Application of flavoxate in the preparation of drugs for treating infectious pneumonia caused by methicillin-resistant Staphylococcus aureus.

Abstract

This invention discloses the application of duracil in the preparation of drugs for treating infectious pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA), relating to the field of pharmaceutical technology. This invention is the first to discover that duracil can significantly reduce the virulence of Staphylococcus aureus by inhibiting the activity of the ClpP protein in MRSA. Furthermore, duracil exhibits significant protective effects in the treatment of MRSA-induced infectious pneumonia. Specifically, duracil not only reduces the degree of lung tissue damage but also reduces the infiltration and aggregation of inflammatory cells in the lungs. These findings indicate that duracil has potential application value in the field of anti-infective therapy. This invention provides a novel therapeutic strategy, namely, using duracil as the active ingredient in the preparation of drugs for treating Staphylococcus aureus-induced infectious pneumonia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more particularly to the application of flavin in the preparation of drugs for treating infectious pneumonia caused by methicillin-resistant Staphylococcus aureus. Background Technology

[0002] In the medical field, methicillin-resistant Staphylococcus aureus (MRSA) pneumonia is becoming an increasingly serious problem. MRSA is a type of Staphylococcus aureus resistant to β-lactam antibiotics (including methicillin), and its resistance significantly increases the difficulty of treating the infection and often leads to poor prognosis. Globally, the incidence of MRSA pneumonia continues to rise, especially in healthcare facilities, where MRSA has become one of the leading causes of hospital-acquired infections. However, the efficacy of traditional antimicrobial drugs in treating MRSA pneumonia is gradually declining, and the long-term, widespread use of antibiotics has further exacerbated the emergence of drug-resistant strains. Therefore, the development of safe, effective, and non-resistant novel anti-MRSA drugs has become an urgent priority.

[0003] The pathogenicity of MRSA is closely related to its virulence factors, especially the casein hydrolase ClpP secreted by Staphylococcus aureus. ClpP plays a crucial role in bacterial protein degradation, virulence regulation, and antibiotic resistance mechanisms. Studies have shown that mutations in the clpP gene lead to various phenotypic changes in Staphylococcus aureus, including increased sensitivity to environmental stress, abnormal cell morphology, cell division arrest, and reduced virulence. Furthermore, inhibiting the activity of ClpP protein has been shown to significantly reduce the pathogenicity of MRSA, including reducing its survival ability in the host and its ability to induce pathological inflammation. Notably, targeted inhibition of ClpP does not directly inhibit the growth of Staphylococcus aureus or kill it, and therefore does not increase the risk of developing drug-resistant strains. This characteristic is particularly important in the context of increasing bacterial antibiotic resistance. With the growing need for the development of novel antimicrobial drugs, ClpP, due to its unique proteolytic function, has become a potential new target for antimicrobial drug development, providing new strategies and hope for anti-infective therapy.

[0004] Against this backdrop, the present invention aims to provide a novel method for treating MRSA-infectious pneumonia, namely, using a drug with strychnine as the active ingredient. Strychnine, also known as strychnine, gentianin, or gentianin, has the chemical formula C. 22 H 22 O 11 Its molecular weight is 462.4, and its molecular structure is as follows: Figure 1As shown, it is a natural flavonoid compound extracted from medicinal plants such as *Belamcanda chinensis*, *Iris tectorum*, and *Swertia spp.*. This compound has been shown to possess various pharmacological activities, including antioxidant, antidiabetic, antidepressant, anti-inflammatory, antiviral, and anti-radiation effects. However, although dansoflavone has shown potential in the treatment of multiple diseases, there are currently no reports, either domestically or internationally, on its use in treating MRSA-induced pneumonia. This indicates that further research and exploration are needed regarding the application of dansoflavone in the field of anti-infectives, especially for pneumonia caused by MRSA. Summary of the Invention

[0005] This invention is the first to discover that flavoxetine can significantly reduce the virulence of Staphylococcus aureus by inhibiting the activity of ClpP protein in MRSA. Furthermore, experiments conducted using this invention demonstrate that flavoxetine exhibits a significant protective effect in the treatment of MRSA-infected pneumonia.

