiting toxicity (dlt), maximum tolerated dose (MTD), and pharmacokinetics (PK) of this drug, a previous clinical trial [Phase I clinical trial of CX-3543, a protein-rDNA quadruplex inhibitor, Journal of Clinical Oncology, Volume 24, Number 18_suppl, https: / / doi.org / 10.1200 / jco.2006.24.18_suppl.3082] screened eligible patients with advanced solid tumors or lymphomas who had progressed with standard therapy or had no standard therapy, and received a continuous dose cohort of Quarfloxin (CX-3543). Specifically, eligible patients with advanced solid tumors received a continuous dose cohort of CX-3543 at doses of 10, 20, 40, 80, and 160 mg / m². 2 The administration was by intravenous infusion over one hour daily for five consecutive days, repeated in three-week cycles. Treatment continued until the patient showed signs of intolerance to CX-3543 or evidence of disease progression. Response according to RECIST criteria was determined after every two cycles. Results showed that 10 patients with solid tumors (3–4 in each group) received intravenous CX-3543. The dose was well tolerated. Seven grade 3 adverse events were reported during the study, but none were related to CX-3543. To date, no objective response has been observed. One patient with advanced refractory prostate cancer had stable disease for more than four months. CX-3543 showed a good linear relationship in PK parameters between dose groups, with a terminal half-life of approximately 12 hours after the first dose. The results of this clinical trial showed that CX-3543 did not exhibit drug-related toxicity and had predictable PKs. No dlt has been observed. Therefore, these results indicate that Quarfloxin is well tolerated within a safe concentration range and can be applied to the development of anti-tuberculosis drugs.

[0076] The above results indicate that Quarfloxin can inhibit the growth of both clinical and drug-resistant strains of Mycobacterium tuberculosis, and its in vitro antibacterial activity experiments showed strong biological activity against Mycobacterium tuberculosis, suggesting its potential efficacy in the treatment of tuberculosis.

[0077] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A composition for preventing and treating tuberculosis, comprising quarfloxin and rifampicin.

2. The composition of claim 1, wherein, It consists of quarfloxin and rifampicin, wherein the mass ratio of quarfloxin to rifampicin is (0.1-0.3):(0.01-0.05).

3. The composition of claim 2, wherein, The mass ratio of the quarfloxin to rifampicin is 0.2:0.

03.

4. Use of the composition according to any one of claims 1 to 3 for the manufacture of a medicament for preventing and treating tuberculosis.

5. Use according to claim 4, characterized in that, The preventing and treating tuberculosis comprises inhibiting the activity of Mycobacterium tuberculosis and / or killing Mycobacterium tuberculosis.

6. Use according to claim 5, characterized in that, The Mycobacterium tuberculosis is drug-resistant or non-drug-resistant.

7. Use according to claim 4, characterized in that, The tuberculosis is human tuberculosis.

8. Use according to claim 7, characterized in that, The tuberculosis is human pulmonary tuberculosis.

9. Use according to claim 7 or 8, characterized in that, The tuberculosis is non-drug-resistant human pulmonary tuberculosis or drug-resistant human pulmonary tuberculosis.

10. A medicament for preventing and treating tuberculosis, comprising a pharmaceutically acceptable adjuvant and the composition according to any one of claims 1 to 3.

11. The medicament according to claim 10, characterized in that, It is a solid dosage form, a liquid dosage form or a gaseous dosage form.

12. The medicament according to claim 11, wherein, The solid dosage form is a tablet, a pill, a granule, a powder, a capsule; The liquid dosage form is an injection, a syrup, a mixture, an emulsion, a suspension, a drop; The gaseous dosage form is an aerosol and / or a spray.

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