A kind of tartrate tyvalocin nanosuspension and its preparation method and application

Abstract

The present invention relates to a tartrate tyvalosin nanosuspension and a preparation method and application thereof. The raw materials for preparing the tartrate tyvalosin nanosuspension include: tartrate tyvalosin, a cosolvent, an alkaline reagent, a stabilizer, a crystallization aid and water; the crystallization aid includes any one of chitosan, povidone, polysorbate or sodium carboxymethyl cellulose or a combination of at least two thereof. The tartrate tyvalosin nanosuspension provided by the present invention is a new dosage form of tartrate tyvalosin, which can significantly improve the solubility, dissolution rate and bioavailability of tartrate tyvalosin, and has good application prospects in the preparation of drugs for treating respiratory and digestive tract diseases of poultry and livestock.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a tyvalocin tartrate nanosuspension and a preparation method and application thereof. Background Art

[0002] Tylosin tartrate, also known as acetylisovaleryl tylosin tartrate, is a third-generation macrolide antibiotic for animals. It is a derivative generated by chemical modification of tylosin A. It mainly acts on Gram-positive bacteria and mycoplasma, and has a significant inhibitory effect on some Gram-negative bacteria. At present, the premix and soluble powder of tylosin tartrate have been approved in China and are widely used in the treatment of respiratory and digestive tract diseases of poultry and livestock.

[0003] Tyvalosin has the advantages of high efficiency, low toxicity, low residue, etc., and will not produce cross-resistance between macrolide antibiotics. However, the solubility of tylvalosin tartrate raw materials in water is difficult to reach the high concentration required by the current large-scale aquaculture using dosing devices. Tylvalosin tartrate dissolves in water, and its water solubility is measured to be around 5000ppm depending on the source of the raw materials. In clinical applications, because tylvalosin tartrate is obtained from raw materials through biological fermentation, floccules will appear in the high-concentration aqueous solution of its preparation, and it takes a long time to stir to dissolve completely. The most common way of drug administration in large-scale farms is through drinking water. The floccules in the preparation will block the water line and the drinking head, which greatly limits the clinical application of the product, and cannot meet the modern aquaculture industry's requirements for the stability of drugs at high concentrations. It is urgent to improve its solubility, compliance and stability.

[0004] Therefore, providing a tartrate tyvalocin nanosuspension to improve the solubility and bioavailability of the drug has good application prospects. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a tylvalosin tartrate nanosuspension and a preparation method and application thereof. The tylvalosin tartrate nanosuspension provided by the present invention is a new dosage form of tylvalosin tartrate, which can significantly improve the solubility, dissolution rate and bioavailability of tylvalosin tartrate, and has good application prospects in the preparation of drugs for treating respiratory and digestive tract diseases of poultry and livestock.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a tylvalosin tartrate nanosuspension, wherein the raw materials for preparing the tylvalosin tartrate nanosuspension include: tylvalosin tartrate, a cosolvent, an alkaline agent, a stabilizer, a crystallization aid and water;

[0008] The crystallization aid includes any one of chitosan, povidone, polysorbate or sodium carboxymethyl cellulose or a combination of at least two thereof.

[0009] The present invention prepares a nanosuspension from tartrate tyvalocin with poor solubility by introducing a crystallization aid, thereby significantly improving the solubility, dissolution and bioavailability of the drug. In addition, water is used as a solvent in the suspension, which effectively avoids the toxicity caused by the introduction of an organic solvent. The suspension is non-irritating to target animals and has little toxic and side effects, and can be used to treat respiratory and digestive tract diseases in poultry and livestock.

[0010] Preferably, the crystallization aid comprises a combination of chitosan and povidone.

[0011] Preferably, the mass ratio of chitosan to povidone is 1.5:1-5:1 (for example, it may be 2:1, 2.5:1, 3:1, 4:1, 4.5:1, etc.).

[0012] In the present invention, after adding chitosan, the system is positively charged, the absolute value of the zeta potential is greater than 30mV, the system is more stable and not easy to agglomerate; povidone as a crystal stabilizer can maintain the crystal size and system stability, and reduce the possibility of crystal aggregation, agglomeration, crystal growth or crystal transformation in the system; the two are compounded in a specific ratio to achieve a synergistic technical effect, which can assist the stable generation of nanocrystals, and the obtained nanocrystals have a smaller particle size and better dispersibility.

