Medical use of peganum harmala alkaloids in the preparation of inhibitors of tmexcd1-toprj1-mediated efflux pumps

Abstract

The present application relates to the application of peganum harmala in the preparation of tmexCD1-toprJ1 mediated efflux pump inhibitors, and the ability of peganum harmala to destroy the function of bacterial efflux pump is verified through chessboard test, growth curve test, bactericidal curve test, EtBr efflux test and mouse Klebsiella pneumoniae infection model, and good anti tmexCD1-toprJ1 positive Klebsiella pneumoniae infection effect is embodied in the in vivo model. This provides a new way and potential lead compound for the prevention and treatment of Klebsiella pneumoniae infection.

Description

Technical Field

[0001] This invention discloses a new use of camel pine resin, and further relates to the medical use of camel pine resin in the preparation of tmexCD1-toprJ1-mediated efflux pump inhibitors, belonging to the field of medical and pharmaceutical technology. Background Technology

[0002] Klebsiella pneumoniae (K. pneumoniae) is one of the most common and challenging zoonotic pathogens causing hospital- and community-acquired infections such as pneumonia, sepsis, and liver abscesses this century. In recent years, due to the long-term irrational use of antibiotics, the pathogenicity of multidrug-resistant (MDR) Klebsiella pneumoniae has been increasing year by year, seriously threatening the health of humans and animals. Tigecycline resistance has been increasingly observed in Klebsiella pneumoniae and has attracted significant attention. The tmexCD1-toprJ1 gene cluster, carried by plasmids from Klebsiella pneumoniae isolated from chickens and patients in my country in recent years, is a novel drug-resistance-nodulation-mitosis (RND) efflux pump gene cluster that can cause multidrug resistance, including to tigecycline.

[0003] Antibiotic adjuvant strategies are cost-effective and promising approaches to prolonging the lifespan of existing antibiotics by inhibiting bacterial resistance or enhancing their cytotoxic effects. In recent years, combination therapies based on tigecycline with other antibiotics have been widely used, but their therapeutic efficacy has been unsatisfactory. Unlike antibiotics, non-antibiotic adjuvant strategies can maximize the antibacterial efficacy of existing antibiotics by enhancing their cytotoxic effects, offering a promising approach to combating antibiotic resistance. To date, some studies on non-antibiotic adjuvants of tigecycline have been reported. Research has shown that the mechanism of tigecycline resistance in tmexCD1-toprJ1-positive bacteria involves the efflux pump on the bacterial membrane, thereby reducing tigecycline accumulation within the bacteria. Therefore, inhibiting the function of the efflux pump in tmexCD1-toprJ1-positive bacteria provides a new avenue for the prevention and treatment of tmexCD1-toprJ1-positive bacterial infections and the discovery of potential lead compounds. Summary of the Invention

[0004] This invention provides a medical use of camel pine in the preparation of tmexCD1-toprJ1-mediated efflux pump inhibitors, discloses the function of camel pine in inhibiting bacterial efflux pumps, and provides a new target drug for the prevention and treatment of tmexCD1-toprJ1-positive Klebsiella pneumoniae infection.

[0005] The camel pine resin described in this invention has the molecular formula: C 13 H 14 N2O, molecular weight: 214.27.

[0006] This invention verifies that Camel Prunil can disrupt the function of bacterial efflux pumps through checkerboard test, growth curve test, bactericidal curve test, EtBr efflux test, and mouse Klebsiella pneumoniae infection model test, and demonstrates good anti-tmexCD1-toprJ1 positive Klebsiella pneumoniae infection effect in the in vivo model. The Klebsiella pneumoniae used in the experiment is Klebsiella pneumoniae T2.

[0007] The positive effects of this invention are as follows:

[0008] This study provides a novel medical use of camel pamoate in the preparation of tmexCD1-toprJ1-mediated efflux pump inhibitors, and discloses the function of camel pamoate in inhibiting bacterial efflux pumps, providing a new approach for the prevention and treatment of tmexCD1-toprJ1-positive bacterial infections and the discovery of potential lead compounds. Attached Figure Description

[0009] Figure 1 This is a checkerboard test diagram of the combination of K. pneumoniae T2 and tigecycline in camel pine resin according to the present invention.

