Application of cacumen platycladi ketone in preparation of gentamicin antibacterial synergist

Abstract

The invention discloses an application of cacumen platycladi ketone in preparation of a gentamicin antibacterial synergist. Through a large number of experimental researches, the inventor accidentally finds that the cacumen platycladi ketone can effectively improve the antibacterial effect of gentamicin on gram-positive bacteria separated from clinical cow mastitis. The discovery provides a brand new direction for improving the antibacterial property of gentamicin, and is expected to play an important role in clinical treatment of cow mastitis.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and particularly relates to the application of cedarone in the preparation of a gentamicin antibacterial synergist. Background Art

[0002] The growing global threat of bacterial resistance urgently calls for the development of new antibiotics. However, the research and development of new antibiotics faces numerous obstacles, with increasingly lengthy development cycles and increasing difficulty. In this challenging environment, exploring methods to improve the antimicrobial activity of existing drugs, broaden their antimicrobial spectrum, or enhance their efficacy has become a proven solution in disease prevention and control.

[0003] Nootkatone, also known as (+)-Nootkatone, possesses unique aromatic properties and is commonly used as a component of flavors and fragrances. Recent studies have demonstrated a wide range of biological activities, including anti-Alzheimer's disease, anti-cell proliferation, and anti-platelet aggregation. In particular, its structural similarity to glutamate suggests that it may interfere with neurotransmission by modulating GABA receptors (such as the Rdl channel in insects), potentially exhibiting potential insecticidal activity. Therefore, existing research has focused on the insecticidal properties of nootkatone and its derivatives. Patent CN111620837A details the use of a modified nootkatone thiazole amide compound for the treatment of ectoparasites.

[0004] Gentamicin (GM) is a commonly used aminoglycoside antibiotic that plays a certain role in clinical treatment. However, it is not a suitable antibiotic for the treatment of clinical Staphylococcus aureus ( Staphylococcus aureus , S. aureus ) infection, its antibacterial efficacy is 5-10% lower than that of cephalosporins. The development of a gentamicin synergist would enhance gentamicin's antibacterial efficacy against Gram-positive bacteria and provide more clinical options. It is worth noting that there are currently no reports of cedarone acting as a gentamicin synergist. Summary of the Invention

[0005] To address the technical issue of gentamicin's poor antibacterial efficacy against Gram-positive bacteria, the inventors, after extensive experimental research, unexpectedly discovered that cedarone can effectively enhance gentamicin's antibacterial efficacy against Gram-positive bacteria isolated from clinically diagnosed dairy cows with mastitis. This discovery provides a new direction for improving gentamicin's antibacterial properties and is expected to play a significant role in the clinical treatment of dairy cow mastitis.

[0006] The first aspect of the present invention provides the use of buciperone or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial enhancer for enhancing the efficacy of gentamicin in resisting bacterial infections.

[0007] The second aspect of the present invention provides the use of bucubitrone or a pharmaceutically acceptable salt thereof in the preparation of a medicament for resisting bacterial infectious diseases.

[0008] The third aspect of the present invention provides an antibacterial composition.

[0009] The antibacterial composition comprises humulone or a pharmaceutically acceptable salt thereof and gentamicin or a pharmaceutically acceptable salt thereof.

[0010] Furthermore, the mass ratio of the cinnamaldehyde or a pharmaceutically acceptable salt thereof to gentamicin or a pharmaceutically acceptable salt thereof is (4-128):1, and can further be (4-64):1, (4-16):1, (8-32):1 or (32-128):1, specifically 4:1, 8:1, 16:1, 32:1, 64:1 or 128:1.

[0011] For example, for anti-Staphylococcus aureus ( Staphylococcus aureus,S. aureus ) (such as cow-derived Staphylococcus aureus), the mass ratio of the cinquefoil or its pharmaceutically acceptable salt to gentamicin or its pharmaceutically acceptable salt is preferably (4-64):1 or (4-16):1.

[0012] For example, for anti-Streptococcus agalactiae ( Streptococcus agalactiae , S. agalactiae ) (such as cow-derived Streptococcus agalactiae), the mass ratio of the cedarone or its pharmaceutically acceptable salt to gentamicin or its pharmaceutically acceptable salt is preferably (8~32):1.