[0013] Preferably, based on the volume of the tylvalosin tartrate nanosuspension as 100 mL, the raw materials for preparing the tylvalosin tartrate nanosuspension include: 1-5 g of tylvalosin tartrate (for example, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g, 4 g, 4.5 g, etc.), 10-30 g of cosolvent (for example, 14 g, 18 g, 22 g, 26 g, etc.), 0.5-2 g of alkaline agent (for example, 0.75 g, 1 g, 1.5 g, 1.75 g, etc.), 0.2-0.8 g of stabilizer (for example, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, etc.), 0.2-1.2 g of crystallization aid (for example, 0.3 g, 0.4 g, 0.5 g, 0.6 g, 0.7 g, 0.8 g, 0.9 g, 1 g, 1.1 g, etc.) and water 75-90 mL (for example, 76 mL, 78 mL, 80 mL, 82 mL, 84 mL, 86 mL, 88 mL, etc.).

[0014] Preferably, the co-solvent comprises any one of acetic acid or hydrochloric acid or a combination of both.

[0015] Preferably, the alkaline agent includes any one of sodium hydroxide or potassium hydroxide or a combination of the two.

[0016] Preferably, the stabilizer comprises any one of polyvinyl alcohol, poloxamer 188 or Tween 80, or a combination of at least two thereof.

[0017] Preferably, the raw materials for preparing the tyvalocin tartrate nanosuspension further include a preservative.

[0018] Preferably, based on the volume of the tylvalosin tartrate nanosuspension being 100 mL, the amount of the preservative used is 0.1-0.3 g (for example, 0.12 g, 0.15 g, 0.18 g, 0.2 g, 0.22 g, 0.25 g, 0.28 g, etc.).

[0019] Preferably, the preservative includes any one of methylparaben or ethylparaben or a combination of the two.

[0020] In a second aspect, the present invention provides a method for preparing the tyvalocin tartrate nanosuspension according to the first aspect, the preparation method comprising the following steps:

[0021] (1) mixing tyvalocin tartrate, a crystallization aid, a cosolvent and water, and stirring to dissolve the mixture to obtain a tyvalocin tartrate solution;

[0022] (2) adding an alkaline reagent to the tartrate tyvalocin solution obtained in step (1) to obtain an initial suspension of tartrate tyvalocin nanocrystals;

[0023] (3) Mixing a stabilizer, a preservative and the initial suspension of tylvalosin tartrate nanocrystals obtained in step (2), and then homogenizing to obtain the tylvalosin tartrate nanosuspension.

[0024] The preparation method of the tylvalosin tartrate nanosuspension provided by the present invention combines acid-base precipitation crystallization and high-pressure homogenization dispersion technology, and the obtained tylvalosin tartrate nanosuspension has a small particle size and good dispersibility, which significantly improves the solubility, dissolution and bioavailability of the drug.

[0025] Preferably, in step (1), the mixing temperature is 35-45°C (for example, 36°C, 38°C, 40°C, 42°C, 44°C, etc.).

[0026] Preferably, the stirring rate is 700-1200 rpm (eg, 800 rpm, 900 rpm, 950 rpm, 1000 rpm, 1100 rpm, etc.).

[0027] Preferably, the stirring time is 15-30 min (for example, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, etc.).

[0028] Preferably, in step (2), the alkaline reagent is added dropwise in the form of an aqueous solution.

[0029] Preferably, the concentration of the alkaline agent in the aqueous solution of the alkaline agent is 1.0-5.0 mol / L (for example, it may be 1.5 mol / L, 2 mol / L, 2.5 mol / L, 3 mol / L, 3.5 mol / L, 4 mol / L, 4.5 mol / L, etc.).

[0030] Preferably, the dripping rate is 0.5-1.5 mL / min (for example, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, etc.).

[0031] Preferably, the dropping temperature is 35-45°C (eg, 36°C, 38°C, 40°C, 42°C, 44°C, etc.).

[0032] Preferably, the dropwise addition is performed under stirring.

[0033] Preferably, the stirring rate is 750-1000 rpm (eg, 800 rpm, 850 rpm, 900 rpm, 950 rpm, etc.).

[0034] Preferably, in step (3), the stabilizer is added in the form of an aqueous solution.

