Use of vinblastine in the preparation of MRSA beta-lactamase inhibitors

Abstract

The application belongs to the technical field of medicine and pharmacy, and particularly relates to application of vincaleukoblastine in preparation of MRSA beta-lactamase inhibitors. The application of the vincaleukoblastine in preparation of MRSA infection treatment drugs, and the vincaleukoblastine can inhibit MRSA biofilm formation. The application discloses that the vincaleukoblastine can effectively restore the antibacterial activity of beta-lactam antibiotics, inhibit MRSA biofilm, and effectively treat MRSA infection mice.

Description

Technical Field

[0001] This invention relates to the field of medical and pharmaceutical technology, specifically to the application of vincristine in the preparation of MRSA β-lactamase inhibitors. Background Technology

[0002] Staphylococcus aureus, a common Gram-positive bacterium, has developed resistance to a variety of antibiotics used clinically for a long time. In particular, the evolution of β-lactamase-producing Gram-positive strains poses a significant threat to global public health in the post-antibiotic era due to their widespread resistance to β-lactam antibiotics. Cephalosporins are often used as a last resort against multidrug-resistant bacteria. However, the emergence of various β-lactamase-producing strains presents a major challenge to their clinical efficacy. Some clinical strains associated with persistent infections, including methicillin-resistant Staphylococcus aureus (MRSA), have shown significant resistance to multiple antibiotics, including β-lactams, primarily due to alterations in penicillin-binding protein (PBP) and the ability to produce β-lactamases. β-lactamases typically contain reactive serine residues and divalent zinc ions, enabling them to hydrolyze β-lactam antibiotics such as cephalosporins and penicillins, effectively neutralizing the therapeutic effects of these drugs and thus posing a significant challenge to clinical treatment. Vincristine is an alkaloid compound extracted from the periwinkle plant (Catharanthus roseus), a member of the Apocynaceae family. It possesses hypotensive and anticancer activities. Currently, there are no research reports on whether vincristine can enhance the efficacy of β-lactam antibiotics by inhibiting β-lactamase; its potential as an antibacterial synergist remains to be explored. Summary of the Invention

[0003] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0004] To address the aforementioned technical problems, according to one aspect of the present invention, the present invention provides the following technical solution:

[0005] Application of a vincristine base in the preparation of MRSA β-lactamase inhibitors.

[0006] As a preferred embodiment of the application of vincristine in the preparation of MRSA β-lactamase inhibitors according to the present invention, the vincristine is used in conjunction with β-lactam antibiotics in the preparation of drugs for treating MRSA infections.

[0007] As a preferred embodiment of the application of vincristine in the preparation of MRSA β-lactamase inhibitors according to the present invention, the vincristine is capable of inhibiting MRSA biofilm formation.

[0008] Compared with the prior art, the beneficial effects of the present invention are: the present invention discloses that vincristine can effectively restore the antibacterial activity of β-lactam antibiotics and inhibit the biofilm formation of MRSA, while effectively treating MRSA-infected mice. Attached Figure Description

[0009] To more clearly illustrate the technical solutions of the embodiments of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and detailed embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0010] Figure 1 This is a graph showing the test results of the checkerboard microdilution method for the use of vincristine combined with cefixime on USA300 according to the present invention.

[0011] Figure 2 This is a graph showing the test results of the checkerboard microdilution method for the effect of vincristine combined with penicillin on USA300 in this invention.

[0012] Figure 3 This is a graph showing the experimental results of the growth curve of vincristine alkali on USA300 according to the present invention;

[0013] Figure 4 The graph shows the inhibitory effects of different concentrations of vincristine on β-lactamase activity according to this invention.

[0014] Figure 5 This is the result of the time-bactericidal curve test of vincristine combined with β-lactam antibiotics against USA300 in this invention;

[0015] Figure 6 The figure shows the experimental results of the inhibition of biofilm formation on USA300 by vincristine alkaloids of the present invention.

[0016] Figure 7 This is a quantitative graph showing the results of the biofilm inhibition test of vincristine alkaloids on USA300 in this invention.

[0017] Figure 8 This image shows the colony colonization results of target organs in a mouse pneumonia model infected with USA300, using vincristine combined with β-lactam antibiotics. Detailed Implementation

[0018] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0019] Secondly, the present invention is described in detail with reference to the schematic diagrams. When detailing the embodiments of the present invention, for ease of explanation, the cross-sectional views illustrating the device structure may be partially enlarged, not according to the usual scale. Furthermore, the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. In addition, actual fabrication should include three-dimensional spatial dimensions of length, width, and depth.

[0020] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0021] This invention proposes the medical use of vincristine in the preparation of methicillin-resistant Staphylococcus aureus β-lactamase inhibitors, providing a new option for the treatment of MRSA infections.

[0022] In this embodiment, methicillin-resistant Staphylococcus aureus USA300 was used as the model bacterium. Through experiments such as checkerboard microdilution method, bactericidal curve, and mouse Staphylococcus aureus pneumonia model, it was verified that vincristine can effectively restore the antibacterial activity of β-lactam antibiotics and improve the in vivo therapeutic effect of β-lactam antibiotics.

[0023] Example 1

[0024] Vincristine can be used in the preparation of methicillin-resistant Staphylococcus aureus β-lactamase inhibitors in any pharmaceutically acceptable carrier.

[0025] Example 2

[0026] Vincristine can be used as a β-lactamase inhibitor of methicillin-resistant Staphylococcus aureus (MRSA) in the preparation of drugs for treating infectious diseases.

[0027] Example 3

[0028] Vincristine can be used as a β-lactamase inhibitor of methicillin-resistant Staphylococcus aureus to treat infectious diseases caused by bacteria.

