Florfenicol preparation capable of reversing drug resistance and preparation method of florfenicol preparation

Abstract

The invention relates to a florfenicol preparation for reversing drug resistance, and every 100 kg of the florfenicol preparation is prepared from the following raw materials by weight: 30 to 50 kg of florfenicol, 5 to 10 kg of puerarin, 0.5 to 1 kg of magnesium stearate, and the balance of silicon dioxide. The florfenicol preparation is subjected to low-temperature crushing treatment, the crystal form of the medicine can be prevented from being converted, the dissolution rate of the medicine is prevented from being reduced, the florfenicol and the puerarin are reasonably proportioned according to the formula, the optimal reverse drug resistance effect is achieved, and meanwhile, the product process is particularly suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of veterinary drug formulation technology, specifically relating to a florfenicol formulation for reversing drug resistance and its preparation method. Background Technology

[0002] Florfenicol, an antibiotic widely used in veterinary clinical practice, faces several significant challenges in its current application. First, single-agent formulations of florfenicol are facing the challenge of rising drug resistance rates, significantly reducing their therapeutic efficacy. Second, to improve the therapeutic effect of florfenicol and reduce the development of resistance, it is often necessary to combine it with traditional Chinese medicine, which not only leads to high costs but also presents significant challenges in quality control.

[0003] Therefore, improving the therapeutic effect of florfenicol, reducing its drug resistance, simplifying the formulation process, reducing costs, and improving quality control are urgent problems that need to be solved.

[0004] Based on this, this application was developed. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a florfenicol formulation for reversing drug resistance. This florfenicol formulation undergoes low-temperature pulverization, which avoids drug crystal transformation and prevents a decrease in drug solubility. Its formulation achieves optimal drug resistance reversal through a rational ratio of florfenicol and puerarin, and the product process is particularly suitable for industrial production.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned florfenicol preparation for reversing drug resistance.

[0007] A third objective of this invention is to provide clinical trial data for the above-mentioned florfenicol formulation that reverses drug resistance.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A florfenicol formulation for reversing drug resistance, wherein each 100 kg of the florfenicol formulation is made from the following raw materials in the following weight ratio: florfenicol 30-50 kg, puerarin 5-10 kg, magnesium stearate 0.5-1 kg, and silica to make up to 100 kg.

[0009] More preferably, the weight ratio of puerarin to florfenicol can be 1:5 to 1:6.

[0010] As a specific technical solution, the florfenicol preparation for reversing drug resistance is made from the following raw materials in the following weight ratio per 100 kg: florfenicol 30 kg, puerarin 6 kg, magnesium stearate 0.5 kg, and silica to make up to 100 kg.

[0011] As a specific technical solution, the florfenicol preparation for reversing drug resistance is made from the following raw materials in the following weight ratio per 100 kg: florfenicol 40 kg, puerarin 7 kg, magnesium stearate 0.8 kg, and silica to make up to 100 kg.

[0012] As a specific technical solution, the florfenicol preparation for reversing drug resistance is made from the following raw materials in the following weight ratio per 100 kg: florfenicol 50 kg, puerarin 9 kg, magnesium stearate 1 kg, and silica to make up to 100 kg.

[0013] A method for preparing the above-mentioned florfenicol formulation for reversing drug resistance includes the following steps: 1) Take florfenicol and pulverize it at low temperature; 2) Dry the puerarin; 3) Take the florfenicol obtained in step 1) and the puerarin obtained in step 2) and grind them in a double cone low-temperature co-grinding mill; 4) Take the mixture obtained in step 3) and add magnesium stearate and silicon dioxide into a V-type mixer and mix evenly (mix at 15~20 rpm for 10~20 minutes) to obtain the product of the present invention.

[0014] Specifically, in step 1), florfenicol is subjected to low-temperature pulverization at 0-20℃ until the particle size is ≤10μm.

[0015] Specifically, in step 2), puerarin is vacuum dried until the moisture content is ≤3%. The vacuum drying conditions are: vacuum degree of -0.08 ~ -0.098 MPa, and drying temperature of 40-60℃.