[0035] Preferably, the method for preparing the aqueous solution of the stabilizer comprises: mixing the stabilizer and water, and obtaining the aqueous solution of the stabilizer after heating and stirring.

[0036] Preferably, the heating temperature is 80-100° C. (for example, 82° C., 85° C., 88° C., 90° C., 92° C., 95° C., 98° C., etc.).

[0037] Preferably, the stirring rate is 500-800 rpm (eg, 550 rpm, 600 rpm, 650 rpm, 700 rpm, 750 rpm, etc.).

[0038] Preferably, the homogenization pressure is 14000-22000 psi (for example, it may be 15000 psi, 16000 psi, 17000 psi, 18000 psi, 19000 psi, 20000 psi, 21000 psi, etc.).

[0039] Preferably, the homogenization is performed 4-12 times (for example, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, etc.).

[0040] Preferably, the preparation method comprises the following steps:

[0041] (1) mixing tylvalosin tartrate, a crystallization aid, a cosolvent and water at 35-45° C., stirring at 700-1200 rpm for 15-30 min to dissolve the mixture, and obtaining a tylvalosin tartrate solution;

[0042] (2) adding a 1.0-5.0 mol / L alkaline reagent aqueous solution to the tartrate tyvalocin solution obtained in step (1) at a temperature of 35-45° C. and a stirring rate of 750-1000 rpm at a rate of 0.5-1.5 mL / min to obtain an initial suspension of tartrate tyvalocin nanocrystals;

[0043] (3) Mixing a stabilizer and water, stirring and dissolving at 80-100° C. and 500-800 rpm to obtain an aqueous solution of the stabilizer; adding the aqueous solution of the stabilizer and a preservative to the initial suspension of tylvalosin tartrate nanocrystals obtained in step (2), and then homogenizing at a pressure of 14000-22000 psi for 4-12 times to obtain the tylvalosin tartrate nanocrystal suspension.

[0044] In a third aspect, the present invention provides a use of the tyvalocin tartrate nanosuspension according to the first aspect in the preparation of a drug for treating bacterial infection and / or mycoplasma disease in poultry and livestock.

[0045] Compared with the prior art, the present invention has at least the following beneficial effects:

[0046] (1) The tylvalosin tartrate nanosuspension provided by the present invention can assist in the stable formation of nanocrystals by rationally compounding the crystallization aids and achieving synergistic effects among the components, so as to make the nanocrystals have a smaller particle size and better dispersibility.

[0047] (2) The preparation method of the tylvalosin tartrate nanosuspension provided by the present invention combines acid-base precipitation crystallization and high-pressure homogenization dispersion technology. The obtained tylvalosin tartrate nanosuspension has a small particle size and good dispersibility, which significantly improves the solubility, dissolution and bioavailability of the drug; within the preferred range, the particle size of the nanocrystals in the obtained nanosuspension can be as low as 359±12.2 nm, and it has a characteristic crystalline structure with a potential of 35.2±3.28 mV.

[0048] (3) The preparation method provided by the present invention does not require the use of organic solvents, is safe and non-toxic throughout the process, is non-irritating to target animals, has little toxic and side effects, and can be used to treat respiratory and digestive tract diseases in poultry and livestock. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is a transmission electron microscopy image of the tartrate tyvalocin nanosuspension provided in Example 3.

[0050] Figure 2 This is the dissolution curve of the tylvalosin tartrate nanosuspension and soluble powder provided in Example 3 in 0.1 mol / L hydrochloric acid solution (pH=1.2).

[0051] Figure 3 The dissolution curves of the tartrate tyvalocin nanosuspension and soluble powder provided in Example 3 in acetic acid-sodium acetate buffer (pH=4.5) are shown.

[0052] Figure 4 The dissolution curves of the tartrate tyvalocin nanosuspension and soluble powder provided in Example 3 in phosphate buffer (pH=6.8) are shown.

[0053] Figure 5 This is the drug-time curve of the tartrate tyvalocin nanosuspension and soluble powder provided in Example 3 in chickens. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0055] The sources of the reagents used in the following examples are as follows:

[0056] Tyvalosin tartrate, purity 87%; glacial acetic acid, purity analytical grade (AR); polyvinyl alcohol purchased from Shanghai MacLean Biochemical Technology Co., Ltd., model 767382; chitosan purchased from Shanghai MacLean Biochemical Technology Co., Ltd., model C804726; povidone purchased from Shanghai MacLean Biochemical Technology Co., Ltd., model 767381;

[0057] Other raw materials are conventional commercially available materials.

