Application of ergolactone in resisting mycobacterial infection

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of ergolactone in resisting mycobacterial infection. According to the research of the applicant, the ergolactone shows good bacteriostatic activity on mycobacterium tuberculosis standard strains and various non-mycobacterium tuberculosis strains; the minimum inhibitory concentration (MIC) of the mycobacterium tuberculosis standard strain can reach 2 [mu] g / mL, and the MIC of the mycobacterium tuberculosis standard strain to other mycobacterium strains is 8-32 [mu] g / mL. In addition, the ergolactone has no obvious cytotoxicity and is expected to become a novel medicine for resisting mycobacterium tuberculosis infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of ergolactone in the treatment of mycobacterial infections. Background Technology

[0002] Mycobacterial infections are a group of diseases caused by bacteria of the genus Mycobacterium. Tuberculosis (TB) is a zoonotic infectious disease caused by infection with the Mycobacterium tuberculosis complex. Its pathogens are known for their unique acid-fast cell walls, slow growth rate, and strong latent infectivity, making it the leading killer among single-source infections, seriously endangering human health and livestock development. According to the World Health Organization, approximately 10.7 million new TB cases were reported globally in 2024, resulting in about 1.23 million deaths. my country, as one of the high-burden countries, ranks fourth globally in the number of cases. Even more serious is the enormous challenge facing TB treatment: with the long-term use of first-line drugs, drug-resistant strains (especially MDR-TB and XDR-TB) are constantly emerging. The lengthy treatment cycle, severe drug side effects, and limitations in diagnostic tools further exacerbate the difficulty of prevention and control, posing a continuous threat to global public health. Given the current severe problem of TB drug resistance and the long and costly development cycle of new drugs, "drug repurposing" has become an important strategy for screening anti-TB drugs. Its core advantages lie in significantly shortening research and development time and reducing costs, as these marketed drugs possess complete pharmacological, toxicological, and safety data, skipping the early stages of research and development; secondly, it can quickly address the drug resistance crisis, with some non-tuberculosis drugs found to have unexpected antibacterial activity against drug-resistant strains (including MDR-TB / XDR-TB), which can be rapidly combined with existing regimens to form new effective therapies; and thirdly, safety risks are more controllable, as their human tolerability and side effects are known, which helps accelerate their clinical translation and application as new indications for anti-tuberculosis, providing an efficient and economical shortcut to conquering tuberculosis. We screened a library of commercially available small molecule compounds and found that ergolactone has a specific killing effect on Mycobacterium spp. Furthermore, its bactericidal effect against Mycobacterium tuberculosis is superior to that of the existing first-line anti-tuberculosis drug rifampin.

[0003] Ergolide, also known as ergolide, CAS: 54999-07-4. Its molecular formula is C2. 17 H 22 O5, with a relative molecular mass of 306.35. Ergonomic lactone is a sesquiterpene lactone natural compound isolated from the dried flowers of Inula Britannica. While ergonomic lactone has been reported to possess anti-inflammatory and antitumor activities, its anti-mycobacterial activity has not been previously reported. Summary of the Invention

[0004] The purpose of this invention is to provide the application of ergolactone in the treatment of Mycobacterium tuberculosis infection. Specifically, this invention reveals for the first time that ergolactone has significant antibacterial and bactericidal activity against standard strains of Mycobacterium tuberculosis, and can produce synergistic or additive effects when used in combination with first-line anti-tuberculosis drugs, providing a new technical path and compound resources for the development of anti-tuberculosis drugs.

[0005] To achieve the above objectives, one of the objectives of this invention is to provide the use of ergolactone in the preparation of drugs for treating mycobacterial infections.

[0006] To further clarify, the mycobacteria mentioned include the standard strain of Mycobacterium tuberculosis and other pathogenic mycobacteria.

[0007] The second objective of this invention is to provide the application of ergolactone in the preparation of anti-tuberculosis drugs.

[0008] To further clarify, the tuberculosis referred to is tuberculosis caused by a standard strain or clinical isolate of Mycobacterium tuberculosis.