[0016] Specifically, the grinding conditions in step 3) are: -10±2℃, 200±50 rpm, 30±10min.

[0017] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: (1) The present invention implements low-temperature pulverization treatment for florfenicol, which can avoid the transformation of drug crystal form and prevent the drug dissolution rate from decreasing; (2) This invention uses a low-temperature co-grinding process to create a low-temperature environment of -10±2℃, which enables molecular-level mixing of heat-sensitive traditional Chinese medicine and Western medicine. This can improve synergistic efficiency and avoid the loss of activity caused by traditional granulation process. (3) The formulation of this invention uses a reasonable ratio of florfenicol and puerarin to achieve the best effect in reversing drug resistance; (4) This invention achieves the reversal of florfenicol resistance by using a simple formula, low temperature process and drug resistance mechanism to target and break through, while the product process is suitable for industrial production. Detailed Implementation

[0018] The technical solution of the present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0019] In the following examples, all raw materials used are common commercially available products that can be purchased directly, or can be prepared using conventional techniques in the art. Example 1

[0020] A florfenicol preparation for reversing drug resistance, the composition of which and the weight of raw materials used in 100kg of the finished florfenicol preparation are: florfenicol 30kg, puerarin 6kg, magnesium stearate 0.5kg, and silica to make up to 63.5kg.

[0021] The preparation method of the above-mentioned florfenicol preparation for reversing drug resistance is as follows: 1. Pulverize florfenicol at -10℃ until the particle size is ≤10μm; 2. Vacuum dry puerarin until the moisture content is ≤3%; the vacuum drying conditions are: vacuum degree -0.09 MPa, drying temperature 50℃; 3. Add florfenicol and puerarin to a double-cone low-temperature co-grinding mill and grind them (-10℃, 200rpm, 30min). 4. Take the mixture obtained in step 3, magnesium stearate, and silicon dioxide and add them to a V-type mixer. Mix (20 rpm) for 10 minutes to obtain the florfenicol preparation product of the present invention. Example 2

[0022] A florfenicol preparation for reversing drug resistance, the composition of which and the weight of raw materials used in 100kg of the finished florfenicol preparation are: florfenicol 40kg, puerarin 7kg, magnesium stearate 0.8kg, and silica to make up to 52.2kg.

[0023] The preparation method of the above-mentioned florfenicol preparation for reversing drug resistance is as follows: 1. Pulverize florfenicol at -10℃ until the particle size is ≤10μm; 2. Vacuum dry puerarin until the moisture content is ≤3%; the vacuum drying conditions are: vacuum degree -0.09 MPa, drying temperature 50℃; 3. Add florfenicol and puerarin to a double-cone low-temperature co-grinding mill and grind them (-10℃, 200rpm, 30min). 4. Take the mixture obtained in step 3, magnesium stearate, and silicon dioxide and add them to a V-type mixer. Mix (20 rpm) for 10 minutes to obtain the florfenicol preparation product of the present invention. Example 3

[0024] A florfenicol preparation for reversing drug resistance, the composition of which and the weight of raw materials used in 100kg of the finished florfenicol preparation are: 50kg florfenicol, 9kg puerarin, 1kg magnesium stearate, and silicon dioxide to a total of 40kg.

[0025] The preparation method of the above-mentioned florfenicol preparation for reversing drug resistance is as follows: 1. Pulverize florfenicol at -10℃ until the particle size is ≤10μm; 2. Vacuum dry puerarin until the moisture content is ≤3%; the vacuum drying conditions are: vacuum degree -0.09 MPa, drying temperature 50℃; 3. Add florfenicol and puerarin to a double-cone low-temperature co-grinding mill and grind them (-10℃, 200rpm, 30min). 4. Take the mixture obtained in step 3, magnesium stearate, and silicon dioxide and add them to a V-type mixer. Mix (20 rpm) for 10 minutes to obtain the florfenicol preparation product of the present invention.

