A long-acting compound preparation of vonimulin, its preparation method and application

Abstract

This invention belongs to the pharmaceutical field, specifically relating to a long-acting compound preparation of vonimuramin, its preparation method, and its application. The long-acting compound preparation of vonimuramin of this invention comprises the following components and their mass percentages: vonimuramin 5%–10%, doxycycline 10%–30%, tartaric acid 0.5%–2.0%, oil phase 10%–20%, emulsifier 10%–30%, co-emulsifier 2%–10%, and the balance being water. This invention, by preparing vonimuramin and doxycycline with corresponding excipients into a nanoemulsion, can effectively delay the release rate of vonimuramin, enabling the two drugs to be eliminated simultaneously in vivo, thus achieving a synergistic antibacterial effect. Furthermore, the drugs are encapsulated in the nanoemulsion core, which improves the solubility of vonimuramin in water while preventing doxycycline from forming insoluble complexes with metal ions in water, thereby improving the stability of the drugs.

Description

Technical Field

[0001] This invention belongs to the pharmaceutical field, specifically relating to a long-acting compound preparation of vonimulin, its preparation method, and its application. Background Technology

[0002] As livestock and poultry farming develops towards intensification, large-scale operation, and high efficiency, the demands and challenges of disease prevention and control place higher demands on veterinary drugs. For example, high-density farming increases the incidence of animal diseases, which spread rapidly and are mainly characterized by mixed infections. This necessitates that drugs be used to achieve prevention and control goals, and that they be highly effective and broad-spectrum. Therefore, in high-efficiency farming, improving efficiency is key, which in turn requires reducing the frequency of drug administration and lowering labor intensity. The rapid development of farming has also brought risks such as food safety and bacterial resistance, which necessitates resolving the prominent contradiction between disease prevention and control and drug use.

[0003] Mixed infections of Gram-positive cocci, Gram-negative bacilli, and mycoplasma are common in livestock farming. However, most drugs have specific antibacterial spectra and few can simultaneously provide highly effective control against different pathogens. Therefore, multiple drugs need to be used in combination for disease prevention and control. However, due to the shortage of clinical veterinarians in livestock farming, accurate drug combinations are rarely performed, leading to a series of problems such as treatment failure, drug waste, increased veterinary drug residues, and even the rapid development of bacterial resistance.

[0004] In recent years, mixed infections of mycoplasma and bacteria have frequently occurred in poultry farming clinical practice, seriously affecting the healthy development of the poultry industry. Vonnemulin is a truncated pleurotin class of animal-specific antibiotics. Its main mechanism of action is to bind to the 50S subunit of the ribosomes of pathogenic microorganisms, inhibiting protein synthesis and thus exerting an antibacterial effect. Vonnemulin has a strong inhibitory effect on various pathogenic mycoplasmas, such as *Mycoplasma gallisepticum* and *Mycoplasma synoviae*, as well as Gram-positive bacteria, but it is ineffective against Gram-negative bacteria. Doxycycline is a tetracycline antibiotic whose mechanism of action is to bind to the 30S subunit of the ribosomes of pathogenic microorganisms, inhibiting the synthesis of pathogenic proteins and achieving a bactericidal effect. Clinically, it is mainly used for the prevention and treatment of Gram-negative bacterial infections. Vonnemulin and doxycycline are complementary in their antibacterial mechanisms and antibacterial spectrum, and can produce a synergistic effect when used together.

[0005] Pharmacokinetic studies have shown that vonnimirin and doxycycline are rapidly absorbed by chickens after oral administration, reaching peak concentrations in 1–2 hours and 2–5 hours, respectively. However, their elimination rates in chickens differ significantly: vonnimirin is eliminated very quickly, with an elimination half-life of 2–4 hours, while doxycycline is eliminated more slowly, with an elimination half-life of 9–13 hours. Therefore, when used in combination, their elimination rates are inconsistent, making it impossible to maintain the same dosing interval. This is a key issue hindering the development of vonnimirin and doxycycline combination formulations. Furthermore, modern high-efficiency farming methods favor convenient drinking water administration. However, the water solubility of vonnimirin is insufficient for high-efficiency drinking water administration requirements, and doxycycline is sensitive to metal ions in water and has poor stability in water. This is another challenge in the development of vonnimirin and doxycycline combination formulations.