[0006] Specifically, in a first aspect, the present invention provides the use of flavoxate in the preparation of drugs for treating Staphylococcus aureus infection.

[0007] Furthermore, the Staphylococcus aureus is methicillin-resistant Staphylococcus aureus.

[0008] Furthermore, the methicillin-resistant Staphylococcus aureus is Staphylococcus aureus USA300.

[0009] This invention provides the use of flavin in the preparation of a drug for treating infectious pneumonia.

[0010] Furthermore, the drug is used to treat methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Doxyflavin not only reduces the degree of lung tissue damage but also decreases the infiltration and aggregation of inflammatory cells in the lungs. These findings suggest that oxyflavin has potential application value in the field of anti-infective therapy.

[0011] Secondly, the present invention also provides the use of flavoxate in the preparation of drugs that inhibit the activity of Staphylococcus aureus casein hydrolase ClpP.

[0012] Thirdly, the present invention also provides a drug for treating Staphylococcus aureus infections, characterized in that the drug reduces the toxicity of MRSA by inhibiting the activity of casein hydrolase ClpP in MRSA, and the drug is prepared into a pharmaceutically acceptable dosage form from flavin as the active ingredient and pharmaceutically acceptable excipients.

[0013] Furthermore, the dosage forms of the drug include, but are not limited to, tablets, capsules, and injections.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] First, this invention validated the efficacy of dandroflavin using a mouse model of MRSA-infected pneumonia. Experimental results showed that dandroflavin possesses significant therapeutic potential in the mouse MRSA-infected pneumonia model, primarily in two aspects: firstly, it significantly improves the survival rate of MRSA-infected mice; and secondly, it effectively reduces the inflammatory response in lung tissue induced by MRSA infection. These findings provide strong experimental evidence for dandroflavin as a potential therapeutic agent against MRSA-infected pneumonia.

[0016] Furthermore, this invention reveals the mechanism of action of dannabisulfonate: dannabisulfonate can inhibit the activity of casein hydrolase ClpP, a key virulence factor in MRSA, effectively reducing the virulence of MRSA and exerting a therapeutic effect. Importantly, compared with conventional antibiotic treatment, dannabisulfonate does not exert survival pressure on Staphylococcus aureus, and therefore does not tend to promote the occurrence and development of bacterial resistance.

[0017] Therefore, the flavin disclosed in this invention has potential application value in the development of safe and effective drugs for the treatment of Staphylococcus aureus pneumonia, and is expected to provide new options for clinical treatment. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0019] Figure 1 This is the molecular structural formula of flavin.

[0020] Figure 2 In Example 1 of this invention, the inhibitory effect of acyclovir on the in vitro activity of Staphylococcus aureus USA300 ClpP is represented by a bar graph.

[0021] Figure 3 The result of the lack of inhibitory effect of flavin on the growth of Staphylococcus aureus USA300 in Example 2 of the present invention is represented by a bar graph.

[0022] Figure 4 In Embodiment 3 of the present invention, the minimum inhibitory concentration (MIC) of adenosine against Staphylococcus aureus USA300 is represented by a bar chart.

[0023] Figure 5 In Example 4 of this invention, the effect of apocytosine on the viability of human lung epithelial cells (A549) is described.

[0024] Figure 6 In Example 5 of this invention, flavin inhibits the invasion of Staphylococcus aureus USA300 into A549 cells.

[0025] Figure 7 In Example 6 of this invention, the protective effect of flavin on lung tissue of mice with infectious pneumonia induced by Staphylococcus aureus USA300 was evaluated, including the results of lung appearance and pathological assessment.

[0026] Figure 8 This is a diagram showing the experimental results of flavin treatment in Example 6 of the present invention, which improved the survival rate of mice with infectious pneumonia induced by Staphylococcus aureus USA300.

