Use of cedrol in the preparation of antibacterial potentiator of apramycin sulfate

Abstract

The application discloses application of cedropion in preparation of anplimycin sulfate antibacterial synergist. Through a large amount of experimental research, the inventors accidentally find that cedropion can effectively improve the bacteriostatic effect of anplimycin sulfate on gram-positive bacteria separated from clinical cow mastitis. The discovery is favorable to exertion of the bacteriostatic effect of anplimycin sulfate on gram-positive bacteria, and also provides more drug selection for treatment of clinical cow mastitis.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to the application of cedarone in the preparation of apramycin sulfate antibacterial synergist. Background Technology

[0002] Globally, bacterial resistance is becoming an increasingly serious problem, urgently calling for the development of new antibiotics. However, the development of new antibiotics faces numerous obstacles, with increasingly longer development cycles and greater difficulties. In this context, exploring methods to improve the antibacterial activity of existing drugs, expand their antibacterial spectrum, or enhance their antibacterial efficacy has become an effective solution in the field of disease prevention and control.

[0003] Cedarone, or (+)-Nootkatone, possesses unique aromatic properties and is commonly used as a component in fragrances and flavorings. Recent studies have shown that cedarone exhibits a wide range of biological activities, primarily including anti-Alzheimer's disease effects, anti-cell proliferation effects, and anti-platelet aggregation effects. In particular, its structure is similar to glutamate, potentially interfering with nerve conduction by regulating GABA receptors (such as the Rdl channel in insects), thus possessing certain insecticidal activity. Therefore, current research largely focuses on the insecticidal functions of cedarone and its derivatives. Patent CN111620837A details the application of a modified cedarone thiazole amide compound in antiparasitic interactions.

[0004] Apramycin Sulfate (AS), an aminoglycoside antibiotic, is primarily used to treat respiratory and intestinal infections caused by Escherichia coli and Salmonella, and is not used to treat clinical Staphylococcus aureus infections. Staphylococcus aureus , S. aureus Apramycin sulfate is the first-line drug for infections, but its antibacterial efficacy is 5-10 times lower than that of cephalosporins. Developing an antibacterial synergist for apramycin sulfate would enhance its antibacterial efficacy against Gram-positive bacteria and provide more treatment options for clinicians. It is worth noting that there are currently no reports on cedarone as an antibacterial synergist for apramycin sulfate. Summary of the Invention

[0005] To address the technical problem of the poor antibacterial effect of apramycin sulfate against Gram-positive bacteria, the inventors, through extensive experimental research, unexpectedly discovered that cedarone can effectively enhance the antibacterial effect of apramycin sulfate against Gram-positive bacteria isolated from clinical bovine mastitis. This discovery provides a new direction for improving the antibacterial properties of apramycin sulfate and is expected to play an important role in the treatment of clinical bovine mastitis.

[0006] A first aspect of the invention provides the use of cedarone or a pharmaceutically acceptable salt thereof in the preparation of an antibacterial synergist that enhances the antibacterial efficacy of apramycin sulfate.

[0007] A second aspect of the invention provides the use of cedarone or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating bacterial infectious diseases.

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

[0009] The antibacterial composition includes cedarone or a pharmaceutically acceptable salt thereof and apramycin sulfate.

[0010] Further, the mass ratio of the cedarone or its pharmaceutically acceptable salt to apramycin sulfate or its pharmaceutically acceptable salt is (8~128):1, and may further be (32~64):1, (8~32):1 or (64~128):1, specifically 8:1, 32:1, 64:1 or 128:1.

[0011] For example, for anti-Staphylococcus aureus ( Staphylococcus aureus,S. aureus (e.g., bovine Staphylococcus aureus), wherein the mass ratio of cedarone or a pharmaceutically acceptable salt thereof to apramycin sulfate or a pharmaceutically acceptable salt thereof is preferably (32~64):1.

[0012] For example, for anti-lactose streptococci ( Streptococcus agalactiae , S. agalactiae (e.g., bovine agalactia streptococci), wherein the preferred mass ratio of cedarone or a pharmaceutically acceptable salt thereof to apramycin sulfate or a pharmaceutically acceptable salt thereof is (8~32):1.

