Application of dapunostat in preparation of medicine for preventing and / or treating mycobacterium tuberculosis infection

Abstract

The invention belongs to the field of biological medicines, and particularly relates to application of dapinustat in preparation of a medicine for preventing and / or treating mycobacterium tuberculosis infection. In-vitro experiments prove that dapunostat has remarkable inhibitory activity on a mycobacterium tuberculosis standard strain, the minimum inhibitory concentration (MIC) is 1 mu g / mL, and the bactericidal effect of dapunostat under high bacterial load is superior to that of rifampicin. The dapinustat and the rifampicin have a synergistic effect. When dapunostat is combined with first-line antituberculosis drugs such as isoniazide, bedaquiline and ethambutol for use, drug effects are added. In addition, the dapunostat has no obvious toxicity to THP-1 human macrophages under an effective concentration. According to the invention, the new application of dapinustat in resisting mycobacterium tuberculosis is found for the first time, a brand new candidate drug and a drug combination scheme with known safety are provided for treatment of tuberculosis, especially drug-resistant tuberculosis, and the dapinustat has important clinical application value and development prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of dapoxetine in the preparation of drugs for the prevention and / or treatment of Mycobacterium tuberculosis infection. Background Technology

[0002] Tuberculosis (TB) is a chronic infectious disease caused by the Mycobacterium tuberculosis complex, which seriously threatens global public health. According to the latest statistics from the World Health Organization, TB remains one of the leading causes of death among single infectious diseases, with approximately 10.7 million new cases and 1.23 million deaths worldwide in 2024. Currently, TB prevention and control face multiple challenges: First, drug resistance is a prominent issue. With the long-term and widespread use of first-line drugs, multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) are constantly emerging. Existing treatment regimens have limited efficacy, long treatment courses, and significant side effects. Second, new drug development is extremely difficult. The discovery and development of novel anti-TB drugs has a very long cycle (often exceeding 10 years), requires huge investment, and has a high failure rate, far from meeting urgent clinical needs. Third, latent infection is difficult to eradicate. The strong latent ability of Mycobacterium tuberculosis means that the risk of relapse persists for a long time.

[0003] Against this backdrop, the "drug repurposing" strategy, with its core advantages of utilizing complete pharmacological and toxicological data of existing compounds, significantly shortening the research and development cycle, reducing development costs, and rapidly addressing the drug resistance crisis, has become a crucial breakthrough in the development of anti-tuberculosis drugs. However, efficiently screening compounds with high activity and good safety against Mycobacterium tuberculosis, especially against drug-resistant strains, from a vast pool of marketed drugs is itself a significant scientific challenge.

[0004] In the existing technology, the biological activity research of daprodusta (CAS No. 960539-70-2) has focused on its therapeutic effects on anemia, blood pressure and other related diseases. To date, no literature or report has revealed or implied that daprodusta has antimicrobial activity, let alone its inhibitory or killing effects on mycobacteria, especially Mycobacterium tuberculosis. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies, such as the limited variety of anti-tuberculosis drugs, the increasingly serious problem of drug resistance, and the long development cycle and high cost of new drugs, by providing a novel pharmaceutical use of the known compound daprodustat in the treatment of mycobacterial infections. Specifically, this invention relates to a novel application of daprodustat in the preparation of drugs for the prevention and / or treatment of Mycobacterium tuberculosis infection.

[0006] To achieve the above objectives, we provide the use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting Mycobacterium tuberculosis.

[0007] To further clarify, the Mycobacterium tuberculosis includes standard strains of Mycobacterium tuberculosis and / or clinically isolated strains of Mycobacterium tuberculosis.

[0008] To further clarify, the product that inhibits Mycobacterium tuberculosis can be a medical product or a non-medical product; the medical product is a drug or pharmaceutical excipient, and the non-medical product is an experimental reagent or antibacterial agent.

[0009] To further clarify, the drug comprises dapoxetine and one or more pharmaceutically acceptable carriers or excipients.

[0010] To further clarify, the dosage form of the drug is selected from one or more of conventional formulations, sustained-release formulations, controlled-release formulations, and microparticle delivery systems.

[0011] The present invention also provides the use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of Mycobacterium tuberculosis infection.

[0012] The present invention also provides the use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of tuberculosis.