[0006] In summary, balancing the half-lives of vornimulin and doxycycline, improving their water solubility, and developing new compound preparations are effective measures to address the needs of prevention and treatment of mixed infectious diseases. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a long-acting compound preparation of vonimoleline, which eliminates vonimoleline and doxycycline at similar rates and improves the solubility and stability of doxycycline and vonimoleline in water, effectively solving the problem of mixed infections in poultry.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A long-acting compound preparation of vonimoleline comprises the following components and their mass percentages: vonimoleline 5%–10%, doxycycline 10%–30%, tartaric acid 0.5%–2.0%, oil phase 10%–20%, emulsifier 10%–30%, co-emulsifier 2%–10%, and the balance being water.

[0010] Vonnemulin and doxycycline readily form flocculent precipitates when mixed and dissolved in water, resulting in poor drug properties when formulated into a compound preparation. During the experimental process, this invention discovered that adding an appropriate amount of tartaric acid significantly improves the compatibility of the two drugs in water, making it easier to formulate a compound preparation. By adding an oil phase, emulsifier, and co-emulsifier, vonnemulin and doxycycline can be encapsulated in a nanoemulsion, which not only improves the solubility and stability of the drugs in water but also effectively slows down the release rate of vonnemulin, allowing the two drugs to be eliminated simultaneously in vivo.

[0011] Preferably, the long-acting compound preparation of vonimoleline is composed of the following components and their mass percentages: 10% vonimoleline, 30% doxycycline, 2.0% tartaric acid, 20% oil phase, 10% emulsifier, 8% co-emulsifier, and the balance being water.

[0012] Preferably, the vonimium is selected from its hydrochloride or tartrate salt; the doxycycline is selected from its hydrochloride salt.

[0013] Preferably, the mass ratio of vonimolecline to doxycycline is 1:3 to 4.

[0014] Preferably, the oil phase is triacetin; the emulsifier is Tween-80; and the co-emulsifier is propylene glycol.

[0015] In the formulation of nanoemulsions, the inventors discovered that oil-phase triacetyl ester is the main component of the hydrophobic core in the nanoemulsion structure. The resulting nanocore can effectively encapsulate vornimulin and doxycycline, achieving a sustained-release and long-lasting effect. When Tween-80 is selected as the emulsifier and propylene glycol as the co-emulsifier, it can react with oil-phase triacetyl ester under high-intensity mechanical force to form a more stable nanoemulsion.

[0016] This invention also provides a method for preparing the aforementioned long-acting compound preparation of Vornimelin, comprising the following steps:

[0017] S1. Add tartaric acid to water and stir until homogeneous. Then add emulsifier and co-emulsifier and continue mixing and stirring until homogeneous to obtain mixture I.

[0018] S2. Add vonnimulin and doxycycline to the mixture I obtained in step S1, stir and mix evenly to completely dissolve the drugs, and obtain mixture II.

[0019] S3. Add the oil phase to the mixture II obtained in step S2, stir and mix evenly to obtain mixture III;

[0020] S4. The mixture III obtained in step S3 is subjected to high-speed shearing to obtain the final product.

[0021] The long-acting compound preparation of vonimium provided by this invention, after the components are mixed evenly, is subjected to high-speed shearing, through centrifugal force and shear force, so that the drug, emulsifier, co-emulsifier and oil phase are fully homogenized and emulsified, so as to form a uniform and stable nanoemulsion.

[0022] Preferably, the mixing conditions in step S1 are mixing at 300-800 rpm for 10-15 minutes; and the conditions for continuing mixing are mixing at 500-800 rpm for 15-20 minutes.