[0013] For example, for anti-Staphylococcus epidermidis ( Staphylococcus epidermidis , S. epidermidis ) (such as Staphylococcus aureus from dairy cows), the mass ratio of the cedarone or its pharmaceutically acceptable salt to gentamicin or its pharmaceutically acceptable salt is preferably 32:1.

[0014] For example, for xylose-resistant Staphylococcus Staphylococcus xylosus , S. xylosus ) (such as Staphylococcus xylosus), the mass ratio of the bubenone or a pharmaceutically acceptable salt thereof to gentamicin or a pharmaceutically acceptable salt thereof is preferably 128:1.

[0015] The antibacterial composition can be used to fight bacterial infectious diseases.

[0016] Furthermore, the antibacterial composition can be used to combat bacterial animal mastitis.

[0017] A fourth aspect of the present invention provides an antimicrobial product.

[0018] The antibacterial product contains the above antibacterial composition and a pharmaceutically acceptable carrier.

[0019] The dosage form of the antibacterial product can be selected from any one of the following: tablets, capsules, syrups, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, injection solutions, and freeze-dried powder injections.

[0020] Pharmaceutically acceptable carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinyl pyrrolidone, and organic acids), poorly soluble carrier materials (such as ethyl cellulose and cholesterol stearate), and enteric-soluble carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose). These materials can be used to formulate the antibacterial products in a variety of dosage forms, including but not limited to tablets, capsules, syrups, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, buccal tablets, and lyophilized powder injections. These formulations can include standard preparations, sustained-release preparations, controlled-release preparations, and various microparticle delivery systems.

[0021] When the composition provided by the present invention is used to prevent and / or treat bacterial infection, an effective amount of the antibacterial composition is administered to the subject.

[0022] The dosage and method of use of the antibacterial composition of the present invention depend on many factors, including the patient's age, weight, sex, natural health status, nutritional status, activity strength of the compound, time of administration, metabolic rate, severity of the disease, and the subjective judgment of the treating physician.

[0023] In the present invention, the term "effective amount" refers to a dose that can achieve the treatment, prevention, alleviation and / or relief of the diseases or conditions described in the present invention in a subject.

[0024] In the present invention, the term "subject" may refer to a patient or other animal that receives the antibacterial composition of the present invention to treat, prevent, alleviate and / or relieve the diseases or conditions described in the present invention, such as cattle, sheep, pigs, poultry, dogs, cats, etc.

[0025] In the present invention, the CAS No. of the chalcogenol is 4674-50-4, and its structural formula is shown in Formula I:

[0026] In the present invention, the bacteria include Gram-positive bacteria and Gram-negative bacteria.

[0027] Furthermore, the bacteria are Gram-positive bacteria, such as Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis, Staphylococcus xylosus, etc.

[0028] Furthermore, the bacteria are bacteria that cause animal mastitis.

[0029] Furthermore, the bacteria are Gram-positive bacteria that cause animal mastitis.

[0030] According to an embodiment of the present invention, the animal mastitis is cow mastitis.

[0031] According to an embodiment of the present invention, the Gram-positive bacteria is Staphylococcus aureus. The Staphylococcus aureus includes S. aureus Standard strains and S. aureus Clinical isolates (i.e., Staphylococcus aureus from clinical dairy cow mastitis).

[0032] According to an embodiment of the present invention, the Gram-positive bacteria is Streptococcus agalactiae. The Streptococcus agalactiae includes S. agalactiae Standard strains and S. agalactiae Clinical isolates (i.e., Streptococcus agalactiae from clinical dairy cow mastitis).

[0033] According to an embodiment of the present invention, the Gram-positive bacteria is Staphylococcus epidermidis. The Staphylococcus epidermidis includes S. epidermidis Standard strains and S. epidermidis Clinical isolates (i.e., Staphylococcus epidermidis from clinical dairy cow mastitis).

[0034] According to an embodiment of the present invention, the Gram-positive bacteria is Staphylococcus xylosus. The Staphylococcus xylosus includes S. xylosus Standard strains and S. xylosus Clinical isolates (i.e., Staphylococcus xylosus from clinical dairy cow mastitis).

[0035] Furthermore, the anti-bacterial infectious disease is anti-bacterial animal mastitis.