[0010] Figure 2 This is a growth curve diagram of K. pneumoniae T2 on camel pine.

[0011] Figure 3 This is a bactericidal curve of K. pneumoniae T2 against camel pine sulfate and tigecycline, alone or in combination, according to the present invention.

[0012] Figure 4 This is a bar chart comparing the fluorescence intensity at 530 / 600 nm for different concentrations of camel pine resin treated according to the present invention.

[0013] Figure 5 This is a bar chart comparing the fluorescence intensity of different concentrations of camel pine and tigecycline, alone or in combination, at 530 / 600 nm.

[0014] Figure 6 This is a comparison of survival rates in the tmexCD1-toprJ1-positive K. pneumoniae T2-infected mouse model, including the infection group, the camel pine pine treatment group alone, the tigecycline treatment group alone, and the combined treatment group of camel pine pine and tigecycline. Detailed Implementation

[0015] The present invention is further illustrated by the following embodiments, but is not intended to limit the invention in any way. Any modifications or alterations made to the present invention that are easily implemented by those skilled in the art without departing from the technical solutions of the present invention shall fall within the scope of the claims of the present invention.

[0016] Example 1

[0017] Vasicine can be used as a tmexCD1-toprJ1-mediated efflux pump inhibitor in any pharmaceutically acceptable carrier.

[0018] Example 2

[0019] Vasicine can be used as a tmexCD1-toprJ1-mediated efflux pump inhibitor for preparing a medicament for treating infectious diseases.

[0020] Example 3

[0021] Vasicine can be used for treating bacterial infectious diseases.

[0022] Test Example 1

[0023] Determination of FIC (fractional inhibitory concentration) value of vasicine in combination with tigecycline against K. pneumoniae T2

[0024] The synergistic activity and fractional inhibitory concentration (FIC) values of vasicine and tigecycline were tested by the checkerboard method. The two preparations were mixed in a 96-well plate to form an 8×8 matrix, followed by adding 1 μL of OD 600nm = 0.1 bacterial suspension to a final concentration of 5×10 5 CFU / mL. The experiment was independently repeated 5 times. After the 96-well plate was incubated in a 37°C incubator for 18 hours, the results were recorded visually. The FIC index (FICI) is expressed by the following formula: FICI = FIC1 + FIC2 = MIC12 / MIC1 + MIC21 / MIC2; FIC1 and FIC2 are the FIC indices of drug 1 and drug 2, respectively; MIC1 and MIC2 are the MIC values of drug 1 and drug 2, respectively, and MIC12 and MIC21 are the MIC values of one drug in combination with the other drug. If FICI ≤ 0.5, the combination is defined as synergistic; if 0.5 < FICI ≤ 4, it is non-synergistic; if FICI > 4, it is antagonistic.

[0025] Conclusion: The FIC of vasicine and tigecycline against K. pneumoniae T2 is 0.425, and the combination of vasicine and tigecycline has a synergistic effect, see Figure 1 .

[0026] Test Example 2

[0027] Determination of growth curve

[0028] Pick a single colony of *K. pneumoniae* T2 and inoculate it into a test tube containing 3 mL of LB broth. Incubate overnight at 37°C with shaking at 180 rpm. The overnight culture is then amplified 1:100 in a 200 mL Erlenmeyer flask containing 150 mL of fresh MHB broth until OD is reached. 600nm Reaching 0.3. OD 600nm The bacterial suspension with a pH of 0.3 was poured into three 50 mL Erlenmeyer flasks, each containing 20 mL, after autoclaving and drying. Camelapordin was added to the flasks at concentrations of 0, 64 μg / mL, and 128 μg / mL, respectively. The OD values ​​were measured using a spectrophotometer every half hour. 600nm The data were recorded at 37℃ for 6 hours, and a growth curve was generated. The experiment was performed in triplicate, with independent replicates.

[0029] Conclusion: Treatment of K. pneumoniae T2 with 64 μg / mL and 128 μg / mL camel carmine had almost no effect on bacterial growth. Figure 2 .