[0058] Example 1

[0059] This embodiment provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0060]

[0061] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0062] (1) Weigh tyvalosin, chitosan and povidone in a small beaker, add 5 mL of sterile water and place on a magnetic stirrer (condition set at 40°C / 1000 rpm). Add acetic acid while stirring continuously. Continue stirring for 10 min to allow the drug to fully dissolve. Then, add 3 mL of 1 mol / L sodium hydroxide dropwise at a rate of 1 mL / min to obtain an initial suspension of tyvalosin tartrate nanocrystals.

[0063] (2) dissolving polyvinyl alcohol in 5 mL of sterile water, stirring and dissolving at 50° C. and 600 rpm to obtain an aqueous solution of polyvinyl alcohol, adding the aqueous solution to the initial suspension of tyvalocin tartrate nanocrystals obtained in step (1), adding methyl paraben, and stirring for 5 minutes to obtain a crude nanocrystal solution;

[0064] (3) Add sterile water to the crude nanocrystal solution obtained in step (2) to 17 mL, and homogenize it six times at a pressure of 15,000 psi using a high-pressure homogenizer to obtain a tartrate tyvalocin nanosuspension.

[0065] Example 2

[0066] This embodiment provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0067] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0068] (1) Weigh tyvalosin, chitosan and povidone in a small beaker, add 5 mL of sterile water and place on a magnetic stirrer (condition set at 40°C / 1000 rpm). Add acetic acid while stirring continuously. Continue stirring for 10 min to allow the drug to fully dissolve. Then, add 3 mL of 1 mol / L sodium hydroxide dropwise at a rate of 1 mL / min to obtain an initial suspension of tyvalosin tartrate nanocrystals.

[0069] (2) dissolving polyvinyl alcohol in 5 mL of sterile water, stirring and dissolving at 50° C. and 600 rpm to obtain an aqueous solution of polyvinyl alcohol, adding the aqueous solution to the initial suspension of tyvalocin tartrate nanocrystals obtained in step (1), adding methyl paraben, and stirring for 5 min to obtain a crude nanocrystal solution;

[0070] (3) Add sterile water to the crude nanocrystal solution obtained in step (2) to 17 mL, and homogenize it six times at a pressure of 15,000 psi using a high-pressure homogenizer to obtain a tartrate tyvalocin nanosuspension.

[0071] Example 3

[0072] This embodiment provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0073] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0074] (1) Take tylvalerin tartrate, chitosan and povidone in a small beaker, add 5 mL of sterile water and place on a magnetic stirrer (conditions set at 40℃ / 1000 rpm), add acetic acid while stirring continuously, continue stirring for 20 min to fully dissolve the drug, and add 3 mL of 1 mol / L sodium hydroxide at a rate of 1 mL / min to obtain an initial suspension of tylvalerin tartrate nanocrystals;

[0075] (2) Add 5 mL of sterile water to poloxamer 188, stir and dissolve at 50°C and 600 rpm to obtain an aqueous solution of poloxamer 188, then add it to the initial suspension of tyvalocin tartrate nanocrystals obtained in step (1), add methyl paraben, and stir for 10 min to obtain a crude nanocrystal solution;

[0076] (3) The crude nanocrystal solution obtained in step (2) was supplemented with sterile water to 17 mL, and high-pressure homogenized 6 times at a pressure of 15,000 psi using a high-pressure homogenizer to obtain a tartrate tyvalocin nanosuspension.

[0077] Example 4

[0078] This embodiment provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0079]

[0080] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0081] (1) Take tylvalerin tartrate, chitosan and povidone in a small beaker, add 5 mL of sterile water and place on a magnetic stirrer (condition set at 40℃ / 1000 rpm), add acetic acid while stirring continuously, continue stirring for 20 min to fully dissolve the drug, and add 3 mL of 1 mol / L sodium hydroxide at a rate of 1 mL / min to obtain an initial suspension of tylvalerin tartrate nanocrystals;

[0082] (2) Add 5 mL of sterile water to Tween 80 and stir to dissolve to obtain an aqueous solution of Tween 80, then add it to the initial suspension of tyvalocin tartrate nanocrystals obtained in step (1), add methyl paraben, and stir for 10 min to obtain a crude nanocrystal solution;

[0083] (3) The crude nanocrystal solution obtained in step (2) was supplemented with sterile water to 17 mL, and high-pressure homogenized 6 times at a pressure of 15,000 psi using a high-pressure homogenizer to obtain a tartrate tyvalocin nanosuspension.