[0036] According to an embodiment of the present invention, the anti-bacterial animal mastitis is anti-bacterial dairy cow mastitis, especially anti-cow mastitis caused by Staphylococcus aureus and / or Streptococcus agalactiae and / or Staphylococcus epidermidis and / or Staphylococcus xylosus.

[0037] Compared with the prior art, the present invention has the following beneficial technical effects: 1. Antibacterial synergistic effect. Cinnamaldehyde can synergize with gentamicin in antibacterial effect by inhibiting the production of bacterial hemolysin and reducing bacterial adhesion to host cells, significantly enhancing the antibacterial activity of gentamicin against Gram-positive bacteria.

[0038] 2. Expand the antibacterial spectrum. In clinical treatment, aminoglycosides are mainly used to treat Gram-negative bacteria. Through antibacterial enhancement, gentamicin expands the sensitivity to Gram-positive bacteria and improves the success rate of treatment.

[0039] 3. Reduce antibiotic usage. Through the combined use of antimicrobial enhancers, the clinical treatment dosage of gentamicin can be reduced by 50% to 80%.

[0040] 4. Reduce the risk of drug side effects.

[0041] 5. Delaying drug resistance: Shifting from a single drug target to multiple targets can further reduce the risk of clinical bacterial resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The results were as follows: S. aureus Growth curve of clinical bacterial isolate SABN27.

[0043] Figure 2 The results show the bacterial culture in host cells under the action of different concentrations of cedarone in Example 4.

[0044] Figure 3 The figure shows the effect of different concentrations of cedarone on the bacterial invasion rate in Example 4. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0046] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0047] Example 1: MIC determination of cedarone against clinical isolates from dairy cows Isolation, identification and preservation of clinical isolates: Milk samples were collected from cows with mastitis at a large-scale dairy farm in Beijing. Traditional bacterial culture methods were used in the laboratory to enrich, culture and purify the bacteria. The identification of clinical isolates was completed through a combination of bacterial biochemical identification and Sanger sequencing. Cow source S. aureus SABN27, dairy cow source S. aureus SABN29, dairy cow sourceS. aureus SABN145, dairy cow source S. aureus SABN-78 is Staphylococcus aureus; Cow source S. agalactiae HB27, dairy cow source S. agalactiae HB31 is Streptococcus agalactiae; Cow source S. epidermidis EB15 is Staphylococcus epidermidis; Cow source S. xylosus XB16 is Staphylococcus xylosus.

[0048] The standard quality control strains are: S. aureus ATCC 29213, S. agalactiae ATCC 13813.

[0049] The single colonies of the 8 clinical isolates from dairy cows and 2 standard quality control strains were inoculated into sterile MH broth and cultured for 12 h. The cultured bacterial solution was diluted to 10 5 CFU / mL. Prepare 12 2mL centrifuge tubes and sequentially add 0.9mL of MH broth and 100µL of 8.0mg / mL cedarwood solution to each tube. Add 500µL of MH broth to tubes 2-12. Use a micropipette to aspirate 500µL of the mixture from the first column. Then, dilute the mixture 2-fold from the first tube to the last. Finally, add 500µL of bacterial solution to each tube. For the positive control well, add only MH broth and bacterial solution, and for the negative control well, add only MH broth. Each concentration is repeated in triplicate. Finally, incubate in a 37°C incubator for 18-24 hours. Calculate the MIC value for each strain.

[0050] The results showed that cedarone had an S. aureus ATCC 29213, S. agalactiae ATCC 13813, dairy cow source S. aureus SABN27, dairy cow source S. aureus SABN29, dairy cow source S. aureus SABN145, dairy cow source S. aureus SABN78, dairy cow source S. agalactiae HB27, dairy cow source S. agalactiae HB31, dairy cow source S. epidermidis EB15, dairy cow source S. xylosus The MICs of eight clinically isolated Gram-positive bacteria, including XB16, were all greater than 1000 μg / mL, indicating that the cedarone monomer had little antibacterial activity.