[0009] The third objective of this invention is to provide the application of ergolactone in the preparation of combined synergistic drug compositions against Mycobacterium tuberculosis.

[0010] To further explain, the combined synergistic drug composition contains ergolactone and one or more first-line anti-tuberculosis drugs, wherein the first-line anti-tuberculosis drugs are selected from isoniazid (INH), rifampin (RFP), bedaquiline (BDQ) or ethambutol (EMB).

[0011] The fourth objective of this invention is to provide an anti-tuberculosis drug, wherein the drug comprises ergolactone as an active ingredient.

[0012] Furthermore, the drug also includes one or more pharmaceutically acceptable carriers or excipients.

[0013] To further clarify, the formulation of the drug is selected from one or more of conventional formulations, sustained-release formulations, controlled-release formulations, and / or various microparticle delivery systems.

[0014] The beneficial effects of this invention are as follows:

[0015] This research team has discovered for the first time that ergonomics possesses specific bactericidal activity against mycobacteria. It exhibits low MIC values ​​(2–32 μg / mL) against various pathogenic mycobacteria, including standard strains of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marineum, and shows no inhibitory effect on common Gram-positive or Gram-negative bacteria, demonstrating species-specific activity. The study also found that ergonomics at effective concentrations has no significant cytotoxicity to THP-1-derived human macrophages, exhibiting good biocompatibility and suitability for long-term use. Ergonomics shows no significant antagonistic effect when used in combination with first-line anti-tuberculosis drugs (such as INH, RFP, BDQ, and EMB), and instead shows an additive effect (FIC value of 0.5–1), enhancing the antibacterial effect without increasing toxicity, demonstrating potential for combination therapy development. Through a "drug repurposing" strategy, utilizing the known pharmacological basis of ergonomics, the clinical translation process of ergonomics as an anti-tuberculosis drug can be significantly accelerated, possessing significant scientific value and clinical application prospects. Attached Figure Description

[0016] Figure 1 The figure shows the experimental results of the in vitro broad-spectrum antibacterial activity assay of ergolactone.

[0017] Figure 2 The figure shows the experimental results of ergolactone combined with INH, RFP, BDQ and EMB to combat Mycobacterium tuberculosis.

[0018] Figure 3 The figure shows the experimental results of the time-kill curve of ergolactone against Mycobacterium tuberculosis.

[0019] Figure 4 This is a graph showing the results of the ergolactone cytotoxicity test. Detailed Implementation

[0020] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0021] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0022] Example 1: This example is a study on the antibacterial spectrum of ergolactone.

[0023] The research methodology is as follows:

[0024] 1. The ergolactone was purchased from MCE. It was dissolved in DMSO to prepare a stock solution with a concentration of 10 mg / mL, then aliquoted into brown EP tubes and stored at -80°C protected from light. When needed, the stock solution was removed from the -80°C freezer.

[0025] 2. Strain activation. Remove the strain from the -80°C freezer and inoculate it into 5 mL of antibiotic-free medium containing the Mycobacterium tuberculosis standard strain. Mycobacterium bovis Marine Mycobacterium Mycobacterium smegmatis Pseudomonas aeruginosa was activated using 7H9 medium (purchased from BD Biosciences). Escherichia coli Staphylococcus aureus Activation culture was performed using MHB medium (purchased from Solarbio Science & Technology Co., Ltd.) at 37°C and 160 rpm. For the standard strains of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marineense, an additional 1% OADC (purchased from Shanghai Jingnuo Co., Ltd.) was added.

[0026] 3. Preparation of bacterial culture before the experiment. Activated Mycobacterium smegmaecum, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus OD200 were prepared. 600 After adjusting the medium to 0.5, dilute it 100 times with the medium. Then, use the activated Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium marineum to adjust the OD... 600 Adjust the concentration to 0.318, then dilute 10-fold. Ensure the initial colony-forming units are approximately 5 x 10⁻⁶. 5 CFU.