[0027] Figure 9 This is a schematic diagram illustrating the mechanism of action of dioxin.

[0028] Significance indicators for each figure: ns: no significant difference, *: P≤0.05, **: P≤0.01, ***: P≤0.001. Detailed Implementation

[0029] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0031] Unless otherwise specified, all experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0032] The *Staphylococcus aureus* MRSA USA300 used in this invention was purchased from the American Center for Type Culture Collection (CAS NO: ATCC BAA-1717). MRSA USA300 is a highly pathogenic and drug-resistant *Staphylococcus aureus* strain, originally discovered in the United States. It is a representative strain of community-acquired MRSA (CA-MRSA) and is one of the leading pathogens causing skin and soft tissue infections worldwide. The USA300 used in this invention... dclpP strain and BL21-pET28a- clpPAll bacterial strains were previously constructed and preserved in the laboratory, and the construction method referred to existing technical literature (Jing Shisong. Study on the inhibitory effect and mechanism of myricetin on Staphylococcus aureus ClpP [D]. Jilin University, 2021. DOI: 10.27162 / d.cnki.gjlin.2021.003326.). The human lung epithelial cells A549 used in this invention were purchased from Wuhan Pronosei Biotechnology Co., Ltd. Dangyao xanthophyll (CAS NO: 6991-10-2) was purchased from Chengdu Dester Biotechnology Co., Ltd.

[0033] The application of the flavin provided by the present invention in the preparation of a drug for treating infectious pneumonia caused by methicillin-resistant Staphylococcus aureus is described in detail below with reference to embodiments.

[0034] Example 1: Determination of the half-maximal inhibitory concentration (IC50) 50 )value

[0035] (1) Purification of ClpP protein

[0036] BL21-pET28a- clpP The strain was inoculated into LB liquid medium containing 50 μg / mL kanamycin and incubated overnight at 37°C. The next day, it was re-inoculated at a 1:100 ratio into LB medium containing 50 μg / mL kanamycin resistance and incubated at 37°C and 200 rpm until OD500. 600 The concentration was increased to 0.8. Isopropyl thiogalactoside (IPTG) was added to the bacterial culture to a final concentration of 0.25 mM, and the culture was induced overnight at 16°C and 180 rpm. The next day, the bacterial cells were collected by centrifugation and resuspended in protein buffer (40 mM Tris, pH = 8.5). The resuspended cells were then sonicated, and the supernatant was collected by centrifugation (10,000 rpm, 50 minutes). Finally, the His-tagged ClpP protein was isolated and purified using Ni-NTA affinity chromatography.

[0037] (2) Determination of ClpP activity

[0038] The activity of ClpP was determined by fluorescence resonance energy transfer (FRET). The experiment was performed in 96-well black plates. The substrate peptide Suc-LY-AMC (0.5 mg / mL) was used as the substrate for internal fluorescence quenching. Each well contained 10 μM of purified ClpP protein, different concentrations (final concentrations of 0, 4, 8, 16, 21, 64, 128, and 256 μg / mL) of dextrin, and reaction buffer, with a final volume of 99 μL. The reaction was incubated at 37°C for 30 min. Then, 1 μL of the substrate peptide was added, and the reaction was incubated at 37°C for 30 min. Finally, the fluorescence signal was read using a microplate reader (excitation wavelength 360 nm, emission wavelength 465 nm). A control group containing only buffer and substrate peptide was used. The inhibition rate of the drug on ClpP protein activity was determined by the following formula: 100% × (CT) / C, where C is the fluorescence value of the untreated group and T is the fluorescence value of the experimental group.

[0039] The results are as follows Figure 2 As shown, when azadirachtin significantly inhibits the activity of ClpP protein, its fluorescence signal decreases in a dose-dependent manner. Nonlinear regression analysis was performed using GraphPad Prism software, and curve fitting was performed on the experimental data to calculate the IC50 of azadirachtin. 50 The value was 32.91 μg / mL.

