Use of echinocandin and rifampin in the preparation of synergistic anti-tuberculosis drugs

Abstract

The application discloses application of ecurin and rifampicin in preparation of a synergistic anti-tuberculosis drug, and belongs to the technical field of anti-tuberculosis drugs. The core of the application is that ecurin and rifampicin are used in combination, the bactericidal activity of rifampicin is significantly enhanced, the minimum inhibitory concentration of rifampicin is reduced, and the development of drug resistance of Mycobacterium tuberculosis is effectively inhibited. The application further provides a pharmaceutical preparation and a medicine box containing the two active ingredients. The combined application scheme can reduce the clinical dosage of rifampicin, thereby reducing the adverse reactions such as hepatotoxicity of rifampicin, and is suitable for the treatment of sensitive and rifampicin-resistant tuberculosis, and has important clinical value and application prospect.

Description

Technical Field

[0001] This invention relates to the application of ecumycin and rifampin in the preparation of a synergistic anti-tuberculosis drug, belonging to the field of anti-tuberculosis drug technology. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis and is one of the leading causes of death worldwide. According to the World Health Organization (WHO), approximately 10 million new cases are diagnosed with TB each year, and about 1.5 million people die from it.

[0003] Rifampin, as a first-line anti-tuberculosis drug, exerts its bactericidal effect by inhibiting bacterial RNA polymerase activity. However, its clinical application faces two major challenges: first, monotherapy or irregular treatment can easily induce bacterial resistance, the resistance mechanism of which is mainly related to rpoB gene mutations; second, rifampin has significant dose-related hepatotoxicity, and long-term or high-dose use may lead to severe drug-induced liver injury, limiting its clinical application.

[0004] Ecumycin is a novel small-molecule inhibitor of the clpc1 protein. In recent years, it has been found to have strong antibacterial activity against Mycobacterium tuberculosis. It exerts its antibacterial effect by inhibiting the function of the bacterial clpc1 protein and interfering with protein degradation. However, the cost of using cumycin alone is high, and its pharmacokinetic characteristics and formulation are difficult, which limits its widespread use as a standalone product.

[0005] Therefore, there is an urgent need in the field for a new treatment regimen that can enhance the efficacy of rifampin, reduce its dosage, decrease its toxic side effects, and effectively delay or overcome the development of drug resistance. Summary of the Invention

[0006] The purpose of this invention is to provide an application of ecumycin and rifampin in the preparation of a synergistic anti-tuberculosis drug. This invention provides a novel application of ecumycin and rifampin that can produce a synergistic anti-tuberculosis effect, thereby significantly enhancing efficacy, reducing dosage, minimizing toxic side effects, and inhibiting the development of drug resistance.

[0007] The technical solution provided by this invention is as follows: the application of ecumycin and rifampin in the preparation of synergistic anti-tuberculosis drugs.

[0008] In the above-mentioned applications, the synergistic anti-tuberculosis effect is manifested by ecumycin enhancing the antibacterial activity of rifampin.

[0009] In the aforementioned applications, the synergistic anti-tuberculosis effect manifests as ecumycin inhibiting the development of rifampicin resistance in Mycobacterium tuberculosis.

[0010] In the aforementioned application, the drug is formulated as a combination drug preparation comprising a therapeutically effective amount of ecumenicalin and a therapeutically effective amount of rifampin.

[0011] In the aforementioned applications, the combined pharmaceutical formulation also includes pharmaceutically acceptable excipients.

[0012] In the aforementioned applications, the excipients include one or more of the following: solvents, dispersants, diluents, wetting agents, binders, disintegrants, lubricants, preservatives, suspending agents, emulsifiers, excipients, flavoring agents, and carriers.

[0013] In the aforementioned applications, the dosage form of the combined drug preparation is a powder, capsule, tablet, or injection.

[0014] In the aforementioned applications, the combined drug formulation is a single compound formulation containing ecumycin and rifampin, or a combination package of ecumycin and rifampin formulations.

[0015] In the aforementioned applications, the tuberculosis includes susceptible tuberculosis and drug-resistant tuberculosis.

[0016] A medicine box for treating tuberculosis, the medicine box comprising: (A) Component 1, which contains a therapeutically effective amount of ecumenium; (B) The second component contains a therapeutically effective amount of rifampin; (C) Instruction manual.

[0017] Compared with the prior art, the present invention has the following significant advantages: 1. This invention is the first to discover and confirm that the combined use of eculomycin and rifampin has a significant synergistic antibacterial effect, which can greatly enhance the bactericidal effect of rifampin and significantly reduce its minimum inhibitory concentration (MIC).