[0027] 4. Gradual dilution of ergolactone. Minimum inhibitory concentration (MIC) tests were performed using 96-well plates. The left two columns of the 96-well plate contained a positive control (diluted bacterial suspension) and a negative control (culture medium), respectively. The right column... Add 200 μL of diluted bacterial culture to each well. Add 400 μL of bacterial culture and 2.56 μL of ergolactone (final concentration 64 μg / mL) to the rightmost column. Transfer 200 μL of the liquid from each well to the left well using a 2-fold serial dilution.

[0028] 5. Culture, color development, and data statistics. Standard strains of Mycobacterium tuberculosis were cultured at 37℃. Mycobacterium bovis After standing for 7 days, marine mycobacteria After standing for 4 days, Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Mycobacterium smegmatis After standing for 1 day, the OD was measured. 600Analyze absorbance to determine the minimum inhibitory concentration (MIC). Alternatively, after incubation, add 10 μL of resporane chromogenic solution (0.04% resporane stock solution) to each well and continue incubation for 24 hours, observing the color change. If the solution in the well changes from blue to pink, it indicates bacterial growth. Calculate the minimum drug concentration required for the solution in the well to be completely blue and plot the results using GraphPad Prism software.

[0029] See results Figure 1 Specifically, the MIC of ergolide against the standard strain of Mycobacterium tuberculosis was 2 μg / mL, against Mycobacterium bovis it was 8 μg / mL, against Mycobacterium marineum it was 32 μg / mL, and against Mycobacterium smegmatis it was greater than 64 μg / mL. This indicates that even among mycobacteria, the inhibitory effect of ergolide on different strains varies considerably. Furthermore, ergolide also showed inhibitory effects against Pseudomonas aeruginosa (PAO1) and Escherichia coli. Staphylococcus aureus The values ​​were all greater than 64 μg / mL, indicating that ergolide has a specific inhibitory effect on pathogenic mycobacteria.

[0030] Example 2: This example is a study on the combined use of ergolide.

[0031] The research methodology is as follows:

[0032] 1. The activation of the strain and the preparation of ergolactone were the same as in Example 1.

[0033] 2. Add ergolide. The left two columns and right two columns of the 96-well plate contain blank culture medium; select the middle column. The area is the experimental area, and 200 μL of bacterial solution is added to each well. Add an additional 200 μL of bacterial culture to the rightmost column of the area, followed by 2 times the MIC of ergolactone (final concentration 4 μg / mL). It is noteworthy that the top right well should contain twice the amount of ergolactone as in the other wells (final concentration 8 μg / mL). After adding ergolactone, perform a serial dilution from right to left using the serial dilution method described in Example 1 until... The second column of the area, i.e. The first column in the region does not contain ergolactone. After ergolactone dilution, in... Add first-line drugs at 2 times the MIC to the first row of the region (isoniazid (INH), final concentration of 0.125 μg / mL), rifampicin (RFP, final concentration of 0.0156 μg / mL), bedaquiline (BDQ, final concentration of 0.0625 μg / mL), ethambutol (EMB, final concentration of 1 μg / mL)), and then perform 2-fold serial dilution from top to bottom according to the method of gradient dilution in Example 1.

[0034] 3. Culture and color development. Culture at 37 °C, let stand for 7 days, and determine the minimum inhibitory concentration by detecting the OD 600 absorbance. After incubation or at the end of the culture, add resazurin chromogenic solution to each well, continue to incubate for 24 hours, observe the color change, calculate the FIC value, and evaluate whether there is a synergistic or antagonistic effect between the two drugs according to the FIC value. Calculation of the FIC index: FIC index = MIC (drug A in combination) / MIC (drug A alone) + MIC (drug B in combination) / MIC (drug B alone). When FIC ≤ 0.5, the interaction mode of the two drugs is synergistic; when 0.5 < FIC ≤ 1, the interaction mode of the two drugs is additive; when 1 < FIC ≤ 2, the interaction mode of the two drugs is irrelevant; when FIC > 2, the interaction mode of the two drugs is antagonistic.