[0013] For example, for anti-Staphylococcus epidermidis ( Staphylococcus epidermidis , S. epidermidis (e.g., bovine Staphylococcus epidermidis), wherein the mass ratio of cedarone or a pharmaceutically acceptable salt thereof to apramycin sulfate or a pharmaceutically acceptable salt thereof is preferably 32:1.

[0014] For example, for anti-Staphylococcus xylose ( Staphylococcus xylosus , S. xylosus (e.g., Staphylococcus xylose), wherein the mass ratio of cedarone or a pharmaceutically acceptable salt thereof to apramycin sulfate or a pharmaceutically acceptable salt thereof is preferably 128:1.

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

[0016] Furthermore, the antibacterial composition can be used to treat bacterial mastitis in animals.

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

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

[0019] The dosage form of the antibacterial product may be selected from any of the following: tablets, capsules, syrups, droplets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, injections, and lyophilized powder for injection.

[0020] The pharmaceutically acceptable carriers include, but are not limited to, water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), and enteric-coated carrier materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.). Using these materials, the above-mentioned antibacterial products can be formulated into various dosage forms, including but not limited to tablets, capsules, syrups, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, lozenges, lyophilized powder injections, etc. These can be conventional formulations, sustained-release formulations, controlled-release formulations, and various microparticle delivery systems.

[0021] When using the compositions provided by this invention to prevent and / or treat bacterial infections, an effective amount of the antibacterial composition is administered to the subject organism.

[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, gender, natural health condition, nutritional status, the activity intensity of the compound, the time of administration, metabolic rate, the severity of the disease, and the subjective judgment of the treating physician.

[0023] In this invention, the term "effective dose" refers to a dose that can achieve treatment, prevention, reduction, and / or relief of the disease or condition described in this invention in a subject.

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

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

[0026]

[0027] In this invention, the bacteria include Gram-positive bacteria and Gram-negative bacteria.

[0028] Furthermore, the bacteria are Gram-positive bacteria, such as Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis, Staphylococcus xylose, etc.

[0029] Furthermore, the bacteria are those that cause mastitis in animals.

[0030] Furthermore, the bacteria are Gram-positive bacteria that cause mastitis in animals.

[0031] According to an embodiment of the present invention, the animal mastitis is bovine mastitis.

[0032] According to an embodiment of the present invention, the Gram-positive bacterium is Staphylococcus aureus. The Staphylococcus aureus includes... S. aureus Standard strains and S. aureus Clinical isolates (i.e. Staphylococcus aureus, the source of clinical bovine mastitis).

[0033] According to an embodiment of the present invention, the Gram-positive bacteria are agalactococci. The agalactococci include... S. agalactiae Standard strains and S. agalactiae Clinical isolates (i.e., Streptococcus agalactiae, the source of clinical bovine mastitis).

[0034] 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 isolate (i.e., Staphylococcus epidermidis, the source of clinical bovine mastitis).

[0035] According to an embodiment of the present invention, the Gram-positive bacterium is *Staphylococcus xylose*. The *Staphylococcus xylose* includes... S. xylosus Standard strains and S. xylosus Clinical isolate (i.e. Staphylococcus xylose, the source of clinical bovine mastitis).

[0036] Furthermore, the antibacterial infectious disease is antibacterial animal mastitis.

[0037] According to an embodiment of the present invention, the antibacterial animal mastitis is antibacterial bovine mastitis, especially antibacterial bovine mastitis caused by Staphylococcus aureus and / or Streptococcus agalactiae and / or Staphylococcus epidermidis and / or Staphylococcus xylose.

[0038] Compared with the prior art, the present invention has the following beneficial technical effects:

[0039] 1. Antibacterial synergistic effect. Cephalosporin exerts a synergistic antibacterial effect with apramycin sulfate by inhibiting the production of bacterial hemolysin and reducing bacterial adhesion to host cells, which can significantly enhance the antibacterial activity of apramycin sulfate against Gram-positive bacteria.