[0040] Example 2: Evaluation of Staphylococcus aureus growth

[0041] To assess whether 128 μg / mL chondromycin would affect the growth of MRSA (USA300 strain), the present invention involves adding overnight cultured USA300 to sterile TSB medium with or without 128 μg / mL chondromycin at a ratio of 1:100 and growing at 37°C with shaking for 24 hours. The group with 128 μg / mL chondromycin was designated as the observation group (USA300 + chondromycin (128 μg / mL) group), and the group without chondromycin was designated as the control group (USA300 group). The absorbance at 600 nm was measured using a UV spectrophotometer at 0, 1, 2, 3, 4, 6, 8, 10, 12, and 24 hours, and growth curves were plotted.

[0042] The results are as follows Figure 3 As shown, the growth rate of USA300 treated with 128 μg / mL quercetin (USA300 + quercetin (128 μg / mL) group) was similar to that of the untreated USA300 group. The results indicate that 128 μg / mL quercetin does not affect the growth of USA300, and treatment of Staphylococcus aureus with 128 μg / mL quercetin is unlikely to induce drug resistance.

[0043] Example 3 Determination of Minimum Inhibitory Concentration (MIC)

[0044] The MICs of different concentrations of daunoflavin (final concentrations from 0 to 1024 μg / mL) against USA300 were determined using the micro-broth dilution method to evaluate its antibacterial activity. The specific steps were as follows: USA300 was revived using TSB medium. The next day, it was inoculated into CAMHB medium at a ratio of 1:1000 and incubated at 37°C until OD... 600 The value was 0.8. The bacterial suspension was then diluted 1:100 in CAMHB medium and inoculated into sterile 96-well plates. The 96-well plates were incubated at 37°C for 16 hours. The USA300 group without scopolamine treatment served as the negative control group. The lowest drug concentration at which scopolamine inhibited bacterial growth at a statistically significant level compared to the negative control group was defined as its minimum inhibitory concentration (MIC).

[0045] The results are as follows Figure 4 As shown, the MIC value of flavin against Staphylococcus aureus USA300 is 512 μg / mL.

[0046] Example 4: Evaluation of the cytotoxicity of flavin to A549 cells

[0047] The potential toxicity of dextrin to human lung epithelial cell line (A549, CAS NO: CL-0016, Wuhan Pronosei Biotechnology Co., Ltd.) was assessed using the MTT assay. The dextrin, 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (trade name: thiazolyl blue), is widely used to assess cell viability. A549 cells in logarithmic growth phase were cultured at 5 × 10⁻⁶ cells / cells. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured for 24 hours. Then, scutellarin (final concentration 0-128 μg / mL) was added to each well, and the plates were cultured for another 24 hours. After culture, the culture medium was removed, and 100 µL of medium and 50 µg of MTT solution were added to each well, followed by incubation for 4 hours. After incubation, the resulting purple crystalline MTT metabolite was dissolved in 200 µL of dimethyl sulfoxide (DMSO), and cell viability was quantified by measuring absorbance at 570 nm.

[0048] The results are as follows Figure 5 As shown, the viability of A549 cells treated with daunoflavin did not change significantly, indicating that daunoflavin had no obvious toxic effect on A549 cells within the concentration range tested in the experiment.