[0084] Example 5

[0085] This embodiment provides a tartrate tyvalocin nanosuspension, which differs from the embodiment 3 only in that it does not contain chitosan, and its weight proportion is allocated to povidone; the preparation method is referred to the embodiment 3.

[0086] Example 6

[0087] This embodiment provides a tartrate tyvalocin nanosuspension, which differs from the embodiment 3 only in that it does not contain povidone, and its weight portion is allocated to chitosan; the preparation method is referred to the embodiment 3.

[0088] Example 7

[0089] This embodiment provides a tylvaleric acid tartrate nanosuspension, which is different from the embodiment 3 only in that the total amount of the crystallization aid is kept unchanged and the mass ratio of chitosan and povidone is adjusted to 1:1; the preparation method is referred to the embodiment 3.

[0090] Example 8

[0091] This embodiment provides a tartrate tyvalocin nanosuspension, which differs from Embodiment 3 only in that the total amount of the crystallization aid is kept unchanged and the mass ratio of chitosan to povidone is adjusted to 6:1; the preparation method is referred to Embodiment 3.

[0092] Comparative Example 1

[0093] This comparative example provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0094] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0095] (1) Add tylvaleric acid tartrate to 5 mL of sterile water at 40°C and 1000 rpm with constant stirring, stir for 10 min to fully dissolve it, add glacial acetic acid to completely dissolve the tylvaleric acid, add 3 mL of sterile water and continue stirring for 5 min;

[0096] (2) Add polyvinyl alcohol to 5 mL of 1 mol / L sodium hydroxide solution, stir at 80°C and 600 rpm to obtain 5 mL of 1 mol / L sodium hydroxide-polyvinyl alcohol solution; add the sodium hydroxide-polyvinyl alcohol solution to the above-mentioned 40°C acidic solution containing tylvaleric acid tartrate at a rate of 1 mL / min under continuous stirring at 40°C and 1000 rpm, and then continue to dropwise add 2 mL of 1 mol / L sodium hydroxide to obtain an initial suspension of tylvaleric acid tartrate nanocrystals;

[0097] (3) Add methyl paraben and stir for 5 min. Add sterile water to the system to make up to 20 mL. Use a high-pressure homogenizer to homogenize 5 times at a pressure of 15,000 psi to obtain a tyvalocin tartrate nanosuspension.

[0098] Comparative Example 2

[0099] This comparative example provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0100]

[0101] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0102] (1) Add tylvaleric acid tartrate to 5 mL of sterile water at 40°C and 1000 rpm with constant stirring, stir for 10 min to fully dissolve it, add glacial acetic acid to completely dissolve the tylvaleric acid tartrate, and then add 3 mL of sterile water and continue stirring for 5 min;

[0103] (2) adding polyvinyl alcohol to 5 mL of 1 mol / L sodium hydroxide solution, stirring at 80°C and 600 rpm to obtain 5 mL of 1 mol / L sodium hydroxide-polyvinyl alcohol solution; adding the sodium hydroxide-polyvinyl alcohol solution to the above-mentioned 40°C acidic solution containing tartrate tartrate at a rate of 1 mL / min while continuously stirring at 40°C and 1000 rpm to obtain an initial suspension of tartrate tartrate nanocrystals;

[0104] (3) Add methyl paraben and stir for 5 min. Add sterile water to the system to make up to 17 mL. Use a high-pressure homogenizer to homogenize 5 times at a pressure of 15,000 psi to obtain a tyvalocin tartrate nanosuspension.

[0105] Comparative Example 3

[0106] This comparative example provides a tartrate tyvalocin nanosuspension, and the raw material composition of the preparation thereof is as follows:

[0107]

[0108] The preparation method of the tylvalosin tartrate nanosuspension comprises the following steps:

[0109] (1) Add 5 mL of sterile water to tartrate of tartrate of tartrate at 40°C and 1000 rpm with constant stirring, and add glacial acetic acid to completely dissolve the tartrate of tartrate.