[0018] 2. The combined drug regimen of the present invention can effectively reduce the frequency of drug-resistant mutations in Mycobacterium tuberculosis under rifampicin pressure, delay or even inhibit the emergence of drug-resistant strains, which is of great significance for controlling the spread and development of drug-resistant tuberculosis.

[0019] 3. Due to the synergistic effect of eculomycin, the dosage of rifampin can be significantly reduced while achieving the same or even better therapeutic effect, which may reduce adverse reactions such as hepatotoxicity and improve patients' tolerance and compliance.

[0020] 4. The combined drug regimen of the present invention is not only effective against sensitive tuberculosis bacteria, but also shows the potential to restore the sensitivity of rifampicin-resistant strains isolated clinically, thus broadening the scope of clinical application and providing new hope for the treatment of drug-resistant tuberculosis. Attached Figure Description

[0021] Figure 1 The in vitro synergistic antibacterial effect of the combination of eculomycin and rifampin against the standard strain H37Ra of Mycobacterium tuberculosis is shown in the figure. Figure 2 The in vitro synergistic antibacterial effect of the combination of eculomycin and rifampin against Mycobacterium smegma mc2155 is shown in the figure. Figure 3 A comparison of the growth inhibition effects of rifampin monotherapy versus the combination of rifampin and eculomycin on the standard strain H37Ra of Mycobacterium tuberculosis; Figure 4 This is a comparison of the half-maximal inhibitory concentrations (IC50) of rifampin monotherapy and eculomycin combination therapy against the standard strain H37Ra of Mycobacterium tuberculosis. Figure 5 A graph showing the effect of different drug treatments on colony-forming units of Mycobacterium tuberculosis; Figure 6 The effect of rifampin monotherapy and rifampin combined with ecumycin on the frequency of drug resistance mutations in Mycobacterium tuberculosis is shown in the figure. Figure 7 Figure showing the effect of different drugs and concentrations on the survival rate of normal human hepatocytes LO2; Figure 8 The figure shows the effect of different drugs and concentrations on the survival rate of normal human hepatocytes THLE-2. Detailed Implementation

[0022] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0023] Example 1: Synergistic antibacterial effect of eculin and rifampin in vitro The checkboard assay was used to determine the effects of ecumicin and rifampicin on Mycobacterium tuberculosis standard strain H37Ra and Mycobacterium smegma mcg. 2 The minimum inhibitory concentration (MIC) was 155. Single-drug groups and groups receiving different proportions of combined drugs were set up, and the results were observed after 7 days of incubation.

[0024] Figure 1 This study demonstrated the synergistic antibacterial effect of the combination of ecumycin and rifampin against the standard strain H37Ra of Mycobacterium tuberculosis in vitro. Figure 2 Demonstrated the effectiveness of the combination therapy of ecumycin and rifampin against Mycobacterium smegmae MC. 2 The in vitro synergistic antibacterial effect of 155. Figure 1 and Figure 2 The color intensity represents the bacterial survival rate (%), with darker colors indicating stronger antibacterial effect (lower bacterial survival rate) and lighter colors indicating weaker antibacterial effect (higher bacterial survival rate). Figure 1 and Figure 2 It can be seen that the combined use of ecumycin and rifampin significantly enhances the antibacterial effect and shows a significant synergistic effect (FIC index < 0.5). This directly proves that the combined use of ecumycin and rifampin can significantly enhance the antibacterial effect against Mycobacterium tuberculosis, providing direct in vitro experimental evidence for the combined use of the two in anti-tuberculosis treatment.

[0025] Example 2: Effect of eculomycin on the MIC value of rifampin Under the condition of fixed ecumycin concentration (0.5 μg / mL), the concentration of rifampin was gradually increased, and its MIC change for H37Ra was determined.

[0026] Figure 3 The study demonstrated the growth-inhibiting effects of rifampicin monotherapy (H37Ra group, black dot curve) and ecumycin combined with rifampicin (H37Ra+ecumycin group, square curve) on the standard strain H37Ra of Mycobacterium tuberculosis. Figure 4 The half-maximal inhibitory concentration (MIC50) of rifampicin monotherapy (H37Ra) and ecumycin combination therapy (H37Ra + ecumycin) is shown. Figure 3 It can be seen that after the combined use of ecumycin, the "effective concentration range" of rifampicin in inhibiting bacterial growth shifted significantly to the left, indicating that ecumycin can enhance the antibacterial effect of rifampicin and significantly inhibit bacterial growth at lower rifampicin concentrations. Figure 4 The results showed that the MIC50 of rifampin against H37Ra was significantly reduced after the combined use of eculomycin (from approximately 0.003 μg / mL to approximately 0.001 μg / mL, a 4-fold decrease), further demonstrating that eculomycin can significantly enhance the antibacterial activity of rifampin and reduce the effective dosage of rifampin. Figure 3 and Figure 4 The results showed that eculomycin could significantly enhance the antibacterial activity of rifampin.