[0023] Preferably, the mixing conditions described in steps S2 and S3 are: stirring at a speed of 500-800 rpm for 15-25 minutes.

[0024] Preferably, the high-speed shearing conditions described in step S4 are homogeneous shearing at a rotation speed of 7000–9000 rpm for 15–20 min.

[0025] This invention also provides the application of the aforementioned long-acting compound preparation of vonimium in the preparation of veterinary drugs adapted to efficient farming models.

[0026] Compared with the prior art, the present invention has the following technical advantages:

[0027] (1) This invention is a compound preparation composed of vonimium and doxycycline. The antibacterial mechanisms and antibacterial spectra of the two are complementary. When used together, they can achieve synergistic antibacterial, broaden the antibacterial spectrum and enhance efficacy. Furthermore, the use of tartaric acid effectively solves the problems of poor water solubility and poor compatibility of vonimium and doxycycline, and significantly improves the drug-likeness of the vonimium-doxycycline compound preparation.

[0028] (2) The present invention prepares vonimium and doxycycline into a compound nanoemulsion, which can effectively delay the release rate of vonimium and enable vonimium and doxycycline to be eliminated simultaneously in the body, thereby achieving the effect of synergistic antibacterial maximization.

[0029] (3) The present invention prepares vonimoleline and doxycycline into a compound nanoemulsion, which not only improves the solubility and stability of vonimoleline in water, but also avoids contact between doxycycline and metal ions in water, thus improving its stability in water.

[0030] (4) The compound nanoemulsion provided by the present invention can be completely dissolved in water in any proportion and can tolerate different water qualities, which can fully meet the needs of modern high-efficiency aquaculture drinking water administration. Attached Figure Description

[0031] Figure 1 This is a graph showing the sample property detection results of Example 1 of the present invention;

[0032] Figure 2 The figures show the morphological detection results of Comparative Example 3 (left) and Comparative Example 4 (right) of this invention. Detailed Implementation

[0033] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention. Where specific techniques or conditions are not specified in this embodiment, they shall be operated in accordance with conventional technical methods and instrument manuals in the art; where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0034] Example 1: A long-acting compound preparation of vonimulin

[0035] The long-acting compound preparation of Vonimulin comprises the following components and their mass percentages: Vonimulin 5%, doxycycline 15%, tartaric acid (DL-tartaric acid, purchased from Hunan Ercon Pharmaceutical Co., Ltd., the same below) 1.0%, triacetin 15%, Tween-80 20%, propylene glycol 10%, and the balance being water.

[0036] The preparation method of the long-acting compound preparation of vonimuramin is as follows: 1.0 g tartaric acid is added to 30 mL of water and stirred at 500 rpm for 10 min until homogeneous. Then, 20 g Tween-80 and 10.0 g propylene glycol are added and stirred at 500 rpm for 10 min until homogeneous. 5.8 g vonimuramin hydrochloride (5.0 g as vonimuramin) and 16.3 g doxycycline hydrochloride (15.0 g as doxycycline) are added and stirred at 500 rpm for 20 min until the drugs are completely dissolved. 15.0 g triacetylglycerol is added and stirred at 500 rpm for 20 min. The mixture is then subjected to high-speed shearing at 8000 rpm for 15 min. The volume is adjusted to 100 mL with water and stirred until homogeneous to obtain the final product.

[0037] Example 2: A long-acting compound preparation of vonimulin

[0038] The long-acting compound preparation of Vornimirin comprises the following components and their mass percentages: Vornimirin 10%, doxycycline 30%, tartaric acid 2.0%, triacetin 20%, Tween-80 10%, propylene glycol 8%, and the balance being water.