[0049] Example 5: When flavin inhibits MRSA invasion of A549 cells

[0050] The experiment was divided into four groups: control group, USA300 infection group, duracil (128 μg / mL) treatment group, and USA300- dclpP Infection group. In short, A549 cells were infected at a rate of 1×10⁻⁶. 5 Cells were carefully seeded at a density of 100 cells / well in 24-well plates and cultured for 24 hours in F-12K medium containing fetal bovine serum, streptomycin, and penicillin. The USA300 infection group and the scopolamine treatment group were treated with 0 μg / mL and 128 μg / mL scopolamine, respectively, and cultured at 37°C with shaking at 220 rpm until the OD of the bacterial culture reached 100%. 600 The value reaches 1.0. Repeat the process in the same way. USA300-dclpP strain cultured to OD 600 The value reached 1.0. After culture, bacteria were collected by centrifugation and resuspended in F-12K medium. Except for the control group, which was directly given 500 μL of F-12K medium, the other groups were given 500 μL of their corresponding bacterial suspension (1×10⁻⁶). 5 CFU / mL of gentamicin was added to each well of a 24-well plate containing A549 cells, and the cells were incubated at 37°C for 2 hours to allow bacterial invasion. After invasion, the cells were washed with PBS to remove uninvaded bacteria, followed by incubation for 1 hour in F-12K medium containing gentamicin (300 μg / mL) to remove extracellular Staphylococcus aureus. The cells were then washed twice with PBS, and finally lysed using 5% Triton X-100. The cell lysate was spread on TSB solid medium containing 5% defibrinated rabbit blood and incubated overnight. The number of colonies formed was observed to assess the number of bacteria invading the A549 cells, and the results were photographed and recorded.

[0051] The results are as follows Figure 6 As shown, no bacteria were detected in A549 cells in the control group. In the untreated USA300 infection group, the number of bacteria within A549 cells was significantly increased, indicating that Staphylococcus aureus had successfully invaded the A549 cells. Compared to the untreated USA300 infection group, the number of bacteria within A549 cells in the USA300 infection group treated with quercetin was significantly reduced. Furthermore, compared to the untreated USA300 infection group, the number of bacteria within A549 cells in the USA300-... dclpP The infection group showed the lowest number of bacteria invading A549 cells. These results indicate that flavoxate significantly inhibits the invasion of MRSA into A549 cells, with an effect similar to that of inhibiting the virulence factor ClpP.

[0052] Example 6: Experimental therapeutic study of flavin on MRSA-infected pneumonia

[0053] A pneumonia infection model was established using 6- to 8-week-old SPF-grade C57BL / 6J male mice. The specific steps were as follows: Mice were anesthetized with isoflurane. Then, 30 μL of the prepared bacterial suspension was inoculated into the left nasal cavity of the mouse. The mouse was kept upright for 30 seconds to allow for sufficient inhalation of the bacterial suspension, and then laid flat to allow for natural awakening. In the survival rate assessment experiment, 30 μL of Staphylococcus aureus resuspension (concentration 2 × 10⁻⁶) was administered intranasally to the mice. 8 CFU); In the histopathological evaluation experiment, 30 μL of Staphylococcus aureus resuspension (concentration of 1×10⁻⁶) was inoculated intranasally. 8 Two hours later, the infected mice were subcutaneously injected with 50 mg / kg of scopolamine or the same volume of DMSO, twice daily.

[0054] (1) Pathological tissue examination

[0055] Mice were randomly divided into four groups (control group, USA300 infection group, senna-flavin (50 mg / kg) group, and USA300- dclpP (Groups, 10 animals per group). Except for the control group, which received an equal volume of PBS, the USA300 infection group and the roximate (50 mg / kg) group received OD... 600 USA300 bacterial suspension with a value of 1.0, USA300- dclpP Group gives OD 600 USA300 with a value of 1.0 dclpP Bacterial liquid (USA300- dclpP The strain used was USA300 with the clpP gene knocked down. Mice were euthanized 24 hours after receiving quercetin treatment. Left lung tissue was harvested from the mice, and the appearance of the lung tissue was observed and images recorded. The tissue was then fixed with 4% paraformaldehyde to prepare histopathological sections, which were stained with hematoxylin and eosin (H&E). Finally, the lung tissue was observed and images were acquired using an optical microscope for subsequent analysis.

[0056] (2) Survival rate experiment

[0057] The grouping and treatment methods for this part of the experiment were the same as those for "Pathological Tissue Examination". Mice mortality was observed and recorded every 12 hours, and survival rate was calculated after 96 hours.

