A compound preparation capable of reducing the drug resistance of pathogenic bacteria to florfenicol and a preparation method thereof

Abstract

The application discloses a compound preparation capable of reducing drug resistance of pathogenic bacteria to florfenicol and a preparation method thereof, and belongs to the technical field of medicines. The compound preparation comprises a composition of florfenicol and potassium clavulanate, wherein the composition comprises 2-60 parts of the florfenicol and 0.5-15 parts of the potassium clavulanate by weight. The application further provides application of the potassium clavulanate in preparation of a product for reducing antibacterial activity of florfenicol to pathogenic bacteria and application of the potassium clavulanate in preparation of a product for reducing drug resistance of pathogenic bacteria to florfenicol. The results of examples of the application show that the potassium clavulanate can reduce drug resistance of florfenicol and enhance antibacterial activity of florfenicol, the compound preparation provided by the application is simple to prepare, raw materials are easy to obtain, and the compound preparation is suitable for industrial large-scale production, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and in particular to a compound preparation capable of reducing the resistance of pathogens to florfenicol and a preparation method thereof. Background Art

[0002] In the technical field where modern medicine and the livestock farming industry are closely intertwined, florfenicol (FF), as a key component of the amide alcohol class of broad-spectrum antibiotics, exhibits a unique pharmacological mechanism of action. Its core principle of action lies in the precise inhibition of peptidyl transferase activity, thereby interrupting the bacterial protein synthesis chain and achieving highly effective antibacterial efficacy. In the 1990s, florfenicol was successfully approved for marketing, standing out for its low toxicity and other characteristics, greatly reducing the potential negative effects of the drug on the physiological functions of livestock bodies, and providing farmers with a wider range of choices. Florfenicol has a broad antimicrobial spectrum and is effective against infections caused by both Gram-positive and Gram-negative bacteria. In addition, florfenicol has the characteristics of rapid absorption. Once applied, it can efficiently penetrate the body's tissue barriers and quickly reach the target site to release its efficacy. At the same time, it is excreted quickly, has a short withdrawal period, and does not accumulate in the animal body. These excellent properties are highly consistent with the rigid demand for rapid turnover in modern animal husbandry, making florfenicol an important choice for many breeding companies in their disease prevention and control strategy layout.

[0003] In aquaculture, the frequency of various bacterial diseases is increasing, resulting in significant economic losses. Florfenicol, with its excellent antibacterial activity, has provided a boost to the robust and thriving development of aquaculture. However, over time, difficulties in its application have gradually emerged. Currently, florfenicol is being abused and misused in aquaculture, accelerating the development of bacterial resistance. Conventional doses used to easily kill pathogens, but now, even increased doses are less effective. These factors have not only led to exponential increases in aquaculture costs, but have also brought the disease prevention and control system to the brink of collapse, plunging the entire aquaculture industry into a quagmire.

[0004] Therefore, how to effectively reduce bacterial resistance and better exert the antibacterial activity of florfenicol through scientific and reasonable drug use strategies, innovative drug combination schemes or new drug dosage form improvements is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The present invention aims to provide a compound preparation and preparation method thereof capable of reducing pathogenic bacteria's resistance to florfenicol, thereby overcoming the problems of the prior art. Potassium clavulanate can significantly reduce drug resistance and enhance the antibacterial activity of florfenicol. The florfenicol and potassium clavulanate compound powder provided by the present invention is effective in treating various infections in aquatic animals.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] Technical Solution 1: A compound preparation capable of reducing the resistance of pathogens to florfenicol, the compound preparation comprising a combination of florfenicol and potassium clavulanate.

[0008] Furthermore, the composition comprises 2-60 parts of florfenicol and 0.5-15 parts of potassium clavulanate in parts by weight.

[0009] Furthermore, the composition comprises 5-15 parts of florfenicol and 2.5-7.5 parts of potassium clavulanate in parts by weight.

[0010] Furthermore, the pathogens include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veseri and drug-resistant Citrobacter freundii.

[0011] Technical Solution 2: A compound powder of florfenicol and potassium clavulanate for aquatic products, wherein the raw materials, calculated by weight, include 2-60 parts of florfenicol, 0.5-15 parts of potassium clavulanate and 25-97.5 parts of starch.