[0039] The preparation process of the long-acting compound preparation of Vornemirin is as follows: 2.0g of tartaric acid is added to 30mL of water and stirred at 500rpm for 10min until it is evenly mixed. Then, 10.0g of Tween-80 and 8.0g of propylene glycol are added and stirred at 500rpm for 10min until it is evenly mixed. Next, 11.6g of Vornemirin hydrochloride (10.0g based on Vornemirin) and 32.6g of doxycycline hydrochloride (30.0g based on doxycycline) are added and stirred at 500rpm for 20min until the drugs are completely dissolved. Then, 20.0g of triacetylglycerol is added and stirred at 500rpm for 20min. The mixture is then subjected to high-speed shearing at 8000rpm for 15min. The volume is adjusted to 100mL with water and stirred evenly to obtain the final product.

[0040] Example 3: A long-acting compound preparation of vonimulin

[0041] The long-acting compound preparation of Vonimulin comprises the following components and their mass percentages: Vonimulin 7%, doxycycline 28%, tartaric acid 1.5%, triacetin 15%, Tween-80 20%, propylene glycol 10%, and the balance being water.

[0042] The preparation process of the long-acting compound preparation of Vornimirin is as follows: 1.5g of tartaric acid is added to 30mL of water and stirred at 500rpm for 10min until it is evenly mixed. Then, 20.0g of Tween-80 and 10.0g of propylene glycol are added and stirred at 500rpm for 10min until it is evenly mixed. Next, 8.1g of Vornimirin hydrochloride (7.0g based on Vornimirin) and 30.4g of doxycycline hydrochloride (28.0g based on doxycycline) are added and stirred at 500rpm for 20min until the drugs are completely dissolved. Then, 15.0g of triacetin is added and stirred at 500rpm for 20min. The mixture is then subjected to high-speed shearing at 8000rpm for 15min. The volume is adjusted to 100mL with water and stirred evenly to obtain the final product.

[0043] Comparative Example 1: A solution of vornimulin hydrochloride

[0044] Weigh 5.8g of vonimium hydrochloride (5.0g as vonimium), add it to 75mL of water, stir at 500rpm for 10min until completely dissolved, and dilute to 100mL with water.

[0045] Comparative Example 2: A Doxycycline Hydrochloride Solution

[0046] Weigh 16.3g of doxycycline hydrochloride (15.0g as doxycycline), add it to 75mL of water, stir at 500rpm for 10min until completely dissolved, and dilute to 100mL with water.

[0047] Comparative Example 3: A combination solution of valerium and doxycycline

[0048] The vonimium-doxycycline complex solution comprises 10% vonimium, 30% doxycycline, and the balance being water.

[0049] The specific preparation process is as follows: Weigh 11.6g of vonimium hydrochloride (10.0g as vonimium hydrochloride) and 32.6g of doxycycline hydrochloride (30.0g as doxycycline), add them to 75mL of water, stir at 500rpm for 20min, and then dilute to 100mL with water.

[0050] Comparative Example 4: A combination solution of valerium and doxycycline

[0051] The vonimium-doxycycline complex solution comprises 10% vonimium, 30% doxycycline, 2.0% citric acid, and the balance being water.

[0052] The specific preparation process is as follows: Weigh 11.6g of vonimium hydrochloride (10.0g as vonimium) and 32.6g of doxycycline hydrochloride (30.0g as doxycycline), add them to 75mL of water, then add 2.0g of citric acid, stir at 500rpm for 20min, and dilute to 100mL with water.

[0053] Comparative Example 5: A combination solution of valerium and doxycycline

[0054] The vonimium-doxycycline complex solution comprises 10% vonimium, 30% doxycycline, 2.0% tartaric acid, and the balance being water.

[0055] The specific preparation process is as follows: Add 2.0g of tartaric acid to 75mL of water, stir at 500rpm for 10min until the mixture is uniform, then add 11.6g of vonimium hydrochloride (10.0g based on vonimium) and 32.6g of doxycycline hydrochloride (30.0g based on doxycycline), stir at 500rpm for 20min to completely dissolve the drugs, and then dilute with water to 100mL.