[0026] Comparative Example 1 By changing the ratio of florfenicol to puerarin, the composition and the weight of raw materials used in 100kg of florfenicol preparation are as follows: florfenicol 30kg, puerarin 7.5kg, magnesium stearate 0.5kg, and silicon dioxide to make up to 62kg.

[0027] The preparation method of the above-mentioned florfenicol preparation is as follows: 1. Pulverize florfenicol at -10℃ until the particle size is ≤10μm; 2. Vacuum dry puerarin until the moisture content is ≤3%; the vacuum drying conditions are: vacuum degree -0.09 MPa, drying temperature 50℃; 3. Add florfenicol and puerarin to a double-cone low-temperature co-grinding mill and grind them (-10℃, 200rpm, 30min). 4. Take the mixture obtained in step 3, magnesium stearate, and silicon dioxide and add them to a V-type mixer. Mix (20 rpm) for 10 minutes to obtain the product.

[0028] Comparative Example 2

[0029] Without using low-temperature pulverization, the composition and raw material weights used in 100kg of florfenicol preparation are as follows: florfenicol 30kg, puerarin 6kg, magnesium stearate 0.5kg, and silica to make up to 63.5kg.

[0030] The preparation method of the above-mentioned florfenicol preparation is as follows: 1. Pulverize florfenicol at room temperature (25±5℃) until the particle size is ≤10μm; 2. Vacuum dry puerarin until the moisture content is ≤3%; the vacuum drying conditions are: vacuum degree -0.09 MPa, drying temperature 50℃; 3. Add florfenicol and puerarin to a double-cone low-temperature co-grinding mill and grind them (-10℃, 200rpm, 30min). 4. Take the mixture obtained in step 3, magnesium stearate, and silicon dioxide and add them to a V-type mixer. Mix (20 rpm) for 10 minutes to obtain the product.

[0031] Comparative Example 3 Without the low-temperature co-grinding process, the composition and raw material weights used in 100kg of florfenicol formulation are as follows: florfenicol 30kg, puerarin 7.5kg, magnesium stearate 0.5kg, and silica to make up to 62kg.

[0032] The preparation method of the above-mentioned florfenicol preparation is as follows: 1. Pulverize florfenicol at -10℃ until the particle size is ≤10μm; 2. Vacuum dry puerarin until the moisture content is ≤3%; the vacuum drying conditions are: vacuum degree -0.09 MPa, drying temperature 50℃; 3. Add florfenicol, puerarin, magnesium stearate, and silica to a V-type mixer and mix (20 rpm) for 10 minutes to obtain this product.

[0033] Comparative Example 4 A commercially available brand of florfenicol powder contains 30% florfenicol.

[0034] Experimental Example 1 1. Test materials

[0035] Florfenicol formulations prepared in Examples 1, 2, and 3.

[0036] Comparative Example 1: The ratio of florfenicol to puerarin was changed.

[0037] Comparative Example 2: No low-temperature pulverization was used.

[0038] Comparative Example 3: No low-temperature co-grinding process.

[0039] Comparative Example 4: A commercially available brand of florfenicol powder.

[0040] 2. Experimental methods and results 2.1 Resistance reversal effect (in vitro antibacterial test).

[0041] For clinically isolated porcine *Escherichia coli* strains resistant to florfenicol (MIC ≥ 128 μg / mL), the drug concentration (florfenicol) was set at 256 μg / mL. The inoculum size was 1 × 10⁻⁶. 5 After incubating at 37℃ for 24 hours with CFU / mL, the diameter of the inhibition zone was measured. The results are shown in Table 1.

[0042] Table 1 Antibacterial test data

[0043] As can be seen from Table 1, the average diameter of the inhibition zone in Examples 1, 2, and 3 reached 18.33 mm, which was not significantly different from Comparative Example 1, but was 19.80%, 39.92%, and 132.03% higher than Comparative Examples 2, 3, and 4, respectively.