[0040] 2. Expanding the antibacterial spectrum. In clinical treatment, aminoglycosides are mainly used to treat Gram-negative bacteria. After antibacterial enhancement, apramycin sulfate expands the sensitivity to Gram-positive bacteria, improving the treatment success rate.

[0041] 3. Reduce antibiotic dosage. The clinical dosage of apramycin sulfate can be reduced by 50%–80% through the combined use of antibacterial potentiators.

[0042] 4. Reduce the risk of drug side effects.

[0043] 5. Delaying drug resistance. Shifting from single-target to multi-target action can further reduce the risk of bacterial resistance in clinical settings. Attached Figure Description

[0044] Figure 1 The effects of the drug combination group, cedarone monoclonal group, apramycin monosulfate group, and no drug group in Example 2 are as follows: S. aureus Growth curve of clinically isolated bacteria SABN27;

[0045] Figure 2 The following is an example of the intracellular bacterial culture of the host cell under different concentrations of cedarone in Example 4;

[0046] Figure 3 The effect of different concentrations of cedarone on bacterial invasion rate in Example 4. Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] Example 1: MIC determination of cedarwood ketone against clinical isolates from dairy bovine strains

[0050] Isolation, identification, and preservation of clinical isolates: Milk samples were collected from cows with mastitis at a large-scale dairy farm in Beijing. The samples were enriched, cultured, and purified in the laboratory using traditional bacterial culture methods. Identification of the clinical isolates was completed through a combination of bacterial biochemical identification and Sanger sequencing. Among these:

[0051] dairy cow source S. aureus SABN27, dairy cow source S. aureusSABN29, dairy cow source S. aureus SABN145, dairy cow source S. aureus SABN-78 is Staphylococcus aureus;

[0052] dairy cow source S. agalactiae HB27, dairy cow source S. agalactiae HB31 is a non-lactate streptococcus;

[0053] dairy cow source S. epidermidis EB15 is Staphylococcus epidermidis;

[0054] dairy cow source S. xylosus XB16 is Staphylococcus xylose.

[0055] The standard quality control strain is: S. aureus ATCC 29213 S. agalactiae ATCC 13813.

[0056] Single colonies of the above 8 clinical isolates from dairy cows and 2 standard quality control strains were inoculated into sterile MH broth and cultured for 12 hours. The cultured bacterial solution was then diluted to 10. 5 CFU / mL. Prepare twelve 2mL centrifuge tubes. Add 0.9mL of MH broth and 100µL of 8.0mg / mL cedarone solution sequentially to each tube. Add 500µL of MH broth to each of the 2nd to 12th tubes. Using a micropipette, pipette 500µL of the mixture from the first column, then perform a 2-fold dilution sequentially from the first tube to the last. Finally, add 500µL of bacterial suspension to each tube. For the positive control wells, add only MH broth and bacterial suspension; for the negative control wells, add only MH broth. Perform triple replicates for each concentration. Incubate at 37℃ for 18-24 hours, then calculate the MIC values ​​for each strain.

[0057] The results show that cedar ketone has an effect on... 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 cedarone monomers basically have no antibacterial activity.

[0058] Example 2: Dairy cow source S. aureus Study on the growth curve of SABN27 bacteria

[0059] Selecting dairy cows used in Example 1 S. aureus A single colony of SABN27 was cultured in 4 ml of MH broth and incubated overnight at 37°C with shaking. The bacterial culture was then diluted to 0.5 McFarland turbidity tubes and then further diluted 1:100 to 10⁻⁶. 6 CFU / mL was prepared for use. Different groups were set up: a combined group (64 μg / mL cedarone + 0.5 μg / mL apramycin sulfate), a cedarone group (64 μg / mL), an apramycin sulfate group (0.5 μg / mL), and an SABN27 group. Subsequently, 200 μL samples were taken into 96-well plates at 0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h, and 24h, and the OD600 values ​​were measured and recorded. During the measurement of growth curves, the culture temperature and rotation speed of each group should be kept consistent, and the samples should be thoroughly mixed before sampling to ensure uniform bacterial concentration.