[0012] Preferably, by weight, the amount of florfenicol is 10-50 parts, the amount of potassium clavulanate is 2.5-12.5 parts, and the amount of starch is 37.5-87.5 parts.

[0013] Preferably, the compound preparation comprises a pharmaceutical preparation and a feed additive.

[0014] Furthermore, the dosage form of the compound preparation includes powder.

[0015] Furthermore, the preparation method of the compound powder comprises the following steps: firstly mixing the florfenicol and potassium clavulanate, then adding the starch and mixing, and sieving to obtain the compound preparation.

[0016] Technical solution three: Use of the compound preparation or the compound powder in enhancing the antibacterial ability of aquatic animals against drug-resistant pathogens.

[0017] Technical Solution 4: Use of the compound preparation or the compound powder in the preparation of medicines for treating aquatic animal diseases, wherein the diseases are diseases caused by infection with drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas welchii or drug-resistant Citrobacter freundii.

[0018] Technical Solution 5: Use of potassium clavulanate in the preparation of a product that reduces the antibacterial activity of florfenicol against pathogens, including drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas freundii, and drug-resistant Citrobacter freundii.

[0019] Technical Solution 6: Use of potassium clavulanate in the preparation of a product for reducing the resistance of pathogens to florfenicol, wherein the pathogens include drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas freundii and drug-resistant Citrobacter freundii.

[0020] The present invention discloses the following technical effects:

[0021] The reason for the generation of florfenicol resistance is that the amide bond hydrolase in bacteria can hydrolyze the amide bond of florfenicol, resulting in a decrease in drug efficacy. Therefore, the bacterial resistance can be reduced by inhibiting the activity of the amide bond hydrolase, thereby improving the antibacterial activity of florfenicol. Potassium clavulanate is an amide bond hydrolase inhibitor that can bind to most amide bond enzymes to generate irreversible conjugates, thereby improving the antibacterial activity of antibiotics. In order to reduce bacterial resistance and better exert the antibacterial activity of florfenicol, the present invention prepares a compound preparation of florfenicol and potassium clavulanate, and uses it for the treatment of aquatic animal diseases. The results show that the compound preparation can well treat disease infections of aquatic animals. In short, potassium clavulanate can greatly reduce the drug resistance of florfenicol and enhance the antibacterial activity of florfenicol; the compound preparation provided by the present invention is simple to prepare, the raw materials are easy to obtain, and it is suitable for industrial large-scale production. Therefore, it has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a chessboard pattern diagram. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] The preparation method of the florfenicol and clavulanate potassium compound powder is as follows: 2-60 parts of florfenicol, 0.5-15 parts of potassium clavulanate, and 25-97.5 parts of starch are prepared for standby use. Specifically, the preparation method comprises the following steps: placing the sifted florfenicol and potassium clavulanate in a mixer and mixing them thoroughly, adding the sifted starch to the mixer, and then sieving to obtain the compound powder. Preferably, the preparation method comprises 10-50 parts of florfenicol, 2.5-12.5 parts of potassium clavulanate, and 37.5-87.5 parts of starch in parts by weight.

[0030] In a specific embodiment, the efficacy of the compound powder in treating bacterial infections in aquatic animals was verified through a challenge experiment using yellow catfish as the experimental subject. The drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas versii, and drug-resistant Citrobacter freundii used in the present embodiment were all clinically isolated from yellow catfish and their species were confirmed by molecular biology. The isolation site was Wuhan, Hubei Province.

[0031] The reagents and biological materials involved in the embodiments of the present invention are as follows:

[0032] Potassium clavulanate (61177-45-5) was purchased from Jiangxi Ruiweier Biotechnology Co., Ltd., with a content of 99%; Florfenicol (73231-34-2) was purchased from Shandong Guobang Pharmaceutical Co., Ltd., API, with a content of 98%;

[0033] The 16S sequence of drug-resistant Aeromonas hydrophila is:

[0034]

[0035] The 16S sequence of drug-resistant Aeromonas vermiformis is:

[0036]

[0037] The 16S sequence of drug-resistant Citrobacter freundii is:

[0038]

[0039] Unless otherwise specified, other experimental materials are conventional materials in this field and can be purchased through conventional channels.