[0110] (2) Add 3 mL of 1 mol / L sodium hydroxide dropwise at a rate of 1 mL / min to the 40°C acidic solution containing tyvalosin at 40°C and 1000 rpm with continuous stirring; add polyvinyl alcohol to 5 mL of sterile water, stir and dissolve at 80°C and 600 rpm, and quickly add the polyvinyl alcohol to the suspension after it is completely dissolved and cooled to room temperature to obtain an initial suspension of nanocrystals;

[0111] (3) Add methyl paraben and stir for 5 min. Add sterile water to the system to make up to 17 mL. Use a high-pressure homogenizer to homogenize 5 times at a pressure of 15,000 psi to obtain a tyvalocin tartrate nanosuspension.

[0112] Test Example 1

[0113] Particle size, dispersion and appearance inspection

[0114] Test sample: Talvalocin tartrate nanosuspension provided in Examples 1-8 and Comparative Examples 1-3

[0115] Test method: The particle size and dispersion of the nanopreparation were investigated using a nanoparticle size analyzer, and the appearance properties of the nanopreparation were investigated according to the relevant requirements of the 2020 edition of the "Pharmacopoeia of the People's Republic of China".

[0116] The test results are shown in Table 1 (mean ± standard deviation, n = 3). For example, the transmission electron microscopy image of the tartrate tyvalocin nanosuspension provided in Example 3 is as follows: Figure 1 shown.

[0117] Table 1

[0118]

[0119] The test results show that:

[0120] (1) It can be seen from Examples 1 to 8 that the tylvalosin tartrate nanosuspension obtained by acid-base precipitation crystallization and auxiliary high-pressure homogenization treatment has a uniform and small particle size, which helps to improve the solubility, dissolution rate and bioavailability of the drug.

[0121] (2) By comparing Example 3 with Examples 5-8, it can be seen that when only one crystallization aid is used or the ratio of the crystallization aid is too high / too low, the particle size of the obtained tartrate tyvalocin nanosuspension becomes larger and the dispersibility becomes worse. It can be concluded that the crystallization aid provided by the present invention can play a role in stabilizing and assisting the formation of nanocrystals through reasonable compounding in a specific ratio.

[0122] (3) By comparing Example 3 with Comparative Examples 1-3, it can be seen that the present invention reduces the particle size of nanocrystals in the tartrate tyvalocin nanosuspension and improves the dispersibility of the product by using a specific crystallization aid and a method of first acid-base precipitation crystallization and then adding a stabilizer.

[0123] Test Example 2

[0124] Encapsulation efficiency and drug loading detection

[0125] Test sample: Talvalocin tartrate nanosuspension provided in Example 3

[0126] Test method: Use ultra-high performance liquid chromatography tandem mass spectrometry for detection. The specific method is as follows:

[0127] (1) Analytical method: A Waters ACQUITY UPLC® HSS T3 column (100×2.1 mm, 1.8 μm) was used; mobile phase A was formic acid: water (0.001:1, v / v), and mobile phase B was formic acid: methanol (0.001:1, v / v); the flow rate was 0.3 mL min -1 ; Column temperature is 40℃.

[0128] UPLC elution procedure:

[0129]

[0130] Mass spectrometry parameters: positive ion mode (ESI+), quantitative ion pair: 1042.40>174.15 m / z, collision energy: 44 eV; qualitative ion pair: 1042.40>814.3 m / z, collision energy: 32 eV.

[0131] (2) Sample pretreatment: Take 1 mL of tylvalosin tartrate nanocrystal suspension, add 4 mL of acetonitrile, shake for 10 min, serially dilute by 2, 5, 20, and 50 times, take 1 mL of the supernatant and conduct detection by ultra-high performance liquid chromatography-tandem mass spectrometry to calculate the mass of the encapsulated tylvalosin tartrate drug.

[0132] The calculation formula is:

[0133] Encapsulation efficiency (%) = (mass of entrapped tartrate tyvalocin drug / initial tartrate tyvalocin drug mass) × 100%

[0134] Drug loading (%) = (mass of tartrate tyvalocin drug encapsulated / total mass of initial input materials) × 100%

[0135] The experimental results showed that the encapsulation efficiency of the tartrate tyvalocin nanosuspension provided in Example 3 was 70.9% and the drug loading was 54.6%.