[0030] Experimental Example 3

[0031] Sterilization curve determination

[0032] A single *K. pneumoniae* T2 colony was selected and inoculated into a test tube containing 3 mL of LB medium. The colonies were incubated overnight at 37°C and 180 rpm using a shaker. In a 96-well plate, 128 μg / mL of *Camelumbellatus*, 4 μg / mL of tigecycline, or a combination thereof were added to each well, with a total volume of 200 μL. At the start of the experiment, 5 × 10⁶ cells / well were inoculated per well. 5 K. pneumoniae T2 at CFU / mL was cultured in 96-well plates at 37°C without shaking and spread onto LB agar plates at 0, 1, 3, 7, 10, 12, and 24 hours. Single colony counts were calculated on each plate the following day. Triple replicates were performed independently. Inhibition was defined as no significant change over 24 hours; synergism was defined as a combined reduction of ≥2 log₂C / mL relative to the most potent single active ingredient. 10 CFU / mL.

[0033] Conclusion: Treatment with camel pine or tigecycline alone did not inhibit bacterial growth, but combined treatment with camel pine and tigecycline significantly increased bacterial growth. 10 The significant reduction in CFU indicates that camel pine resin is an adjuvant that enhances the efficacy of tigecycline. (See...) Figure 3 .

[0034] Test Example 4

[0035] Ethidium bromide efflux test

[0036] To evaluate the inhibitory effect of camel embrittlement on the multidrug efflux pump, we conducted an ethidium bromide (EtBr) efflux assay based on previous studies. The samples were washed with PBS during the logarithmic growth phase (OD). 600nm =0.6) bacteria were extracted three times. The extracts were aliquoted into 1.5 mL centrifuge tubes, 1 mL per tube. Different concentrations of camel pine sulfate, tigecycline, and their combinations were added to the bacterial culture, and the mixtures were incubated at 37°C for 1 hour. 10 μM EtBr was added, and the mixture was incubated at 37°C for 1 hour. Subsequently, the efflux of EtBr from the cells was monitored using an excitation wavelength of 530 nm and an emission wavelength of 600 nm. The experiment was performed in triplicate, with independent replicates.

[0037] Conclusion: Treatment with camel pine alone significantly increased fluorescence intensity. When camel pine and tigecycline were used in combination, the fluorescence intensity increased significantly in a dose-dependent manner compared to tigecycline alone. (See [link to article]). Figure 4 , 5 .

[0038] Experimental Example 5

[0039] Mouse pneumonia infection model experiment

[0040] To conduct survival analysis, mice were infected with a dose of 1.6 × 10⁻⁶ via nasal instillation. 10 CFUs were synthesized from K. pneumoniae T2. Mice were then randomly assigned to four groups (n=10 per group): infection group, tigecycline monotherapy group (10 mg / kg), camel pine monotherapy group (20 mg / kg), and tigecycline combined with camel pine monotherapy group (10 + 20 mg / kg). Camel pine monotherapy was administered by gavage, and tigecycline was administered by subcutaneous injection, every 12 hours. Mice survival was monitored for 7 consecutive days.

[0041] Conclusion: The survival rate in the combination therapy group of camel pine tincture and tigecycline was significantly higher than that in the other three groups. In vivo results strongly demonstrate the adjuvant potential of camel pine tincture and tigecycline in combating infectious diseases caused by tmexCD1-toprJ1 positive pathogens. See Figure 6 .

Claims

1. The medical use of camel pine in the preparation of tmexCD1-toprJ1-mediated efflux pump inhibitors, characterized in that, The medical use refers to the preparation of drugs for treating infections caused by tmexCD1-toprJ1 positive Klebsiella pneumoniae.

2. The medical use of Camel Prunil according to claim 1 in the preparation of tmexCD1-toprJ1-mediated efflux pump inhibitors, characterized in that, The tmexCD1-toprJ1-mediated efflux pump inhibitor reduces bacterial resistance to tigecycline by inhibiting bacterial efflux pump function.

3. The medical use of camel pine resin according to claim 1 in the preparation of tmexCD1-toprJ1-mediated efflux pump inhibitors, characterized in that, The drug, used in combination with tigecycline, is used to prepare a composition for treating infections caused by tmexCD1-toprJ1-positive Klebsiella pneumoniae.

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