Mequinidox nano sustained-release injection and preparation method thereof

Abstract

The invention relates to a mequinidox nano sustained-release injection and a preparation method thereof. The mequinidox nano sustained-release injection is prepared from Tween 80, Span 80, isopropyl myristate, absolute ethyl alcohol, polyvinylpyrrolidone K30, mequinidox and water. In every 1000 mL of the mequinidox nano sustained-release injection, the content of the Tween 80 is 60 to 180 mL, the content of the Span 80 is 12 to 36 mL, the content of the isopropyl myristate is 24 to 72 mL, the content of the absolute ethyl alcohol is 40 to 120 mL, the content of the polyvinylpyrrolidone K30 is 2 to 6 g, and the content of the mequinidox is 10 to 30 g. The mequinidox nano sustained-release injection disclosed by the invention has certain drug loading capacity, stability and sustained-release performance.

Description

Technical Field

[0001] The invention relates to a mequidoxine nano sustained-release injection and a preparation method thereof, and belongs to the field of veterinary drugs. Background Art

[0002] In order to improve the level of animal health care, the development of new veterinary drugs with broad spectrum, high efficiency and low toxicity, the development of new preparations and new drug delivery technologies have become important issues facing the majority of veterinary medicine workers. Drugs need to be suitable for medical and preventive applications in certain dosage forms, and the clinical efficacy of drugs is directly related to whether the dosage form used is reasonable. Developing new dosage forms, using modern new technologies and new materials to improve traditional drugs and expand their uses is much cheaper and shorter than developing new veterinary drugs, and has a higher economic return rate. Nano sustained-release preparations are currently receiving widespread attention.

[0003] Nanoemulsion is a kind of nano sustained-release preparation with a particle size between 10 and 100 nm. It is a stable, transparent, low-viscosity, isotropic and thermodynamically stable dispersion system formed by an oil phase, an aqueous phase, a surfactant and a co-surfactant in appropriate proportions. As long as the composition of the four phases is appropriate, a uniform, transparent or slightly opalescent liquid can be formed, which is a thermodynamically stable system. Due to the outstanding targeting and sustained-release effects of nanoemulsions and their strong solubilization effect on poorly soluble drugs, the research on nanoemulsion drug delivery systems has been widely concerned by scholars at home and abroad in recent years. In addition, as a new type of drug carrier, nanoemulsion is divided into different modes of administration according to the characteristics of the drug carried and the different types of emulsions: transdermal administration, oral administration, mucosal administration, injection administration, etc. At the same time, advanced nanoemulsion preparation instruments, experimental design schemes, detection equipment, computer software, etc. are also used in the preparation of nanoemulsions and are being further developed. As a new type of drug carrier system, nanoemulsion has a strong solubilization effect on poorly soluble drugs, and also has the advantages of obvious sustained-release effect, targeting and high bioavailability, and has broad application prospects in the field of pharmacy.

[0004] Mequindox is currently the first choice for treating serious intestinal infections in livestock and poultry. Due to its toxicity, animals have a certain sensitivity to the drug. In recent years, incidents of livestock and poultry poisoning due to improper use have occurred repeatedly in veterinary clinics. At the same time, the drug is easily absorbed when taken orally, distributed in various tissues throughout the body, disappears quickly in the body, and has a very short blood half-life (T1 / 2 is 2h), which limits its clinical use. At present, the main products of mequindox in clinical practice are powders, premixes, and ordinary injections. There are also related mequindox nanoemulsion research reports, but most of them are oral and transdermal preparations, and there are no related products on the market. There are currently no research reports on mequindox nano sustained-release injections, and according to pharmacological principles, the same drug, the effect of injection is often better than oral and transdermal administration. Especially in the case of severe livestock and poultry conditions, the advantages of injectable medication can be more prominent.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] In view of the shortcomings of the prior art, one of the purposes of the present invention is to provide a mequidoxine nano sustained-release injection having good drug loading capacity, stability and sustained-release performance; a second purpose of the present invention is to provide a method for preparing the above-mentioned mequidoxine nano sustained-release injection, which is simple and suitable for large-scale production.

[0007] In order to solve the above technical problems, the technical solution of the present invention is as follows:

[0008] A mequidox nano sustained-release injection, comprising Tween 80, Span 80, isopropyl myristate, anhydrous ethanol, polyvinylpyrrolidone K 30 , mequindox and water; in each 1000mL of mequindox nano sustained-release injection, the content of Tween 80 is 60-180mL, the content of Span 80 is 12-36mL, the content of isopropyl myristate is 24-72mL, the content of anhydrous ethanol is 40-120mL, and the content of polyvinylpyrrolidone K is 24-72mL. 30 The content of is 2-6g, and the content of mequidoxine is 10-30g.

[0009] Further, in each 1000mL of mequindox nano sustained-release injection, the content of Tween 80 is 80-150mL, the content of Span 80 is 18-30mL, the content of isopropyl myristate is 36-60mL, the content of anhydrous ethanol is 60-100mL, and the content of polyvinylpyrrolidone K is 100-150mL. 30 The content of is 3-5g, and the content of mequidoxine is 15-25g.