[0136] Test Example 3

[0137] Dissolution Testing

[0138] Test samples: the tylvalosin tartrate nanosuspension provided in Example 3 and commercially available tylvalosin tartrate soluble powder.

[0139] Test method: The release rate of tylvalosin tartrate nanosuspension was determined by the paddle method. The test referred to the relevant requirements of the Appendix of Part IV of the 2020 edition of the Veterinary Pharmacopoeia of the People's Republic of China and the USP39-NF34 edition of the United States Pharmacopoeia. Three dissolution media were set: pH 1.2 dilute hydrochloric acid solution to simulate the acidic environment of gastric fluid; pH 4.5 acetic acid-sodium acetate buffer to simulate the environment of the drug in the upper intestine after gastric emptying; pH 6.8 phosphate buffer to simulate the intestinal fluid environment. Accurately measure 3 portions of 5 mL of tylvalosin tartrate nanosuspension with an initial concentration of 3 mg / mL and 5 mL of tylvalosin tartrate soluble powder solution with the same initial concentration, and inject them into 6 dissolution cups containing 900 mL of dissolution medium respectively. The temperature was set at 37±0.5℃, and the speed of the stirring paddle was set to 25 rpm / min. After adding the sample, 5 mL of sample was taken from each dissolution cup at 5, 10, 15, 30, 45, 60, 120 and 240 min, and the dissolution medium of equal volume and temperature was added to the dissolution cup after sampling. The sample was filtered through a 0.22 μm filter membrane, and the drug concentration was determined by high performance liquid chromatography to draw the dissolution curve.

[0140] Test results: Release rate Figure 2 , Figure 3 , Figure 4As shown, the results show that the tylvalosin tartrate nanosuspension provided by the present invention can be quickly released in solutions under acidic, weakly acidic and neutral conditions, and the nano preparation significantly improves the solubility of tylvalosin tartrate.

[0141] Test Example 4

[0142] Pharmacokinetic testing

[0143] Test samples: the tylvalosin tartrate nanosuspension provided in Example 3 and commercially available tylvalosin tartrate soluble powder.

[0144] Test method:

[0145] Experimental animals: 16 healthy white-feathered broilers weighing 3±0.5 kg were purchased from Baoding Jiufu Food Co., Ltd. They were acclimatized to the environment and routinely raised during the experiment, with free access to drinking water and complete feed.

[0146] Animal experiment: After acclimating to the environment, 16 white-feathered broilers were randomly divided into two groups, 8 in each group, and they were administered with a dose of 25 mg / kg (in terms of tylvalosin tartrate). At the predetermined time points (0, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24h) after oral administration, 1.5 mL of blood was collected through the toe vein, and the plasma was separated to detect the blood concentration of tylvalosin tartrate.

[0147] Data processing: WinNonlin software was used to fit the pharmacokinetic parameters of tyvalocin tartrate in chickens according to the non-compartmental model.

[0148] The main pharmacokinetic parameters (mean ± standard deviation, n=8) of the tylvalosin tartrate nanosuspension provided in Example 3 and the commercially available tylvalosin tartrate soluble powder when administered orally to chickens are shown in Table 2.

[0149] Table 2

[0150]

[0151] *Significant difference compared with nanosuspension, p﹤0.05.

[0152] Test results: The drug-time curves and pharmacokinetic parameters of tartrate tartrate nanosuspension and commercially available tartrate tartrate soluble powder in chickens are as follows: Figure 5As shown in Table 2. After oral administration of the nanosuspension, the maximum blood concentration of 402.99 ng / mL of tartrate tyvalocin rapidly reached 0.66 h, and then slowly decreased. The commercially available soluble powder reached a maximum blood concentration of 287.19 ng / mL at 1.88 h, and then rapidly decreased, falling below the detection line at 12 h. Pharmacokinetic parameter studies have shown that the nanosuspension can maintain an effective blood concentration for a long time, and the nanosuspension increases the oral bioavailability and elimination half-life of tartrate tyvalocin by 3.1 and 9.28 times, respectively. The above results show that the preparation provided by the present invention can promote the absorption of tartrate tyvalocin in chickens and prolong the drug's action time in the body.