[0044] Therefore, it can be inferred that increasing the ratio of puerarin to florfenicol does not significantly improve the antibacterial properties of the product. From a cost control perspective, the ratio used in the examples is more reasonable; it also shows that the low-temperature pulverization and low-temperature co-grinding methods in the process of this invention have the effect of enhancing antibacterial ability.

[0045] 2.2 Process Activity Retention Rate Sample preparation: Weigh the final product of each example, use methanol-water (volume ratio 70:30) as the extraction solvent, perform ultrasonic extraction at ≤30℃ for 30 minutes, and then filter it using a 0.45μm filter membrane.

[0046] Chromatographic conditions: Chromatographic column: C18 column (4.6×250mm, particle size 5μm) was selected. Mobile phase: Acetonitrile-0.1% phosphoric acid aqueous solution (volume ratio 25:75); The flow rate was set to 1.0 mL / min; the detection wavelength was determined to be 250 nm (for the detection of puerarin).

[0047] calculate: Puerarin retention rate (%) = Puerarin content in the final product / Puerarin amount in the feed × 100%.

[0048] Differential scanning calorimetry (DSC) was used to detect the crystal form of florfenicol and the degree of molecular mixing among its components. The results are shown in Table 2.

[0049] Table 2 Process Activity Retention Rate

[0050] As shown in Table 2, in Comparative Example 2, the florfenicol was not subjected to a low-temperature pulverization process, and its crystal form easily transformed from α-type to β-type. Compared to α-type florfenicol, the dissolution rate of β-type florfenicol decreased by 30%. In Comparative Example 3 (without a low-temperature co-grinding process), the florfenicol crystal form did not transform, but the mixing uniformity of the components was poor, and molecular-level mixing was not achieved.

[0051] 2.3 Clinical efficacy verification Fifty-six three-way crossbred piglets (Duroc × Landrace × Large White) weighing 20±2kg were selected. After preliminary testing, their mycoplasma antibody test was negative and their body temperature was below 39.5℃.

[0052] Florfenicol-resistant porcine Streptococcus pneumoniae (MIC ≥ 128 μg / mL) was isolated from clinical samples and inoculated onto tryptone-soy agar (TSA) blood plates containing 5% sheep blood. After 24 hours of anaerobic incubation at 37°C, colonies were washed off with physiological saline, and the bacterial concentration was adjusted to 1 × 10⁻⁶. 9 Colony forming units (CFU) / mL were injected into the sample pigs for infection. Within 48 hours post-infection, the sample pigs exhibited symptoms such as body temperature ≥40.5℃, respiratory rate >50 breaths / min, and cough score ≥2.

[0053] The sample pigs were randomly divided into 7 groups of 8 pigs each. In Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4, florfenicol was administered at a dose of 20 mg / kg and mixed into the basal diet, ensuring that the pigs consumed the feed within 2 hours.

[0054] The following experimental groups were set up: Blank control group (8 animals): No infection treatment or treatment was given; Infection control group (8 animals): Infection treatment was performed, but no further treatment was given; Example 1 group (8 heads): Infection treatment was performed, and the preparation of Example 1 was administered; Comparative Example 1 (8 animals): Infection treatment was performed, and the Comparative Example 1 preparation was administered. Comparative Group 2 (8 animals): Infection treatment was performed, and the Comparative Group 2 formulation was administered. Comparative Group 3 (8 animals): Infection treatment was performed, and the Comparative Group 3 preparation was administered. Comparative Group 4 (8 animals): Infection treatment was performed, and commercially available florfenicol powder was administered.

[0055] The monitoring indicators and methods are as follows: The body temperature, respiratory rate and cough index of pigs were measured daily, and their mental state and appetite were observed; computed tomography (CT) scans were performed before administration, on the 3rd day after administration and on the 7th day after administration; necropsy was performed on the 7th day after administration to detect the bacterial load (CFU / g) in the lung tissue.

[0056] The scoring criteria are as follows: Cough Index: A 0-3 rating system is used (0 indicates no cough symptoms, 3 indicates persistent cough).