[0056] Comparative Example 6: A Vornemirin Compound Formulation

[0057] The vonimulin compound preparation comprises the following components and their mass percentages: vonimulin 10%, doxycycline 30%, tartaric acid 2.0%, glyceryl stearate 20%, Tween-80 10%, glycerol 8%, and the balance being water.

[0058] The preparation process of the vonimium compound preparation is as follows: 2.0g tartaric acid is added to 30mL of water and stirred at 500rpm for 10min until it is evenly mixed. Then, 10.0g Tween-80 and 8.0g glycerol are added and stirred at 500rpm for 10min until it is evenly mixed. Next, 11.6g vonimium hydrochloride (10.0g based on vonimium) and 32.6g doxycycline hydrochloride (30.0g based on doxycycline) are added and stirred at 500rpm for 20min until the drugs are completely dissolved. Then, 20.0g glyceryl stearate is added and stirred at 500rpm for 20min. The mixture is then subjected to high-speed shearing at 8000rpm for 15min. The volume is adjusted to 100mL with water and stirred evenly to obtain the final product.

[0059] Test Example 1: Sample Clarity Detection

[0060] 1. Test samples: Products obtained from Examples 1-3 and Comparative Examples 1-6.

[0061] 2. Test method: Take 30 mL of each of the above product solutions and place them in beakers. The entire process is aseptic. After standing for 24 hours, observe the color change of each sample.

[0062] 3. Experimental results: The specific experimental results are shown in Table 1 below.

[0063] Table 1. Results of morphological investigation of samples from different embodiments and comparative examples.

[0064] Serial Number Sample properties Properties after standing for 24 hours Example 1 Yellow clear solution Figure 1 ) Yellow clear solution Example 2 Yellow clear solution Yellow clear solution Example 3 Yellow clear solution Yellow clear solution Comparative Example 1 colorless clear solution A white precipitate formed at the bottom. Comparative Example 2 Yellow clear solution Yellow clear solution Comparative Example 3 Yellowish turbid solution with white floating matter on the surface. / Comparative Example 4 Yellow turbid solution with white floating matter on the surface. / Comparative Example 5 Yellow clear solution Yellow clear solution Comparative Example 6 Yellow clear solution Yellow clear solution

[0065] As shown in Table 1 above, after vornimulin hydrochloride dissolves in water, a white precipitate forms after 24 hours, indicating that it is unstable in water (Comparative Example 1); when vornimulin hydrochloride and doxycycline hydrochloride are mixed in water, they do not completely dissolve, forming a yellow turbid solution with white floating matter on the surface (Comparative Example 3). Figure 2 (Left figure); After replacing tartaric acid with citric acid, the solution stability was slightly better than that of Comparative Example 3, but still much worse than that of the Example group, and white floating matter could be clearly seen (Comparative Example 4). Figure 2 (Right figure); Adding an appropriate amount of tartaric acid can significantly improve the solubility and compatibility of vornimulin hydrochloride and doxycycline hydrochloride in water, and both can be completely dissolved (Comparative Example 5 and Comparative Example 6). After vornimulin hydrochloride and doxycycline hydrochloride are made into a compound nanoemulsion, the sample is a clear yellow solution and is relatively stable. No precipitation or other issues were observed after long-term storage (Examples 1 to 3).

[0066] Test Example 2: Dilution Tolerance in Water

[0067] Considering the complexity of clinical water quality in aquaculture, the dilution tolerance of Examples 1-3 was investigated in purified water and standard hard water to prevent drug precipitation after dilution, which could affect efficacy and clog drinking water pipes, thus impacting drug use in aquaculture. Comparative Examples 1, 2, 5, and 6 were used as control groups for comparison.