[0010] Furthermore, in each 1000 mL of mequindox nano sustained-release injection, the content of Tween 80 is 120 mL, the content of Span 80 is 24 mL, the content of isopropyl myristate is 48 mL, the content of anhydrous ethanol is 80 mL, and the content of polyvinylpyrrolidone K is 100 mL. 30 The content of is 4g, and the content of mequidoxine is 20g.

[0011] Furthermore, in the mequidox nano sustained-release injection, the volume ratio of Tween 80, Span 80, and isopropyl myristate is 4-6:1:1-3. The applicant has found that by controlling the volume ratio of Tween 80, Span 80, and isopropyl myristate within a specific range, it is helpful to obtain an excellent emulsification effect and obtain a uniform and stable injection with small particle size and high drug concentration.

[0012] Furthermore, in the mequidoxine nano sustained-release injection, anhydrous ethanol and polyvinyl pyrrolidone K 30The applicant found that by controlling the ratio of anhydrous ethanol and polyvinyl pyrrolidone K 30 The ratio is within a specific range, which helps to improve the stability of the injection, making the emulsification effect stable and without stratification.

[0013] Furthermore, the mequidoxine nano sustained-release injection is an oil-in-water nanoemulsion preparation.

[0014] Furthermore, in the mequidoxine nano sustained-release injection, the average particle size of the emulsion droplets is 40-100 nm, and further 50-90 nm.

[0015] Preferably, the water is purified water, and may be distilled water.

[0016] The emulsion droplets in the mequidoxine nano sustained-release injection of the present invention are of nanometer size, have high drug loading and stability, are quality controllable, have sustained-release and synergistic functions, can overcome the shortcomings of existing injections and fat emulsions, and are suitable for conventional injection methods such as subcutaneous injection, intramuscular injection, and intravenous injection.

[0017] The mequidox nano sustained-release injection of the present invention has obvious sustained-release synergistic function compared with existing preparations. Through in vitro release tests and clinical trials, the mequidox nano sustained-release injection of the present invention has better sustained-release performance and therapeutic effect than ordinary mequidox injections. The in vitro release rate of the mequidox nano sustained-release injection of the present invention is 3-4 times that of the original one, which can reduce the number of medications and the amount of medication, save drug costs, reduce veterinary drug residues in animals, and reduce toxic side effects.

[0018] The mequidoxine injection of the present invention adopts nanoemulsion as a drug carrier, which can increase the solubility of the poorly water-soluble drug mequidoxine, so that the mequidoxine can be well dispersed and rapidly absorbed, thereby improving the bioavailability.

[0019] The preparation method of the mequidoxine nano sustained-release injection as described above comprises the following steps:

[0020] S1. Mix Tween 80, Span 80, isopropyl myristate and mequidoxine in a certain ratio to obtain solution A.

[0021] According to the ratio of anhydrous ethanol, polyvinyl pyrrolidone K 30 Mix well to obtain liquid B;

[0022] S2, mixing the liquid A and the liquid B evenly to obtain an oil phase;

[0023] S3. Add water dropwise into the oil phase according to the ratio, stirring while adding water, so that the solution changes from clear to turbid and then from turbid to clear, to obtain a finished product of mequidox nano sustained-release injection.

[0024] Furthermore, in S1, Tween 80, Span 80 and isopropyl myristate are firstly mixed uniformly to obtain a composite emulsion; then mequindox is mixed with the composite emulsion and heated to 20-80° C. (preferably 40-60° C., more preferably 45-55° C.) to fully dissolve mequindox to obtain liquid A.

[0025] Furthermore, in S2, during mixing, the temperature of liquid A and liquid B is the same. The temperature of the two liquids needs to be kept at the same state, which can effectively avoid crystallization during emulsification, thereby causing a blocky or rough uneven emulsion.

[0026] Furthermore, in S2, the liquid B is preheated to 20-80°C (preferably 40-60°C, more preferably 45-55°C), and then mixed evenly with the liquid A to obtain an oil phase.

[0027] Adopting a specific temperature is helpful to improve the mutual solubility and dispersion effect of Tween 80, Span 80, isopropyl myristate and mequidoxine.

[0028] Preferably, in S3, the oil phase is placed on a constant temperature magnetic stirrer, and the water phase is added dropwise into the oil phase in a constant temperature stirring state under the drainage of a constant flow pump.

[0029] Furthermore, in S3, during the dropping process, the temperature of the oil phase is maintained at 20-80°C, preferably 40-60°C, and more preferably 45-55°C.

[0030] Using a specific temperature helps polyvinyl pyrrolidone K 30 Dissolve fully in ethanol.

[0031] At a specific temperature, adding a water phase to the oil phase helps to smoothly obtain an oil-in-water (O / W) nanoemulsion preparation, promote its dispersion, and reduce the particle size.

[0032] The applicant has found that temperature is an important factor affecting the preparation of mequindox nano sustained-release injection. When the temperature of the oil phase in S3 is too high, the drug solution will change color and affect the state observation of nanoemulsion formation. When the temperature is too low, nanoemulsion cannot be formed and only a jelly-like substance is formed.