[0027] Example 3: Evaluation of the efficacy of combination therapy in a mouse tuberculosis model Establish a BALB / c mouse model of tuberculosis infection (aerosol inhalation infection 10) 6 -10 7 Mice were then randomly divided into four groups for subcutaneous injection of cFU (eculomycin monotherapy) and rifampin monotherapy (10 mg / kg). After four weeks of treatment, mice were sacrificed, and lung tissue was collected for colony count (CFU).

[0028] Figure 5This study demonstrates the effects of different drug treatments on colony-forming units (CFU / mL) of Mycobacterium tuberculosis, used to evaluate the in vivo and in vitro bactericidal efficacy of the combination of eculomycin and rifampin. ****: Extremely significant difference (p < 0.0001); **: Significant difference (p < 0.01); ns: No significant difference (p > 0.05). Graphical elements: Each "diamond region" represents the distribution range of the data for that group, with black dots inside representing the measurement value of a single sample, and the horizontal line representing the mean within the group. Compared with the control group, the CFU / mL of the eculomycin monotherapy group, rifampin monotherapy group, and combination group were all significantly reduced, indicating that all three treatments had bactericidal effects. Compared with the rifampin monotherapy group, the CFU / mL of the combination group was further significantly reduced, and the difference was extremely significant compared with the eculomycin monotherapy group, proving that the bactericidal effect of the combination of eculomycin and rifampin was significantly better than that of the monotherapy, and that the two had a synergistic antibacterial effect. This result, from the quantitative perspective of "the number of surviving bacteria," directly verifies that eculomycin can enhance the bactericidal effect of rifampin on Mycobacterium tuberculosis, providing key experimental evidence for the combined use of the two in anti-tuberculosis treatment.

[0029] Example 4: Drug Resistance Induction Experiment H37Ra strain was continuously passaged at the same inhibitory concentration (see checkerboard experiment results). Rifampicin monotherapy group (0.015625 μg / ml) and combination therapy group (eculomycin 0.125 μg / ml + rifampicin 0.0002441 μg / ml) were set up. The bacterial culture was extracted from each generation to detect the drug resistance mutation frequency (fluctuation assay).

[0030] Figure 6 This study demonstrated the effects of rifampin monotherapy and rifampin combined with ecumycin on the frequency of drug resistance mutations in Mycobacterium tuberculosis. The results showed that the frequency of drug resistance mutations was significantly lower in the rifampin combined with ecumycin group compared to the rifampin monotherapy group, proving that ecumycin can effectively inhibit the development of rifampin resistance in Mycobacterium tuberculosis. This provides direct molecular-level evidence for the combined use of these two drugs in anti-tuberculosis treatment and for delaying drug resistance.

[0031] Example 5: Effects of combined drug therapy on hepatotoxicity Normal human hepatocytes (LO2 and THLE-2) were seeded in 96-well plates. A blank control group, different concentrations of rifampicin monotherapy (1, 5, 10, 20 μg / mL, abbreviated as Rif1, Rif5, Rif10, and Rif20), ecumycin monotherapy (0.1, 0.5, 1 μg / mL, abbreviated as Ecu0.1, Ecu0.5, and Ecu1), and a combination therapy group (rifampicin 5 μg / mL + ecumycin 0.5 μg / mL, abbreviated as Rif5 + Ecu0.5) were established. Cell viability was assessed using the CCK-8 assay after 24 hours of culture.

[0032] Figure 7 The study demonstrated the effects of different drugs and concentrations on the survival rate of normal human hepatocytes (LO2). Figure 8 This study demonstrated the effects of different drug treatments and concentrations on the survival rate of normal human hepatocytes THLE-2. The results showed that the cell survival rates in the combined treatment groups were 100.3±0.6711% and 100.4±0.3%, respectively. These results were not significantly different from those in the high-dose rifampicin monotherapy group (20 μg / mL, survival rates 95.47±5.533% and 95.5±5.167%, respectively) (p>0.05), indicating that the combined treatment had no significant toxicity to hepatocytes. This result provides direct evidence from a cytotoxic perspective for the safety of the combined use of eculomycin and rifampicin in anti-tuberculosis treatment.