[0068] 1. Test samples: Products prepared from Examples 1-3, Comparative Examples 1-2, and Comparative Examples 5-6;

[0069] 2. Experimental procedure: Samples from Examples 1, 2, 3, Comparative Examples 1, 2, 5, and 6 were dissolved in purified water and standard hard water at ratios of 1:10, 1:100, and 1:500, respectively. The solutions were stirred thoroughly at 500 rpm and allowed to stand overnight at room temperature. The dilution tolerance of the sample solutions in purified water and standard hard water was then observed.

[0070] 3. Test results: The specific test results are shown in Tables 2 to 4 below.

[0071] Table 2 Results of dilution tolerance test in water (1:10)

[0072]

[0073] Table 3 Results of dilution tolerance test in water (1:100)

[0074]

[0075]

[0076] Table 4 Results of dilution tolerance test in water (1:500)

[0077]

[0078] As shown in Tables 2-4 above, after dilution with water, Comparative Example 1 samples all became turbid after being left overnight, indicating that the dilution properties of vornimulin hydrochloride aqueous solution in water are poor. Although Comparative Examples 2 and 5 can be diluted in purified water at any ratio, these samples all became turbid or precipitated after dilution in standard hard water, indicating that these samples have poor tolerance to water quality and cannot meet the complex requirements of aquaculture clinical water. Although Comparative Example 6 also added oil phase, emulsifier, and co-emulsifier, it also became turbid or precipitated due to the change in composition. In contrast, Examples 1-3 can dissolve in purified water and standard hard water at any ratio, and no turbidity or precipitation was observed after standing overnight at room temperature, indicating that preparing vornimulin hydrochloride and doxycycline hydrochloride into a nanoemulsion can improve their stability in water and tolerance to different water qualities.

[0079] Experimental Example 3: Stability Test in Water

[0080] 1. Test samples: the long-acting compound preparations of Vonimulin prepared in Examples 1-3;

[0081] 2. Experimental procedure: Following the method in Experiment 2, the drug content in the diluents of Examples 1 to 3 with different water, different times and different dilutions was measured, and the changes in drug content in the nanoemulsion were observed.

[0082] 3. Experimental results: The specific experimental results are shown in Table 5.

[0083] Table 5. Results of drug content in water stability study (unit: %)

[0084]

[0085] As can be seen from the results in Table 5 above, after being diluted with purified water and standard hard water at different dilution concentrations in Examples 1 to 3, the drugs showed good stability, and no obvious degradation reaction was observed after being left overnight.

[0086] Test Example 4: Drug Stability

[0087] In accordance with the stability testing guidelines in the Chinese Veterinary Pharmacopoeia (2020 edition), stability evaluation tests were conducted on the vonimuramin-doxycycline compound nanoemulsions in Examples 1 and 2, with Comparative Examples 5 and 6 serving as control groups for comparison.

[0088] 4.1 High Temperature Test

[0089] Samples from Examples 1, 2, 3, Comparative Example 5, and Comparative Example 6 were aliquoted, sealed, and placed at 60°C for 10 days. Samples were taken on day 0 and day 10 to determine the drug content. The results are shown in Table 6 below.

[0090] Table 6. Results of drug content in high-temperature tests (unit: %)

[0091]

[0092] As shown in Table 6 above, after the vornimulin hydrochloride and doxycycline hydrochloride composite aqueous solution was placed under high temperature conditions for 10 days, the drug content of both vornimulin and doxycycline decreased by more than 4% (Comparative Example 5); in the nanoemulsion obtained by emulsification with other emulsifiers, the drug content of both vornimulin and doxycycline decreased by about 3%; while in the vornimulin hydrochloride and doxycycline hydrochloride composite nanoemulsion, after being placed under the same high temperature conditions for 10 days, the decrease in the content of both drugs was less than 1% (Examples 1 to 3). Therefore, it can be seen that the nanoemulsion prepared according to the formulation of the present invention can effectively improve the drug stability of vornimulin and doxycycline.