[0013] The present invention also provides the use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of combination pharmaceutical compositions for the treatment of mycobacterial tuberculosis infection.

[0014] To further explain, the combination drug composition comprises dapoxetine and at least one first-line anti-tuberculosis drug selected from isoniazid, rifampin, bedaquiline and ethambutol.

[0015] The present invention also provides the use of the above-described dapoxetine in the preparation of a pharmaceutical formulation for inhibiting pathogenic bacteria; wherein the pathogenic bacteria is Staphylococcus aureus; the minimum inhibitory concentration for inhibiting Staphylococcus aureus is 8 µg / mL, and the minimum inhibitory concentration for its pathogenic strain NEWMAN / Methicillin-resistant S. aureus is 16 µg / mL.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention is the first to discover and confirm that dapoxetine possesses excellent anti-tuberculosis activity. As a marketed drug, dapoxetine has relatively complete pharmacological, toxicological, and safety data. The "drug repurposing" strategy can significantly shorten its development cycle as an anti-tuberculosis drug, reduce costs, and accelerate clinical translation. Dapoxetine exhibits strong inhibitory and bactericidal effects on standard strains of Mycobacterium tuberculosis and Mycobacterium bovis, with a minimum inhibitory concentration (MIC) as low as 1 μg / mL. Furthermore, its bactericidal kinetics at high bacterial loads are superior to those of the first-line drug rifampin, demonstrating its potential to eliminate refractory bacterial populations. Dapoxetine shows a synergistic effect with existing first-line anti-tuberculosis drugs such as rifampin, and an additive effect when used in combination with existing first-line anti-tuberculosis drugs (such as isoniazid, bedaquiline, and ethambutol). This provides experimental evidence for constructing novel combination therapy regimens including dapoxetine to address multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) tuberculosis. Our research team's cytotoxicity experiments on dapoxetine showed that dapoxetine has no significant toxicity to mammalian cells at effective antibacterial concentrations and has good biosafety, laying the foundation for its further in vivo studies and clinical development.

[0018] In summary, this invention has creatively discovered a novel anti-tuberculosis activity of dapoxetine that is completely unexpected by existing technologies. This not only adds a highly promising new member to the family of anti-tuberculosis drugs, but also provides a new, efficient, economical, and risk-controllable path to solve the core technical challenges currently facing the field of tuberculosis treatment: the threat of drug resistance and the shortage of effective new drugs. Attached Figure Description

[0019] Figure 1 The figure shows the experimental results of the in vitro broad-spectrum antibacterial activity assay of dapoxetine.

[0020] Figure 2 The figure shows the experimental results of the time-dependent killing curve of dapoxetine against Mycobacterium tuberculosis.

[0021] Figure 3 The figure shows the experimental results of dapoxetine combined with INH, RFP, BDQ, and EMB to combat Mycobacterium tuberculosis.

[0022] Figure 4 The image shows the results of the dapoxetine cytotoxicity test. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] Example 1: This example is a study on the antibacterial spectrum of daprodustat.

[0026] The research methodology is as follows:

[0027] 1. The dapoxetine was purchased from MCE. It was dissolved in DMSO to prepare a stock solution with a concentration of 5 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.

[0028] 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 Staphylococcus aureus Methicillin-resistant Staphylococcus aureus (MRSA) was activated and cultured using MHB medium (purchased from Solarbio) 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 Company) was added.

[0029] 3. Preparation of bacterial culture before the experiment. Activated Mycobacterium smegmaecum, Pseudomonas aeruginosa, Escherichia coli, and Staphylococcus aureus OD200 were prepared. 600 Adjust the medium to 0.5, then dilute it 100 times with the medium. Add the activated Mycobacterium tuberculosis and Mycobacterium bovis to the medium to measure 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.

[0030] 4. Gradient dilution of dapoxetine.

[0031] The minimum inhibitory concentration (MIC) was determined using a 96-well plate. The left two columns of the 96-well plate contained a positive control (diluted bacterial suspension) and a negative control (culture medium), respectively. 200 μL of diluted bacterial suspension was added to each of the 6*10 wells on the right. 400 μL of bacterial suspension and 2.56 μL of dapoxetine (final concentration 32 μg / mL) were added to the rightmost column. 200 μL of the liquid from each well was transferred from right to left to the left wells, performing a 2-fold serial dilution.