[0040] Example 1 Potassium clavulanate increases the bactericidal ability of florfenicol against drug-resistant Aeromonas hydrophila

[0041] 1. Use the microdilution method to determine the minimum inhibitory concentrations of clavulanate potassium and florfenicol against drug-resistant Aeromonas hydrophila. First, pipette 200 μL of drug working solution into the well with the preset maximum drug concentration. Then, add 100 μL of sterile MH broth to each of the remaining wells. Then, pipette 100 μL of drug working solution from the well with the maximum drug concentration into the second well. Repeat by pipetting five times to mix thoroughly, then pipette 100 μL into the third well. Perform doubling dilutions in succession, discarding the 100 μL aspirated from the last well. At this point, each well contains 100 μL of drug-containing broth, with drug concentrations ranging from 512 μg / mL to 0.002 μg / mL.

[0042] 2. Chessboard method for determining the combined effect of clavulanate potassium and florfenicol

[0043] See the chessboard pattern diagram Figure 1 Specifically, a 96-well bacterial culture plate was prepared, with row Y1 and column X1 designated for drug A, potassium clavulanate, and drug B, florfenicol, respectively. 100 μL of standard drug solution with concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of row Y1; 100 μL of standard drug solution with concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of column X1. 50 μL of standard drug solution with concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of rows Y2-7; 50 μL of standard drug solution with concentrations of 1 / 4, 1 / 2, 1, 2, 4, and 8 MIC was added to wells 2-7 of columns X2-7. 100 μL of bacterial suspension was added to each well except well X1Y1, and 200 μL of bacterial suspension was added to well X1Y1. At this time, the total volume of the liquid in the 96-well microplate is 200μL. Place the 96-well microplate with the sample in a constant temperature incubator (28°C) and culture for 24-48 hours. The lowest concentration of the clear well is the MIC for combined drug use. The results are determined by the equivalent midpoint method: each tube along the 0-point angle bisector is an equivalent midpoint tube. The lowest concentration of sterile growth read in the combined result is 1 point. The point on the X-axis corresponding to this point is MICA, and the point on the Y-axis corresponding to this point is MICB. The partial concentration inhibition index (FIC) is used as the basis for judging the combined drug sensitivity test, and the calculation formula is as follows:

[0044]

[0045] Among them, FIC index ≤ 0.5 is synergistic; 0.5 <FIC指数≤1为相加作用;1<FIC指数≤2为无关作用;FIC指数> 2 is antagonistic effect.

[0046] Results: The MIC of clavulanate potassium against drug-resistant Aeromonas hydrophila was 64 μg / mL, and the MIC of florfenicol against drug-resistant Aeromonas hydrophila was 16 μg / mL. The minimum concentrations of clavulanate potassium and florfenicol for sterile growth were 16 μg / mL for clavulanate potassium and 8 μg / mL for florfenicol, respectively. The calculated FIC index was 0.75, indicating an additive effect.

[0047] Example 2 Potassium clavulanate increases the bactericidal ability of florfenicol against drug-resistant Aeromonas viridis

[0048] The determination method is the same as that of Example 1.

[0049] Results: The MIC of clavulanate potassium against drug-resistant Aeromonas vermiformis was 32 μg / mL, and the MIC of florfenicol against drug-resistant Aeromonas vermiformis was 16 μg / mL. The minimum concentrations of clavulanate potassium and florfenicol for sterile growth were 4 μg / mL for clavulanate potassium and 4 μg / mL for florfenicol, respectively. The calculated FIC index was 0.375, indicating synergistic activity.

[0050] Example 3 Clavulanic acid increases the bactericidal ability of florfenicol against drug-resistant Citrobacter freundii

[0051] The determination method is the same as that of Example 1.