[0058] The protective effect of flavoxate on lung tissue of mice with Staphylococcus aureus USA300-induced infectious pneumonia was as follows: Figure 7As shown, the lungs of uninfected mice (control group) appeared pale pink and spongy; while the lungs of mice in the USA300 infected group were dark red and showed obvious congestion. Compared with the untreated infected group (USA300 infected group), the lung tissue congestion of the digoxin treatment group (digoxin (50 mg / kg) group) was improved.

[0059] Pathological section results as follows Figure 7 As shown, pathological sections of the lungs of mice in the USA300 infection group revealed a large accumulation of inflammatory cells in the alveolar cavities; compared with the untreated infection group (USA300 infection group), the inflammatory cell infiltration in the alveolar cavities of mice in the difugoxin treatment group (difugoxin (50 mg / kg) group) was significantly reduced. These results fully demonstrate that difugoxin can alleviate lung damage in USA300 infected mice.

[0060] When flavin was used to treat Staphylococcus aureus USA300-induced infectious pneumonia in mice, the results were as follows: Figure 8 As shown, the survival rate of mice in the USA300-infected group was only 10% at 96 hours. dclpP The survival rate of mice in the infected group was 70%. Compared with the USA300 infected group, the survival rate of mice treated with 50 mg / kg quercetin for 96 hours increased to 50%, with the same effect as USA300. dclpP The infection group was similar. These results strongly confirm that flavin can significantly improve the survival rate of mice infected with Staphylococcus aureus.

[0061] In summary, this invention validated the efficacy of dandroflavin using a mouse model of MRSA-infected pneumonia. Experimental results show that dandroflavin has significant therapeutic potential in the mouse model of MRSA-infected pneumonia, mainly in two aspects: firstly, it significantly improves the survival rate of MRSA-infected mice; secondly, it effectively reduces the inflammatory response in lung tissue induced by MRSA infection. These findings provide strong experimental evidence for dandroflavin as a potential therapeutic agent against MRSA-infected pneumonia. Furthermore, this invention elucidates the mechanism of action of dandroflavin, such as... Figure 9As shown, daunorubicin can inhibit the activity of casein hydrolase ClpP, a key virulence factor in MRSA, effectively reducing the virulence of MRSA and exerting a therapeutic effect. Daunorubicin not only reduces the degree of lung tissue damage but also reduces the infiltration and aggregation of inflammatory cells in the lungs. Importantly, compared with conventional antibiotic treatment, daunorubicin does not exert survival pressure on Staphylococcus aureus, thus not tending to promote the occurrence and development of bacterial resistance. Therefore, this invention provides a novel treatment strategy, namely, using daunorubicin as the active ingredient in the preparation of drugs for treating Staphylococcus aureus pneumonia. The daunorubicin disclosed in this invention has potential application value in the development of safe and effective drugs for treating Staphylococcus aureus pneumonia and is expected to provide new options for clinical treatment.

[0062] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The application of aflatoxin in the preparation of drugs for treating Staphylococcus aureus infections, characterized in that, The Staphylococcus aureus mentioned is methicillin-resistant Staphylococcus aureus.

2. The application according to claim 1, characterized in that, The methicillin-resistant Staphylococcus aureus is Staphylococcus aureus USA300.

3. The application according to claim 1, characterized in that, The drug is used to treat infectious pneumonia.

4. The application according to claim 1, characterized in that, The drug is used to treat pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA).

5. The application of flavin in the preparation of drugs that inhibit the activity of Staphylococcus aureus casein hydrolase ClpP, characterized in that, The Staphylococcus aureus mentioned is methicillin-resistant Staphylococcus aureus.

6. The application according to any one of claims 1-5, characterized in that, The drug reduces the toxicity of MRSA by inhibiting the activity of casein hydrolase ClpP, and the drug is prepared in a pharmaceutically acceptable dosage form from dioxin as the active ingredient and pharmaceutically acceptable excipients.

7. The application according to any one of claims 1-5, characterized in that, The dosage forms of the drug include, but are not limited to, tablets, capsules, and injections.

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