[0057] Mental state / appetite: A 0-2 level rating system is used (0 indicates normal, 2 indicates complete loss of appetite).

[0058] Imaging (CT scan) scoring: Consolidation is scored from 0 to 4 points (based on the proportion of lung lobe affected); Ground-glass opacity is scored from 0 to 3 points (based on density and extent); Air bronchogram is scored from 0 to 2 points (based on the number of branches).

[0059] Lung tissue bacterial load: 1g of tissue from the central region of the right apical lobe was taken and tested using serial dilution and plate culture counting methods. The results are expressed as CFU / g (colony forming units per gram).

[0060] Cure rate calculation criteria: On the 7th day after the end of drug administration, the pigs' body temperature was below 39.5℃, the cough index was 0, and the CT score improvement was >80%. The results are shown in Table 3.

[0061] Table 3 Clinical trial data

[0062] As shown in Table 3, on the 7th day after administration of the product of the present invention, the body temperature of pigs returned to normal, the bacterial load in lung tissue decreased by 30.56%, 34.21%, and 56.89% compared with Comparative Examples 2, 3, and 4, respectively, the total CT score decreased by 21.05%, 28.57%, and 81.01% compared with Comparative Examples 2, 3, and 4, respectively, and the cure rate increased by 26.08%, 40%, and 133% compared with Comparative Examples 2, 3, and 4, respectively.

[0063] Therefore, the florfenicol preparation of this invention exhibits significant advantages in terms of antipyresis, sterilization, improvement in imaging, and cure rate. Although Comparative Example 1 also shows obvious advantages, the present invention is the optimal solution from a cost perspective.

Claims

1. A florfenicol formulation for reversing drug resistance, characterized in that, Each 100 kg of the florfenicol preparation is made from the following raw materials in the indicated weight ratios: florfenicol 30-50 kg, puerarin 5-10 kg, magnesium stearate 0.5-1 kg, and silica to make up to 100 kg.

2. The florfenicol formulation for reversing drug resistance as described in claim 1, characterized in that, The weight ratio of puerarin to florfenicol is 1:5 to 1:

6.

3. The florfenicol formulation for reversing drug resistance as described in claim 1, characterized in that, Each 100 kg of the florfenicol preparation is made from the following raw materials in the indicated weight ratios: florfenicol 30 kg, puerarin 6 kg, magnesium stearate 0.5 kg, and silica to make up to 100 kg.

4. The florfenicol formulation for reversing drug resistance as described in claim 1, characterized in that, Each 100 kg of the florfenicol preparation is made from the following raw materials in the indicated weight ratios: florfenicol 40 kg, puerarin 7 kg, magnesium stearate 0.8 kg, and silica to make up to 100 kg.

5. The florfenicol formulation for reversing drug resistance as described in claim 1, characterized in that, Each 100 kg of the florfenicol preparation is made from the following raw materials in the indicated weight ratios: 50 kg of florfenicol, 9 kg of puerarin, 1 kg of magnesium stearate, and silica to make up to 100 kg.

6. The method for preparing the florfenicol formulation for reversing drug resistance according to any one of claims 1 to 5, characterized in that, Includes the following steps: 1) Take florfenicol and pulverize it at low temperature; 2) Dry the puerarin; 3) Grind the florfenicol obtained in step 1) and the puerarin obtained in step 2); 4) Take the mixture obtained in step 3) and mix it evenly with magnesium stearate and silicon dioxide to obtain the final product.

7. The method for preparing the florfenicol formulation for reversing drug resistance as described in claim 6, characterized in that, In step 1), florfenicol is subjected to low-temperature pulverization at 0-20℃ until the particle size is ≤10μm.

8. The method for preparing the florfenicol formulation for reversing drug resistance as described in claim 6, characterized in that, In step 2), puerarin is vacuum dried until the moisture content is ≤3%.

9. The method for preparing the florfenicol formulation for reversing drug resistance as described in claim 6, characterized in that, The grinding conditions in step 3) are: -10±2℃, 200±50 rpm, 30±10min.

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