[0051] Example 2: Cow Source S. aureus Study on the growth curve of SABN27 bacteria Pick the cow source used in Example 1 S. aureus A single colony of SABN27 was cultured in 4 ml of MH broth and placed in a 37°C incubator with shaking overnight. The bacterial solution was diluted to 0.5 μg / ml using a McFadden turbidimetric tube and then diluted 1:100 to 10 μg / ml. 6 The CFU / mL was set aside. Different groups were set up: a combination group (32 μg / mL of cedarwood ketone and 0.5 μg / mL of gentamicin), a cedarwood ketone group (32 μg / mL), a gentamicin group (0.5 μg / mL), and a SABN27 group. Subsequently, 200 μL of samples were collected from a 96-well plate at 0, 1, 2, 4, 6, 8, 10, 12, and 24 hours, and the OD600 values ​​were measured and recorded. During growth curve measurement, the incubation temperature and rotation speed should be consistent across all groups, and samples should be mixed thoroughly to ensure uniform bacterial concentration.

[0052] The results are as follows Figure 1 As shown in the results: Compared with the SABN27 group, the cedarone group did not affect the growth of Staphylococcus aureus and entered the exponential growth phase at 4 hours, while the gentamicin group entered the exponential growth phase at 6 hours. Both groups did not inhibit the growth of Staphylococcus aureus in the exponential and plateau phases, while the cedarone + gentamicin group could effectively inhibit the colony count of Staphylococcus aureus in the exponential phase, and its antibacterial effect was significantly better than that of the cedarone or gentamicin group alone.

[0053] Example 3: Cinnamaldehyde to cow-derived S. aureus Hemolytic inhibition test of SABN27 Take fresh defibrinated rabbit blood and centrifuge it at 1000 rpm for 10 minutes in a 10 mL centrifuge tube to separate the plasma and red blood cells. Discard the upper plasma and retain the red blood cell pellet. Wash with sterile PBS and centrifuge 3 times until the supernatant becomes colorless. Finally, suspend the red blood cells in PBS to make a 2% red blood cell suspension for later use. Dissolve cinnamone in an appropriate solvent (such as DMSO or sterile water) to prepare compound solutions of different concentrations. Dilute the SABN27 in the logarithmic growth phase to 10 7 CFU / mL, add equal volumes of natural compounds at varying concentrations, incubate at 37°C for 12 hours, centrifuge at 12,000 rpm for 5 minutes, remove 0.5 mL of the supernatant and add 0.5 mL of a 2% red blood cell suspension. The positive control is 0.1% Triton, and the negative control is PBS. Incubate at 37°C for 4 hours, centrifuge at 3,000 rpm for 5 minutes, remove 200 μL of the supernatant, and measure the OD at 543 nm. The positive control should show complete hemolysis, while the negative control should show no hemolysis.

[0054]

[0055] The results (see Table 1) showed that cinnamaldehyde exhibited significant hemolytic activity against rabbit erythrocytes at concentrations ranging from 3.9 μg / mL to 2000 μg / mL, with inhibition rates ranging from 79.5% to 99.26%. This indicates that cinnamaldehyde can effectively inhibit the production of α-hemolysin by Staphylococcus aureus, significantly reducing bacterial virulence.

[0056]

[0057] Example 4: Cinnamaldehyde reduces the S. agalactiae HB31 host cell invasion test The culture medium of bovine mammary epithelial cells was DMEM medium containing 10% FBS and 1% double antibody, and the cells were cultured in a 37°C, 5% carbon dioxide incubator. 6 / well, inoculated into a 24-well plate; picked a single colony of Streptococcus agalactiae HB31 and placed it in 5 mL of broth at 37 ° C for 24 h, took 2 mL of bacterial solution, centrifuged at 6000 rpm for 5 min, and resuspended in PBS, and then used McFadden turbidimetric tube to adjust the bacterial solution concentration to 1×10 7 CFU / mL; cedarone was diluted with PBS to the appropriate concentrations of 512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, and 16 μg / mL), and then the concentration of HB31 was adjusted to 1×10 7 CFU / mL, the solvent control group was DMSO, and the blank control group was PBS. After incubation for 4 hours, the complete culture medium in the 24-well plate was removed and washed three times with PBS. At the same time, the solution after incubation was centrifuged at 3000rpm for 5 minutes, resuspended with an equal volume of DMEM culture medium without antibiotics, and added to the washed 24-well plate. Six replicate wells were set up and placed in a 37°C incubator for infection for 2 hours. At this time, the MOI (ratio of the number of infected bacteria to cells) was 10; then the culture medium in the 24-well plate was discarded, washed once with PBS, and a PBS solution containing 100 μg / mL gentamicin was added, and the plate was incubated in a 37°C incubator for 2 hours; 200 of 1% Triton was added to each well to lyse the cells for 5 minutes; the plate count method was used to count the plates.