[0029] Experimental Example 1

[0030] Chessboard microdilution method experiment:

[0031] Drug susceptibility testing was performed using the microbroth dilution method, following the guidelines of the Clinical and Laboratory Standards Institute (CSIS). In a 96-well plate, 5 × 10⁵ cells were seeded per well. 5CFU / mL bacterial culture was prepared, and serially diluted vincristine and antibiotics were added to achieve final concentrations of 0–128 μg / mL and 0–512 μg / mL, respectively. The cultures were incubated at 37°C for 16 hours, and the results were recorded. FICI = (MIC of the compound used in combination / MIC of the compound used alone) + (MIC of the compound used in combination / MIC of the antibiotic used alone).

[0032] The results showed that vincristine, when used in combination with β-lactam antibiotics, exhibited a synergistic antibacterial effect against methicillin-resistant Staphylococcus aureus (SMRSA) USA300 (FICI < 0.5). Figure 1 and Figure 2 (As shown).

[0033] Experimental Example 2

[0034] Growth curve of methicillin-resistant Staphylococcus aureus USA300:

[0035] Methicillin-resistant Staphylococcus aureus (MRSA) USA300 bacterial cultures were divided into four groups, and different concentrations of vincristine (0, 8, 16, and 32 μg / mL) were added to each group. The growth of bacterial cultures in each group was monitored by measuring the absorbance at 600 nm at different time points.

[0036] The results showed that vincristine did not affect the growth of methicillin-resistant Staphylococcus aureus USA300 within the concentration range of 8-32 μg / mL (e.g., Figure 3 (As shown).

[0037] Experimental Example 3

[0038] Assay for inhibition of methicillin-resistant Staphylococcus aureus β-lactamase activity:

[0039] The purified β-lactamase protein was incubated with different concentrations of vincristine (0, 8, 16, 32 μg / mL) at 37 °C for 15 minutes, and then 25 μM nitrophenol substrate was added to continue the reaction for 30 minutes. Finally, the absorbance of each group of samples at 492 nm was measured.

[0040] The results showed that increasing vincristine concentration could effectively inhibit β-lactamase activity (e.g., Figure 4 (As shown).

[0041] Test Example 4

[0042] Time-bacterial control curve test of vincristine combined with cefixime against methicillin-resistant Staphylococcus aureus USA300:

[0043] Methicillin-resistant Staphylococcus aureus (MRSA) USA300 was diluted to 5 × 10⁵ CFU / mL, and the bacterial suspensions were divided into four groups: control group (DMSO solvent), vincristine group (32 μg / mL), cefixime group (32 μg / mL), and combined drug group. Samples from each group were collected at different time points, diluted, and plated on TSB agar plates. Colony counts were calculated to evaluate the bactericidal effect, and time-kill curves were plotted.

[0044] The results showed that, based on the time-bactericidal curve, vincristine combined with cefixime had a bactericidal effect on methicillin-resistant Staphylococcus aureus USA300 within 12 h (e.g., Figure 5 (As shown).

[0045] Experimental Example 5

[0046] Inhibition test of vincristine on biofilm formation of methicillin-resistant Staphylococcus aureus USA300:

[0047] Methicillin-resistant Staphylococcus aureus USA300 (OD) 600 =0.1) and different concentrations of vincristine (0, 8, 16, 32 μg / mL) were added to 24-well plates and incubated at 37°C for 24 hours. A blank control group was set up: TSB medium containing methicillin-resistant Staphylococcus aureus USA300 without vincristine was added. After 24 hours, the plates were stained with 0.1% crystal violet for 1 hour, followed by washing three times with PBS. Destaining was performed with 33% acetic acid solution, and the staining was measured at OD using a spectrophotometer. 600nm The absorbance value was measured.

[0048] The results showed that vincristine could inhibit the formation of biofilms from methicillin-resistant Staphylococcus aureus USA300 (e.g., Figure 6 and Figure 7 (As shown).

[0049] Experimental Example 6

[0050] Methicillin-resistant Staphylococcus aureus USA300-infected mouse pneumonia model and drug therapeutic effects:

[0051] The experimental mice were divided into four groups: a solvent control group, a vinblastine treatment group, a cefixime treatment group, and a vinblastine combined with cefixime treatment group. After mild anesthesia, the mice were infected with methicillin-resistant Staphylococcus aureus USA300 (2 × 10⁻⁶) via nasal drop. 8 CFU). Administer the medication every eight hours, three times daily, continuing treatment until the day after infection. Mice were anesthetized before sacrifice, and bacterial load was determined in each group of samples using a serial dilution and plate inoculation method.

[0052] The results showed that, compared with the antibiotic monotherapy group, vincristine combined with cefixime effectively reduced bacterial colonization in the lungs of methicillin-resistant Staphylococcus aureus (MRSA)-infected mice, exerting a good therapeutic effect (e.g., Figure 8 (As shown).

[0053] Although the present invention has been described above with reference to embodiments, various modifications can be made and components can be replaced with equivalents without departing from the scope of the invention. In particular, as long as there is no structural conflict, the features in the disclosed embodiments can be combined with each other in any manner. The lack of an exhaustive description of these combinations in this specification is merely for the sake of brevity and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. The application of a vincristine base in the preparation of MRSA β-lactamase inhibitors.

2. The application of vincristine according to claim 1 in the preparation of MRSA β-lactamase inhibitors, characterized in that, The application of vincristine synergistic with β-lactam antibiotics in the preparation of drugs for treating MRSA infection.

3. The application of vincristine according to claim 1 in the preparation of MRSA β-lactamase inhibitors, characterized in that, The vincristine base can inhibit the formation of MRSA biofilm.

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