[0035] The results are shown in Figure 2 , specifically as follows. The FIC values of ergolide combined with four first-line anti-tuberculosis drugs, isoniazid (INH), rifampicin (RFP), bedaquiline (BDQ), and ethambutol (EMB), against the standard strain of Mycobacterium tuberculosis are 1, 0.75, 1, and 1, respectively, all within the range of 0.5 - 1, indicating an additive effect of the drug efficacy. This shows that when ergolide is combined with INH, RFP, BDQ, and EMB, a drug efficacy superposition effect can be produced, that is, the antibacterial effect of the combined drug is equivalent to the sum of the individual effects of each drug.

[0036] This additive effect is of great significance in anti-tuberculosis treatment: on the one hand, the combined use of drugs can reduce the dosage of single drugs while ensuring the antibacterial effect, thereby reducing the potential toxic and side effects caused by long-term drug use; on the other hand, by synergistically (additively) acting with first-line drugs, it is expected to enhance the ability to clear Mycobacterium tuberculosis, providing new ideas for optimizing anti-tuberculosis treatment regimens. Thus, ergolide has potential application value in anti-tuberculosis combination therapy and is worthy of further research on the combined use of drugs against clinically isolated strains of Mycobacterium tuberculosis to verify its broader potential for synergistic drug efficacy.

[0037] Example 3: This example is a study on the time-kill curve of ergolide

[0038] The research method is as follows:

[0039] 1. The activation of the strain and the preparation of ergolactone were the same as in Example 1. The starting colony-forming units were adjusted to 1 x 10⁻⁶ using fresh liquid culture medium. 7 CFU.

[0040] 2. Add ergolide. Dispense the diluted bacterial culture into PA bottles, and set up a negative control (DMSO), a positive control (4-fold MIC rifampicin), and experimental groups with 2-fold MIC ergolide and 4-fold MIC ergolide, respectively. Each group had three independent biological replicates. Incubate at 37°C on a shaker at 80 rpm.

[0041] 3. Sampling and Plating. Collect 100 μL of bacterial culture at 0h, 4h, 8h, 12h, 24h, 2d, 4d, 8d, and 14d intervals by centrifugation. Wash three times with PBS and resuspend in an equal volume of PBS. Perform serial dilutions on each sample. Spot 4 μL of each sample onto a solid medium containing 7H10 + 5% OADC. Calculate the CFU. Incubate the plates upside down at 37℃ for 14 days, observe the cell counts, and calculate the CFU. GraphPad Prism software is used to plot the results.

[0042] See results Figure 3 Specifically, Ergolide exhibits significant bactericidal effects against standard strains of Mycobacterium tuberculosis. Compared to the traditional first-line anti-tuberculosis drug RFP, Ergolide has a slower bactericidal time, requiring 24 hours to show a noticeable bactericidal effect. However, Ergolide's bactericidal efficacy is better, even at 10 hours. 7 CFU can still completely kill bacteria, while RFP's bactericidal effect weakens in the later stages, making it difficult to completely kill all bacteria. This indicates that Ergolide has stronger bactericidal persistence and thoroughness, and can still completely eliminate Mycobacterium tuberculosis even in infection states with high bacterial load, demonstrating potential therapeutic advantages for high bacterial load tuberculosis infections.

[0043] Example 4: This example is a study on the cytotoxicity of ergolide.

[0044] The research methodology is as follows:

[0045] 1. Thawing and Passaging of THP-1 Cells. Remove the THP-1 cell cryovials from liquid nitrogen and quickly place them in a 37°C water bath, agitating continuously until completely thawed (approximately 1-2 minutes). Thoroughly wipe the outer wall of the cryovials with an alcohol swab and transfer them to a clean bench. Slowly add the cell suspension dropwise to a 15 mL centrifuge tube containing 5-10 mL of pre-warmed RPMI-1640 medium, mixing gently. Centrifuge at 1000 r / min for 5 min, carefully discarding the supernatant. Add 5 mL of fresh RPMI-1640 medium to resuspend the cells and transfer to a culture flask. Incubate at 37°C in a 5% CO2 incubator. After 24 hours, centrifuge the cells and replace with fresh medium. Passage the cells at a 1:3 ratio, adding 1 mL of cell suspension to a cell culture flask containing 3 mL of pre-warmed RPMI-1640 medium at 37°C, and incubate in a 5% CO2 incubator, changing the medium every 2 days until the cells reach confluence.