[0093] 4.2 Accelerated Testing:

[0094] Samples from Examples 1, 2, 3, Comparative Example 5, and Comparative Example 6 were aliquoted, sealed, and placed under accelerated testing conditions of 40℃±2℃ and 75%±5% relative humidity for 6 months. Samples were taken at months 0, 1, 3, and 6 for drug content testing. The test results are shown in Table 7 below.

[0095] Table 7 Results of accelerated drug content in the test (unit: %)

[0096]

[0097]

[0098] As shown in Table 7 above, after 6 months of accelerated testing, the content of both vonimium hydrochloride and doxycycline in the aqueous solution of vonimium hydrochloride decreased by more than 5% (Comparative Example 5); in the nanoemulsion obtained by emulsification with other emulsifiers, the content of both vonimium hydrochloride and doxycycline decreased by more than 3%; while in the vonimium hydrochloride and doxycycline hydrochloride compound nanoemulsion, after 10 days of storage under the same high temperature conditions, the decrease in the content of both drugs was less than 2% (Examples 1-3). Compared with the aqueous solution of vonimium hydrochloride and doxycycline hydrochloride, the vonimium hydrochloride and doxycycline hydrochloride nanoemulsion showed better stability under accelerated testing conditions.

[0099] Experimental Example 5: Pharmacokinetic Test

[0100] 1. Test samples: Samples prepared in Example 2, Comparative Examples 1-2 and Comparative Examples 5-6;

[0101] 2. Experimental animals: Commercial broiler chickens, weighing 1.30-1.50 kg, half male and half female, with no obvious adverse reactions, a total of 50 chickens, were randomly divided into 5 groups;

[0102] 3. Experimental Procedure: Animals were randomly divided into four groups: Example 2 experimental group, Comparative Example 1 experimental group, Comparative Example 2 experimental group, Comparative Example 5 experimental group, and Comparative Example 6 experimental group. Ten animals were included in each experimental group. All animals were administered medication orally via gavage according to the dosing regimen in Table 8.

[0103] Table 8 Dosing regimens for pharmacokinetic studies

[0104]

[0105]

[0106] Blood samples were collected from each experimental group before administration and at 0.25h, 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 16h, 18h, 24h, and 36h after administration. Plasma was separated and extracted with 50% acetonitrile. The concentrations of vornimulin and doxycycline in plasma at different time points were determined by high performance liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were fitted using WinNonlin software. The results are shown in Table 9.

[0107] Table 9. Main pharmacokinetic parameters of each experimental group

[0108]

[0109] The results showed that the pharmacokinetic parameters of the vornimulin hydrochloride aqueous solution (Comparative Example 1) and the doxycycline hydrochloride aqueous solution (Comparative Example 2) experimental groups were not significantly different. Compared with the vornimulin hydrochloride aqueous solution (Comparative Example 1) experimental group, the time to peak concentration (T0) of vornimulin hydrochloride in chicken plasma was significantly shorter after vornimulin hydrochloride was formulated into a nanoemulsion (Example 2). max ) and elimination half-life (T 1 / 2β The time to peak concentration (T0) was significantly prolonged, resulting in a long-lasting effect and a 26.41% increase in bioavailability. Compared to the doxycycline hydrochloride aqueous solution (Comparative Example 2) experimental group, the time to peak concentration (T0) was significantly longer after doxycycline hydrochloride was formulated into a nanoemulsion (Example 1). max ) and elimination half-life in chickens (T 1 / 2βThe time to peak concentration (Tc) of vornimulin hydrochloride and doxycycline hydrochloride in chicken plasma was also prolonged, and the bioavailability increased by 10.62%. Similarly, compared with the vornimulin hydrochloride and doxycycline hydrochloride combined aqueous solution (Comparative Example 5) experimental group, the time to peak concentration (Tc) of vornimulin and doxycycline in chicken plasma was longer in the vornimulin hydrochloride and doxycycline hydrochloride combined nanoemulsion experimental group (Example 1) experimental group. max ) and elimination half-life (T 1 / 2β The bioavailability was significantly prolonged, with increases of 28.85% and 15.62%, respectively. Compared to Comparative Example 6, the half-life (T0) of vornimulin hydrochloride and doxycycline hydrochloride compound nanoemulsion prepared in Example 2 was significantly extended. 1 / 2β It also showed a significant extension, and bioavailability increased by 17.71%.