[0032] 5. Culture and staining. The standard strain of Mycobacterium tuberculosis was cultured at 37℃. Mycobacterium bovis After standing for 7 days, marine mycobacteria After standing for 4 days, Mycobacterium smegma Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Staphylococcus aureus Methicillin-resistant Staphylococcus aureus After standing for 1 day, the OD was measured. 600 Analyze absorbance to determine the minimum inhibitory concentration (MIC). Alternatively, after incubation, add resveratrol chromogenic 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 turn completely blue and plot the results using GraphPadPrism software.

[0033] See results Figure 1 Specifically, the MIC (micron activity) of daprodustat against the standard strain of Mycobacterium tuberculosis is 1 μg / mL, against Mycobacterium bovis it is 1 μg / mL, against Mycobacterium marineum it is 8 μg / mL, and against Mycobacterium smegmatis it is 8 μg / mL. This indicates that daprodustat has a certain bactericidal ability against all mycobacteria, and it has a significant specific bactericidal effect against the standard strain of Mycobacterium tuberculosis. Daprodustat also has an effect against Staphylococcus aureus. The MIC is 8 μg / mL, which is effective against pathogenic Staphylococcus aureus. Methicillin-resistant Staphylococcus aureus The MIC was 16 μg / mL, indicating that Daprodustat has a certain killing effect on Staphylococcus aureus. It also showed activity against Pseudomonas aeruginosa (PAO1) and Escherichia coli (Escherichia coli). The fact that certain strains showed no antibacterial activity indicates that Daprodustat only has an inhibitory effect on some pathogens.

[0034] Example 2: This example is a study on the time-kill curve of daprodustat at high bacterial load.

[0035] The research methodology is as follows:

[0036] 1. The activation of the strain and the preparation of dapoxetine 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.

[0037] 2. Add daprodustat. Aliquot the diluted bacterial culture into PA bottles and set up a negative control (DMSO), a positive control (4-fold MIC rifampin), and experimental groups with 2-fold MIC daprodustat and 4-fold MIC daprodustat, respectively. Each group should have 3 independent biological replicates. Incubate at 37°C on a shaker at 80 rpm.

[0038] 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 for plotting.

[0039] See results Figure 2 Specifically, daprodustat showed superior bactericidal efficacy against standard strains of Mycobacterium tuberculosis compared to rifampin, not only due to its rapid bactericidal kinetics but also its ability to completely eliminate high bacterial loads. Rifampin struggles to completely eradicate Mycobacterium tuberculosis, while daprodustat more effectively eliminated these resistant bacterial subgroups, achieving complete eradication of high bacterial loads. This provides a powerful candidate for overcoming the clinical challenge of high bacterial load tuberculosis.

[0040] Example 3: This example is a study on the combined use of daprodustat.

[0041] The research methodology is as follows:

[0042] 1. The activation of the strain and the preparation of dapoxetine were the same as in Example 1.

[0043] 2. Add Daprodustat. The left two columns and the right two columns of the 96-well plate are blank culture media. Select the middle 8*8 area as the experimental area, and add 200 μL of bacterial solution to each well. An additional 200 μL of bacterial solution is added to the rightmost column of the 8*8 area, and then 2-fold MIC of Daprodustat (final concentration is 2 μg / mL) is added. It should be noted that the top right well should be added twice as much Daprodustat as the other wells (final concentration is 4 μg / mL). After adding Daprodustat, perform serial dilution from right to left according to the method of serial dilution in Example 1 until the second column of the 8*8 area, that is, the first column of the 8*8 area does not add Daprodustat. After the dilution of Daprodustat is completed, add 2-fold MIC of first-line drugs (isoniazid INH, final concentration is 0.125 μg / mL), rifampicin (RFP, final concentration is 0.0156 μg / mL), bedaquiline (BDQ, final concentration is 0.0625 μg / mL), ethambutol (EMB, final concentration is 1 μg / mL)) to the first row of the 8*8 area, and then perform 2-fold serial dilution from top to bottom according to the method of serial dilution in Example 1.

[0044] 3. Culture and color development. Incubate at 37°C, let stand for 7 days, and judge the minimum inhibitory concentration by detecting OD 600 absorbance. After culturing or at the end of the culture, add resazurin color developing 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.