[0058] The results are as follows Figure 2 and Figure 3 As shown: Before invasion, HB31 bacteria grew well (lower right); after invasion, the number of bacteria invading cells in the group without cypermethrin was higher (upper right), indicating that the cow-derived S. agalactiaeHB31 has a strong ability to invade host cells. In the treatment groups with different concentrations of cedarone, such as 16μg / mL to 512μg / mL, cedarone can significantly reduce the number of bacteria invading host cells compared with the control group. At concentrations above 16μg / mL, the bacterial invasion rate can be reduced to below 30%, significantly reducing the pathogenicity of Streptococcus agalactiae ( Figure 3 ).

[0059] Example 5: Antibacterial test results of cedarone combined with gentamicin (MIC determination) The FICI assay is a method used to evaluate antimicrobial drug interactions. It can determine the inhibitory or killing effects of different drugs or drug combinations on microorganisms and help determine synergistic, additive, or antagonistic interactions between drugs. The FICI assay is based on the minimum inhibitory concentration (MIC), the lowest concentration of a drug that can inhibit microbial growth. The FICI index is calculated by measuring the MIC values ​​of individual drugs and drug combinations.

[0060] Judgment criteria: FICI index <0.5 indicates synergistic effect; 0.5-1 indicates additive effect; 1-2 indicates irrelevant effect; >2 indicates antagonistic effect.

[0061] The FICI index of cypermethrin against Staphylococcus aureus (ATCC29213) was determined by combining cypermethrin with gentamicin, kanamycin, and apramycin. The results are shown in Table 2. The results showed that cypermethrin exhibited synergistic effects with gentamicin and apramycin, but not with kanamycin, indicating that cypermethrin does not have a synergistic effect with all aminoglycosides.

[0062] Then, cinnamyl acetate was combined with gentamicin to measure the FICI index of standard bacteria and clinical isolates. The results are shown in Table 2. For the 8 clinically isolated Gram-positive strains and 2 standard strains (ATCC 29213 and ATCC 13813) isolated and identified from the dairy farm in Example 1, cinnamyl acetate and gentamicin showed high synergy, with FICI indexes of <0.5. S. aureus Taking SABN145 as an example, the minimum inhibitory concentration (MIC) of gentamicin alone is 4 μg / mL. When used in combination with 8 μg / mL of cypermethrin, the minimum inhibitory concentration (MIC) of gentamicin is reduced to 0.5 μg / mL, and the antibacterial ability is increased by 8 times. Clinical use can significantly reduce the dosage of gentamicin.

[0063]

[0064] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Use of bucubinone or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial synergist for enhancing the efficacy of gentamicin in resisting bacterial infections.

2. The use according to claim 1, characterized in that: The bacteria are Gram-positive bacteria; and / or the bacteria are bacteria that cause animal mastitis.

3. The use according to claim 2, characterized in that: The Gram-positive bacteria include at least one of the following: Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis and Staphylococcus xylosus.

4. An antibacterial composition comprising humbertone or a pharmaceutically acceptable salt thereof and gentamicin or a pharmaceutically acceptable salt thereof.

5. The antibacterial composition according to claim 4, characterized in that: The mass ratio of the cinnamone or a pharmaceutically acceptable salt thereof to gentamicin or a pharmaceutically acceptable salt thereof is (4-128):1 or (4-64):1 or (4-16):1 or (8-32):1 or (32-128):

1.

6. The antibacterial composition according to claim 4 or 5, characterized in that: The antibacterial composition is used for preventing bacterial infectious diseases.

7. The antibacterial composition according to claim 6, characterized in that: The antibacterial composition is used for preventing bacterial animal mastitis.

8. The antibacterial composition according to claim 7, characterized in that: The bacteria are Gram-positive bacteria; and / or the bacteria are bacteria that cause animal mastitis.

9. The antibacterial composition according to claim 8, characterized in that: The Gram-positive bacteria include at least one of the following: Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis, and Staphylococcus xylosus.

10. An antibacterial product comprising the antibacterial composition according to any one of claims 4 to 9.

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