[0060] The results are as follows Figure 1 As shown: Compared with the SABN27 group, the cedarone monotherapy group and the apramycin monotherapy group did not affect the growth of Staphylococcus aureus. They entered the exponential growth phase at 4 hours. Neither group inhibited the growth of Staphylococcus aureus during the exponential or plateau phases. However, the cedarone + apramycin sulfate group significantly inhibited the number of Staphylococcus aureus colonies during the exponential phase. Its antibacterial effect was significantly better than that of cedarone or apramycin sulfate alone.

[0061] Example 3: Effects of cedarone on dairy cow samples S. aureus SABN27 inhibition of hemolysis test

[0062] Fresh defibrinated rabbit blood was centrifuged in a 10 mL centrifuge tube at 1000 rpm for 10 min to separate plasma and red blood cells. The supernatant plasma was discarded, and the red blood cell pellet was retained. The pellet was washed with sterile PBS and centrifuged three times until the supernatant became colorless. The red blood cells were then resuspended in PBS to prepare a 2% red blood cell suspension for later use. Cedarone was dissolved in a suitable solvent (such as DMSO or sterile water) to prepare compound solutions of different concentrations. SABN27 in the logarithmic growth phase was diluted to 10... 7 CFU / mL of natural compounds of varying concentrations were added, and the mixture was incubated at 37°C for 12 h. After centrifugation at 12000 rpm for 5 min, 0.5 mL of the supernatant was added to 0.5 mL of 2% erythrocyte suspension. The positive control was 0.1% Triton, and the negative control was PBS. The mixture was incubated at 37°C for 4 h, and then centrifuged at 3000 rpm for 5 min. 200 μL of the supernatant was collected, and the OD value was measured at 543 nm. The positive control group should show complete hemolysis, while the negative control group should show no hemolysis.

[0063]

[0064] The results (see Table 1) showed that cedarwood ketone exhibited good inhibitory hemolytic activity against rabbit erythrocytes at concentrations ranging from 3.9 μg / mL to 2000 μg / mL, with inhibition rates between 79.5% and 99.26%. This indicates that cedarwood ketone can effectively inhibit the production of α-hemolysin by Staphylococcus aureus, thereby significantly reducing bacterial virulence.

[0065]

[0066] Example 4: Cedarone reduces dairy cow calf ... S. agalactiae HB31 host cell invasiveness assay

[0067] Bovine mammary epithelial cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-dextrose antibiotics at 37°C in a 5% CO2 incubator. Cells were then cultured at a rate of 1×10⁶ cells / year. 6 Inoculate one colony per well into a 24-well plate; pick a single colony of *Streptococcus agalactiae* HB31 and place it in 5 mL of broth, incubate at 37°C for 24 h, take 2 mL of bacterial suspension, centrifuge at 6000 rpm for 5 min, resuspend in PBS, and then use a McFarland turbidimetric tube to adjust the bacterial concentration to 1 × 10⁻⁶. 7 CFU / mL; cedar ketone was diluted with PBS to appropriate concentrations (512 μg / mL, 256 μg / mL, 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL), and then the concentration of HB31 was adjusted to 1×10⁻⁶ CFU / mL. 7 CFU / mL, with DMSO as the control solvent and PBS as the blank control, incubated for 4 h. After removing the complete culture medium from the 24-well plate, wash three times with PBS. Simultaneously, centrifuge the incubated solution at 3000 rpm for 5 min, resuspend in an equal volume of antibiotic-free DMEM culture medium, and add to the washed 24-well plate with 6 replicates. Incubate at 37°C for 2 h, at which point the MOI (infected bacteria to cell ratio) is 10. Then discard the culture medium in the 24-well plate, wash once with PBS, add PBS solution containing 100 μg / mL apramycin sulfate, and incubate at 37°C for 2 h. Add 200 μL of 1% Triton solution to each well to lyse the cells for 5 min. Plate count is performed using the plate counting method.