[0153] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A tylvalosin tartrate nanosuspension, characterized in that: Based on the volume of the tylvalosin tartrate nanosuspension being 100 mL, the raw materials for preparing the tylvalosin tartrate nanosuspension include: 1-5 g of tylvalosin tartrate, 10-30 g of a cosolvent, 0.5-2 g of an alkaline agent, 0.2-0.8 g of a stabilizer, 0.2-1.2 g of a crystallization aid, and 75-90 mL of water; The co-solvent includes any one of acetic acid or hydrochloric acid or a combination of the two; The stabilizer includes any one of polyvinyl alcohol, poloxamer 188 or Tween 80 or a combination of at least two thereof; The crystallization aid comprises a combination of chitosan and povidone; the mass ratio of the chitosan to povidone is 1.5:1-5:

1.

2. The tylvalosin tartrate nanosuspension according to claim 1, characterized in that: The alkaline agent includes any one of sodium hydroxide and potassium hydroxide or a combination of the two.

3. The tylvalosin tartrate nanosuspension according to claim 1, characterized in that: The raw materials for preparing the tylvalosin tartrate nanosuspension also include a preservative; Based on the volume of the tartrate tyvalocin nanosuspension being 100 mL, the amount of the preservative used is 0.1-0.3 g; The preservative includes any one of methylparaben or ethylparaben or a combination of the two.

4. A method for preparing the tylvalosin tartrate nanosuspension according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: (1) mixing tyvalocin tartrate, a crystallization aid, a cosolvent and water, and stirring to dissolve the mixture to obtain a tyvalocin tartrate solution; (2) adding an alkaline reagent to the tartrate tyvalocin solution obtained in step (1) to obtain an initial suspension of tartrate tyvalocin nanocrystals; (3) Mixing a stabilizer, a preservative and the initial suspension of tylvalosin tartrate nanocrystals obtained in step (2), and then homogenizing to obtain the tylvalosin tartrate nanosuspension.

5. The preparation method according to claim 4, characterized in that In step (1), the mixing temperature is 35-45° C., the stirring rate is 700-1200 rpm, and the stirring time is 15-30 min.

6. The preparation method according to claim 4, characterized in that: In step (2), the alkaline reagent is added dropwise in the form of an aqueous solution; The concentration of the alkaline reagent in the aqueous solution of the alkaline reagent is 1.0-5.0 mol / L, the rate of the dropping is 0.5-1.5 mL / min, and the temperature of the dropping is 35-45° C.; The dropwise addition is performed under stirring at a rate of 750-1000 rpm.

7. The preparation method according to claim 4, characterized in that: In step (3), the stabilizer is added in the form of an aqueous solution; The preparation method of the aqueous solution of the stabilizer comprises: mixing the stabilizer and water, heating and stirring to obtain the aqueous solution of the stabilizer; the heating temperature is 80-100° C., and the stirring rate is 500-800 rpm; The homogenization pressure is 14000-22000 psi, and the homogenization times are 4-12 times.

8. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: (1) mixing tylvalosin tartrate, a crystallization aid, a cosolvent and water at 35-45° C., stirring at 700-1200 rpm for 15-30 min to dissolve the mixture, and obtaining a tylvalosin tartrate solution; (2) adding a 1.0-5.0 mol / L alkaline reagent aqueous solution to the tartrate tyvalocin solution obtained in step (1) at a temperature of 35-45° C. and a stirring rate of 750-1000 rpm at a rate of 0.5-1.5 mL / min to obtain an initial suspension of tartrate tyvalocin nanocrystals; (3) Mixing a stabilizer and water, stirring and dissolving at 80-100° C. and 500-800 rpm to obtain an aqueous solution of the stabilizer; adding the aqueous solution of the stabilizer and a preservative to the initial suspension of tylvalosin tartrate nanocrystals obtained in step (2), and then homogenizing at a pressure of 14000-22000 psi for 4-12 times to obtain the tylvalosin tartrate nanocrystal suspension.

9. Use of the tylvalosin tartrate nanosuspension according to any one of claims 1 to 3 in the preparation of a drug for treating bacterial infection and / or mycoplasma disease in poultry and livestock.

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