[0045] The results are shown in Figure 3 , specifically as follows: The FIC value of the combined use of Daprodustat and rifampicin (RFP) against the standard strain of Mycobacterium tuberculosis is 0.5, showing a synergistic effect, that is, when Daprodustat and rifampicin (RFP) are used in combination, the dosage of single drug can be reduced on the premise of ensuring the antibacterial effect. At the same time, the FIC values of the combined use of Daprodustat with the three first-line anti-tuberculosis drugs isoniazid (INH), bedaquiline (BDQ), and ethambutol (EMB) against the standard strain of Mycobacterium tuberculosis are 1, 1, and 1 respectively, and the drug effects are additive, indicating that when Daprodustat is used in combination with INH, BDQ, and EMB, a drug effect superposition effect can be produced, that is, the antibacterial effect of the combined use is equivalent to the sum of the individual effects of each drug.

[0046] Example 4: This example is a study on the cytotoxicity of daprodustat.

[0047] The research methodology is as follows:

[0048] 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.

[0049] 2. Induction of THP-1 cell differentiation. Collect THP-1 cells of good growth 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, obtain THP-1 human macrophages, wash twice with serum-free RPMI-1640 medium at 37°C. Remove the medium, add RPMI-1640 medium for starvation treatment for 8–12 h. After starvation treatment, remove the medium again, add RPMI-1640 complete medium containing 10% Australian fetal bovine serum (FBS), and set aside.

[0050] 3. Cytotoxicity assay of THP-1 human macrophages. THP-1 human macrophages 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 dapoxetine solution in cell culture medium, with 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. 4Cells 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.

[0051] See results Figure 4 Specifically, different concentrations of Daprodustat showed no statistically significant difference in cytotoxicity against THP-1 human macrophages, indicating good biocompatibility.

[0052] In summary, the experiments demonstrate that daprodustat exhibits significant in vitro inhibitory activity against standard strains of Mycobacterium tuberculosis and Mycobacterium bovis (MIC 1 μg / mL), showing superior bactericidal efficacy compared to the first-line drug rifampin, and achieving rapid and thorough bactericidal action even at high bacterial loads. It also shows synergistic and additive effects when used in combination with first-line anti-tuberculosis drugs such as isoniazid, rifampin, bedaquiline, and ethambutol. Furthermore, it demonstrates no significant toxicity to THP-1 human macrophages within its effective concentration range, exhibiting good biocompatibility. These findings collectively indicate that daprodustat, as a marketed compound with known safety profiles, possesses significant potential for development into novel anti-tuberculosis drugs or combination therapies, providing a novel candidate for addressing the current severe drug resistance crisis in tuberculosis.

[0053] 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. The use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of products for inhibiting Mycobacterium tuberculosis.

2. The application according to claim 1, characterized in that, The Mycobacterium tuberculosis includes standard strains of Mycobacterium tuberculosis and / or clinically isolated strains of Mycobacterium tuberculosis.

3. The application according to claim 1, characterized in that, The product that inhibits Mycobacterium tuberculosis can be a medical product or a non-medical product; the medical product is a drug or pharmaceutical excipient, and the non-medical product is an experimental reagent or antibacterial agent.

4. The application according to claim 3, characterized in that, The drug comprises dapoxetine and one or more pharmaceutically acceptable carriers or excipients.

5. The application according to claim 4, characterized in that, The dosage form of the drug is selected from one or more of the following: conventional formulations, sustained-release formulations, controlled-release formulations, and microparticle delivery systems.

6. Use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention and / or treatment of Mycobacterium tuberculosis infection.

7. Use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of medicines for the prevention and / or treatment of tuberculosis.

8. Use of dapoxetine or a pharmaceutically acceptable salt thereof in the preparation of combination pharmaceutical compositions for the treatment of mycobacterial tuberculosis infection.

9. The application according to claim 8, characterized in that, The combination drug composition comprises dapoxetine and at least one first-line anti-tuberculosis drug selected from isoniazid, rifampin, bedaquiline and ethambutol.

10. The use of dapoxetine according to claim 1 in the preparation of a pharmaceutical formulation for inhibiting pathogenic bacteria; wherein the pathogenic bacteria is Staphylococcus aureus; and the minimum inhibitory concentration for inhibiting Staphylococcus aureus is 8 µg / mL, for its pathogenic strains. The minimum inhibitory concentration is 16 µg / mL.

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