[0110] In the experimental group of Example 2, compared with doxycycline hydrochloride, the peak time and elimination half-life of vornimulin hydrochloride after being made into a nanoemulsion were significantly prolonged in vivo. The reasons may be: (1) Doxycycline hydrochloride has better solubility in water than vornimulin hydrochloride, and it is easier to dissolve and release in vivo; (2) The hydroxyl groups on the four-membered ring of doxycycline hydrochloride are easy to form intramolecular hydrogen bonds, which are sterically hindered; while the amino groups on the side chain of vornimulin hydrochloride have less steric hindrance, which is more conducive to its tight binding with the excipients through intermolecular hydrogen bonding or van der Waals forces, which is conducive to its slow release from the lipophilic nanocore formed by the excipients, thus achieving the goal of longer effect.

[0111] The pharmacokinetic parameters above show that when vonimoleline and doxycycline are formulated into a compound nanoemulsion, the elimination half-life of vonimoleline in vivo is significantly prolonged. The elimination half-lives of the two are closer, which is more conducive to maximizing their synergistic antibacterial effect.

[0112] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A long-acting compound preparation of vonimulin, characterized in that, The product comprises the following components and their mass percentages: 5%~10% vonnimirin, 10%~30% doxycycline, 0.5%~2.0% tartaric acid, 10%~20% oil phase, 10%~30% emulsifier, 2%~10% co-emulsifier, and the balance being water; vonnimirin is selected from its hydrochloride or tartrate salt; doxycycline is selected from its hydrochloride salt; the oil phase is triacetin; the emulsifier is Tween-80; and the co-emulsifier is propylene glycol.

2. The long-acting compound preparation of Vornimelin as described in claim 1, characterized in that, It consists of the following components and their mass percentages: 10% vonnimirin, 30% doxycycline, 2.0% tartaric acid, 20% oil phase, 10% emulsifier, 8% co-emulsifier, and the balance being water.

3. The long-acting compound preparation of Vornimelin as described in claim 1 or 2, characterized in that, The mass ratio of vonimolecline to doxycycline is 1:3~4.

4. A method for preparing a long-acting compound preparation of Vornimelin as described in any one of claims 1-3, characterized in that, The process includes the following: S1. Add tartaric acid to water and stir until homogeneous. Then add emulsifier and co-emulsifier and continue mixing and stirring until homogeneous to obtain mixture I. S2. Add vonnimulin and doxycycline to the mixture I obtained in step S1, stir and mix evenly to completely dissolve the drugs, and obtain mixture II. S3. Add the oil phase to the mixture II obtained in step S2, stir and mix evenly to obtain mixture III; S4. The mixture III obtained in step S3 is subjected to high-speed shearing to obtain the final product.

5. The preparation method according to claim 4, characterized in that, The mixing conditions described in step S1 are to stir at a speed of 300-800 rpm for 10-15 minutes; the conditions for continuing mixing and stirring are to stir at a speed of 500-800 rpm for 15-20 minutes.

6. The preparation method according to claim 4, characterized in that, The mixing conditions described in steps S2 and S3 are to stir at a speed of 500-800 rpm for 15-25 minutes to achieve uniform mixing.

7. The preparation method according to claim 4, characterized in that, The high-speed shearing conditions described in step S4 are homogeneous shearing at a speed of 7000~9000 rpm for 15~20 min.

8. The use of the long-acting compound preparation of vonimium as described in any one of claims 1-3 in the preparation of a veterinary drug for the prevention and treatment of mixed mycoplasma and bacterial infections.

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