[0068] The results are as follows Figure 2 and Figure 3 As shown: Before invasion, HB31 bacteria grew well (bottom right); after invasion, the number of bacteria invading cells was higher in the group without cedarone (top right), indicating that the bacteria were of dairy cow origin. S. agalactiaeHB31 exhibits a strong invasive ability against host cells. However, in treatment groups with different concentrations of cedarwood ketone, ranging from 16 μg / mL to 512 μg / mL, compared to the control group, cedarwood ketone significantly reduced the number of bacteria invading host cells. At concentrations above 16 μg / mL, it reduced the bacterial invasion rate to below 30%, significantly decreasing the pathogenicity of *Streptococcus agalactiae*. Figure 3 ).

[0069] Example 5: Antibacterial test results (MIC determination) of cedarone combined with apramycin sulfate

[0070] The FICI assay is a method used to assess antimicrobial interactions. It determines the inhibitory or killing effects of different drugs or drug combinations on microorganisms and helps identify synergistic, additive, or antagonistic effects between drugs. The principle of the FICI assay is based on the minimum inhibitory concentration (MIC), which is the lowest concentration at which a drug can inhibit microbial growth. The FICI index is calculated by testing the MIC values ​​of drugs used alone and drug combinations.

[0071] Judgment criteria: FICI index <0.5 indicates synergistic effect; 0.5-1 indicates additive effect; 1-2 indicates no effect; >2 indicates antagonistic effect.

[0072] The FICI index of cedarone against Staphylococcus aureus (ATCC 29213) was determined by combining it with apramycin, kanamycin, or gentamicin, and the results are shown in Table 2. The results showed that cedarone exhibited synergistic effects with apramycin and gentamicin, but no synergistic effect with kanamycin, indicating that cedarone does not have a synergistic effect with all aminoglycoside drugs.

[0073] Subsequently, cedarone was combined with apramycin, and their FICI indices against standard and clinical isolates were determined. The results are shown in Table 2. For the eight clinically isolated Gram-positive strains and two standard strains (ATCC 29213 and ATCC 13813) identified from the dairy farm in Example 1, cedarone and gentamicin showed high synergy, with FICI indices all <0.5. (Based on dairy cow origin...) S. aureus Taking SABN145 as an example, the minimum inhibitory concentration (MIC) of apramycin sulfate alone is 2 μg / mL. When used in combination with 16 μg / mL cedarone, the MIC of apramycin sulfate is reduced to 0.5 μg / mL, and the antibacterial ability is increased by 4 times. Clinical use can significantly reduce the dosage of apramycin sulfate.

[0074]

[0075] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. Use of cedrene or a pharmaceutically acceptable salt thereof in the manufacture of an antibacterial potentiator for enhancing the antibacterial efficacy of apramycin sulfate against bacterial infection; the bacteria are bovine-derived gram-positive bacteria causing animal mastitis; the bovine-derived gram-positive bacteria are selected from at least one of Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis and Staphylococcus xylosus; the mass ratio of the cedrene or a pharmaceutically acceptable salt thereof to apramycin sulfate is (8-128):

1.

2. An antibacterial composition comprising cedrene or a pharmaceutically acceptable salt thereof and apramycin sulfate; the mass ratio of the cedrene or a pharmaceutically acceptable salt thereof to apramycin sulfate is (8-128): 1; the antibacterial composition is used against bacterial animal mastitis; the bacteria are bovine-derived gram-positive bacteria causing animal mastitis; the bovine-derived gram-positive bacteria are selected from at least one of Staphylococcus aureus, Streptococcus agalactiae, Staphylococcus epidermidis and Staphylococcus xylosus.

3. The antimicrobial composition according to claim 2, characterized in that: the mass ratio of the cedrene or a pharmaceutically acceptable salt thereof to apramycin sulfate is (32-64):

1.

4. The antimicrobial composition according to claim 2, characterized in that: the mass ratio of the cedrene or a pharmaceutically acceptable salt thereof to apramycin sulfate is (8-32):

1.

5. The antimicrobial composition according to claim 2, wherein: the mass ratio of the cedrene or a pharmaceutically acceptable salt thereof to apramycin sulfate is (64-128):

1.

6. An antibacterial product containing the antibacterial composition of any one of claims 2-5.

Images