[0052] Results: The MIC of potassium clavulanate against drug-resistant Citrobacter freundii was 64 μg / mL, and the MIC of florfenicol against drug-resistant Citrobacter freundii was 8 μg / mL. The minimum concentrations of potassium clavulanate and florfenicol for sterile growth were 32 μg / mL for potassium clavulanate and 1 μg / mL for florfenicol, respectively. The calculated FIC index was 0.625, indicating an additive effect.

[0053] Example 4 Clinical Trial

[0054] 150 yellow catfish were randomly divided into five groups, with 30 fish in each group.

[0055] Blank control: no challenge, no treatment;

[0056] Negative control: challenged with drug-resistant Aeromonas vermiformis from yellow catfish without treatment;

[0057] Treatment group 1: The fish were challenged with drug-resistant Aeromonas vermiformis from yellow catfish and treated with florfenicol at a dose of 15 mg / kg twice a day for three consecutive days.

[0058] Treatment group 2: The resistant Aeromonas vermiformis from yellow catfish was used as the challenge agent and the rats were given potassium clavulanate at a dose of 15 mg / kg twice a day for three consecutive days.

[0059] Treatment group 3: The yellow catfish-derived drug-resistant Aeromonas vermiformis was used for infection and the animals were treated with a combination powder of florfenicol and clavulanate potassium at a dose of 15 mg / kg (calculated as florfenicol) twice a day for three consecutive days.

[0060] After 7 days of treatment, the mortality rate of each group of yellow catfish was calculated.

[0061] The preparation method of a florfenicol and clavulanate potassium compound powder comprises the following steps: 20 parts of florfenicol, 5 parts of potassium clavulanate, and 75 parts of starch are prepared for standby use. The preparation method comprises the following steps: sifting the florfenicol and potassium clavulanate in a mixer and thoroughly mixing them; adding the sifted starch to the mixer; and sifting again to obtain the compound powder.

[0062] Table 1 Test results

[0063] Group Number of dead animals Mortality rate Blank control group 1 3.33％ Negative control group 30 100％ Treatment group 1 (florfenicol) 25 83.33％ Treatment group 2 (potassium clavulanate) 24 80.00％ Treatment group 3 (florfenicol and potassium clavulanate compound powder) 1 3.33％

[0064] According to the mortality results of each group in Table 1, the compound powder prepared by florfenicol and clavulanate potassium can be well used to treat disease infections in aquatic animals.

[0065] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. A compound preparation capable of reducing the resistance of pathogens to florfenicol, characterized in that: The active ingredients of the compound preparation consist of florfenicol and potassium clavulanate.

2. The compound preparation according to claim 1, characterized in that By weight, the florfenicol is 2-60 parts, and the potassium clavulanate is 0.5-15 parts.

3. The compound preparation according to claim 1, characterized in that By weight, the florfenicol is 5-15 parts, and the potassium clavulanate is 2.5-7.5 parts.

4. The compound preparation according to claim 1, characterized in that The pathogens are drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veseri and drug-resistant Citrobacter freundii.

5. A compound powder of florfenicol and potassium clavulanate for aquaculture, characterized in that: The raw materials include 2-60 parts of florfenicol, 0.5-15 parts of potassium clavulanate and 25-97.5 parts of starch in parts by weight; wherein the active ingredients are composed of florfenicol and potassium clavulanate.

6. The compound powder according to claim 5, characterized in that The preparation method of the compound powder comprises the following steps: firstly mixing the florfenicol and potassium clavulanate, then adding the starch and mixing, and sieving to obtain the compound preparation.

7. Use of the compound preparation according to any one of claims 1 to 4 or the compound powder according to claim 5 or 6 in the preparation of a medicament for treating aquatic animal diseases, characterized in that: The disease is caused by infection with drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas welchii or drug-resistant Citrobacter freundii.

8. Use of potassium clavulanate in the preparation of a product for reducing the antibacterial activity of florfenicol against pathogenic bacteria, characterized in that: The pathogens are drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas veseri and drug-resistant Citrobacter freundii.

9. Use of potassium clavulanate in the preparation of a product for reducing the resistance of pathogens to florfenicol, characterized in that: The pathogens are drug-resistant Aeromonas hydrophila, drug-resistant Aeromonas welchii and drug-resistant Citrobacter freundii.

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