[0046] 2. Induction of THP-1 cell differentiation. Collect THP-1 cells of good growth condition and appropriate density by centrifugation, wash with pre-warmed RPMI-1640 medium at 37°C to remove cell debris. Seed the washed cells into 100×200 mm cell culture dishes, add PMA to a final concentration of 100 ng / mL, mix gently, and incubate at 37°C in a 5% CO2 incubator for 48 h. After induction, wash twice with serum-free RPMI-1640 medium at 37°C. Remove the medium and starve the cells with RPMI-1640 medium for 8–12 h. After starvation, remove the medium again and add RPMI-1640 complete medium containing 10% Australian fetal bovine serum (FBS) for later use.

[0047] 3. THP-1 cytotoxicity assay. THP-1 cells grown to an appropriate density were dispersed evenly, resuspended in fresh RPMI-1640 medium, and diluted to 1×10⁻⁶. 5 cell / mL. Add 100 μL of fresh RPMI-1640 medium to each well of a sterile 96-well cell plate. Perform 2-fold serial dilutions of the ergolactone solution to be tested in the cell culture medium. Include a solvent control containing 0.1% DMSO. Add 100 μL of the diluted cell suspension (1×10⁻⁶ cells / mL) to each well of the 96-well cell plate. 4 Cells were cultured in a 37°C, 5% CO2 incubator for 48 h. After adding 10 µL of CCK-8 solution and incubating for 2 h, cell viability was determined by UV spectrophotometry at 450 nm. Cell viability was expressed as a percentage of untreated cells. All experiments were performed in triplicate.

[0048] See results Figure 4Specifically, at bactericidal concentrations, there was no statistically significant difference in the cytotoxic effect of Ergolide on THP-1 human macrophages. Figure 4 It has good biosafety.

[0049] In summary, this invention systematically verified the significant antibacterial activity, synergistic effect, and sustained bactericidal ability of ergolactone against Mycobacterium tuberculosis through a series of experiments, including in vitro antibacterial assays, combined drug trials, and time-bactericidal curve analysis. The results show that ergolactone not only possesses broad-spectrum anti-mycobacterial properties but also exhibits an additive effect when used in combination with existing first-line drugs, demonstrating significant development potential as a novel anti-tuberculosis drug or a component of combination therapies. This invention provides a new source of compounds and technical solutions for the treatment of tuberculosis, possessing significant theoretical value and promising clinical application prospects.

[0050] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Application of ergolactone in the preparation of antimycobacterial drugs.

2. The application as described in claim 1, characterized in that, The mycobacteria mentioned include Mycobacterium tuberculosis and various pathogenic mycobacterial strains.

3. Application of ergolactone in the preparation of anti-tuberculosis drugs.

4. The application as described in claim 3, characterized in that, The Mycobacterium tuberculosis mentioned refers to the standard strain of Mycobacterium tuberculosis and various clinical isolates of Mycobacterium tuberculosis.

5. Application of ergolactone in the preparation of combined synergistic drug compositions against Mycobacterium tuberculosis.

6. The application as described in claim 5, characterized in that, The combined synergistic drug composition contains ergolactone and one or more first-line anti-tuberculosis drugs, wherein the first-line anti-tuberculosis drugs are selected from isoniazid (INH), rifampin (RFP), bedaquiline (BDQ) or ethambutol (EMB).

7. An anti-tuberculosis drug, characterized in that, The drug contains ergolactone as its active ingredient.

8. The anti-tuberculosis drug as described in claim 7, characterized in that, The drug also includes one or more pharmaceutically acceptable carriers or excipients.

9. The anti-tuberculosis drug as described in claim 7, characterized in that... The formulation of the drug is selected from one or more of conventional formulations, sustained-release formulations, controlled-release formulations, and / or various microparticle delivery systems.

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