[0033] Example 6: Antibacterial activity against rifampicin-resistant strains Two rifampicin-resistant Mycobacterium tuberculosis strains isolated clinically (with S450L and S432L mutations in the rpoB gene confirmed by sequencing) and two drug-resistant strains H37Ra obtained from laboratory screening (with H445Y and D435V mutations in the rpoB gene confirmed by sequencing) were analyzed using the microdilution method to determine their MIC values. The results are shown in Table 1. Strain Rifampin MIC (μg / ml) Rifampin MIC (μg / ml) H37Rv (clinical strain S450L) ＞32 4 H37Rv (clinical strain S432L) ＞32 2 H37Ra (laboratory screening strain H445Y) ＞32 4 H37Ra (laboratory screening strain D435V) ＞32 1 Table 1 shows that without cucurbitacin, the MICs of rifampicin for all four drug-resistant strains were >32 μg / mL (far higher than the clinically effective concentration), proving that they were rifampicin-resistant strains. After the addition of cucurbitacin, the MICs of rifampicin significantly decreased to 1-4 μg / mL (within the clinically effective concentration range), indicating that cucurbitacin can effectively reverse the drug resistance of drug-resistant Mycobacterium tuberculosis to rifampicin, especially showing a significant effect on restoring sensitivity to drug-resistant strains carrying the rpoB gene mutation, providing a new strategy for the treatment of rifampicin-resistant tuberculosis.

[0034] Example 7: This example provides a medicine box for treating tuberculosis, which facilitates accurate and convenient combined medication administration for patients or medical staff.

[0035] Composition of a medicine box The medicine box includes the following components: The first component consists of tablets packaged in a blister pack, each containing a therapeutically effective amount of ecumenical as the active ingredient, along with pharmaceutically acceptable excipients.

[0036] The first component consists of tablets packaged in a blister pack, each containing a therapeutically effective amount of rifampin as the active ingredient, along with pharmaceutically acceptable excipients.

[0037] Instruction manual: A printed document that details how to use the medicine box.

[0038] The first component and its blister pack are placed together in an outer cardboard box. The instruction manual is folded and placed inside the box. The medication date and / or serial number are clearly printed on the blister pack or each tablet compartment for identification. The instruction manual includes at least the following information: indications, dosage, treatment duration, and precautions.

[0039] In summary, the combination therapy of ecumycin and rifampin provided by this invention has significant synergistic antibacterial effects and anti-drug resistance potential, and is suitable for the clinical treatment of tuberculosis, especially for patients with rifampin resistance or poor tolerance.

[0040] The above description is merely a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention's specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. Application of ecumicin and rifampin in the preparation of synergistic anti-tuberculosis drugs.

2. The application according to claim 1, characterized in that: The synergistic anti-tuberculosis effect is manifested by ecumycin enhancing the antibacterial activity of rifampin.

3. The application according to claim 1, characterized in that: The synergistic anti-tuberculosis effect is manifested in the fact that ecumycin inhibits the development of rifampicin resistance in Mycobacterium tuberculosis.

4. The application according to any one of claims 1-3, characterized in that: The drug is formulated as a combination drug preparation containing a therapeutically effective amount of ecumycin and a therapeutically effective amount of rifampin.

5. The application according to claim 4, characterized in that: The combined drug formulation also includes pharmaceutically acceptable excipients.

6. The application according to claim 5, characterized in that: The excipients include one or more of the following: solvent, dispersant, diluent, wetting agent, binder, disintegrant, lubricant, preservative, suspending agent, emulsifier, excipient, flavoring agent, and carrier.

7. The application according to claim 4, characterized in that, The dosage form of the combined drug preparation is powder, capsule, tablet or injection.

8. The application according to claim 4, characterized in that, The combined drug formulation is a single compound formulation containing ecumycin and rifampin, or a combination package of ecumycin and rifampin formulations.

9. The application according to claim 1, characterized in that, The tuberculosis mentioned includes susceptible tuberculosis and drug-resistant tuberculosis.

10. A medicine box for treating tuberculosis, characterized in that, The medicine box includes: (A) Component 1, which contains a therapeutically effective amount of ecumicin; (B) The second component contains a therapeutically effective amount of rifampin; (C) Instruction manual.

Images