[0033] Furthermore, it also includes S4, that is, after the finished product of mequindox nano sustained-release injection is filtered through a filter membrane, an inert gas is introduced for sealing, and sterilized at 119-123°C for 10-20 minutes; preferably, the pore size of the filter membrane is less than 1 μm, preferably less than 0.5 μm, and more preferably less than 0.2 μm. In this way, the obtained nanoemulsion can be effectively sterilized by filtering and sterilizing at high temperature and high pressure to obtain a uniform and stable nanoemulsion, and the obtained nanoemulsion can be effectively prevented from becoming turbid or even demulsified to cause drug precipitation.

[0034] Preferably, the sterilization is performed at 120-122°C for 12-18 min, and more preferably, the sterilization is performed at 121°C for 15 min.

[0035] The preparation method of the mequidoxine nano sustained-release injection of the invention has a simple process, adopts an oil-in-water (O / W) method to prepare mequidoxine nano-microspheres, and delays the release rate of the drug in the body by modifying the drug dosage form, thereby extending the in vivo half-life of the drug and improving the bioavailability of the drug.

[0036] The applicant found in the study that due to the poor solubility of mequindox itself, the drug concentration directly affects the stability of the nanoemulsion. When the drug concentration is too low, the ideal therapeutic effect cannot be achieved. When the concentration is too high, the nanoemulsion is prone to precipitation. By adding a specific amount of ethanol, mequindox can be better dissolved and is not prone to precipitation at low temperatures, which helps to increase the drug loading and stability.

[0037] The mequidoxine nano sustained-release injection of the present invention has a simple preparation process, high drug loading and stability, and controllable quality, overcomes the shortcomings of existing injections and fat emulsions, and the particle size of the emulsion droplets in the preparation is controlled at the nanometer level, which can be directly used not only for intramuscular and subcutaneous injections, but also for intravenous injections. Generally speaking, the use of nano drug preparations with small particle sizes is helpful to improve the targeting and sustained-release effects of drugs.

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

[0039] The mequindox nano sustained-release injection of the present invention can effectively improve the blood half-life and bioavailability of mequindox in animals. The nanoemulsion prepared by the oil-in-water method has targeting and sustained-release effects. Since some inflammatory phagocytes in animals have the habit of identifying and engulfing microparticles, a part of the drug nanoemulsion entering the body is engulfed by them, and arrives at the inflammation site with the inflammatory phagocytes, playing a certain targeting and synergistic anti-inflammatory effect. This engulfment and re-release and long-distance raid to the inflammation site also prolong the time of drug release in the body to a certain extent, that is, the nano preparation has a certain sustained-release effect. Through in vitro release test and clinical trial, the mequindox nano sustained-release injection obtained by the present invention has an in vitro release rate of 3-4 times that of the original, which can reduce the number of medications and the dosage, thereby saving the drug cost, reducing the residual amount of the drug in the animal body, and reducing the toxic and side effects to the animal body.

[0040] The present invention successfully solves the stability problem of the existing mequidoxine common injection. Mequidoxine itself is difficult to dissolve in water. Even if it is dissolved by a certain cosolvent, the drug will precipitate after a period of time, resulting in inability to use it normally. The present invention uses nanoemulsion as a drug carrier, increases the solubility of mequidoxine, a water-insoluble drug, effectively prevents drug oxidation and hydrolysis, and prolongs the effective period of drug use.

[0041] The mequindox nanoemulsion that has been developed so far does not have an injection administration method, and is generally administered orally or transdermally. It is well known that the same drug has a faster, better and more stable effect when administered by injection than when administered orally or transdermally. The present invention has successfully developed a mequindox nano sustained-release injection, which is an innovation in the administration method of mequindox nanoemulsion.

[0042] The preparation method of the invention is low-carbon, environmentally friendly, and cost-saving. The mequidoxine nanoemulsion is mainly prepared by an oil-in-water method (O / W). The preparation method and process are simple and easy to learn, greatly saving production costs and being suitable for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 1 is a diagram showing the particle size distribution of the nanoemulsion particles in the mequidoxine injectable sustained-release nanoemulsion preparation obtained in Example 5.

[0044] Figure 2 These are the in vitro release test results of the injectable sustained-release nanoemulsion preparation of mequidoxine (y2) obtained in Example 5 and the ordinary mequidoxine injection (y1).

[0045] Figure 3 This is a TEM image of the mequidoxine injectable sustained-release nanoemulsion preparation obtained in Example 5. DETAILED DESCRIPTION

[0046] The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0047] The relevant experimental equipment and materials of each embodiment and comparative example are as follows:

[0048] 1. Experimental Equipment

[0049] 78HW-1 constant temperature magnetic stirrer (Hangzhou Instrument and Motor Factory); constant flow pump (Shanghai Huxi Analytical Instrument Factory Co., Ltd.); transmission electron microscope (Analytical Testing Center of Hunan Agricultural University); precision electronic analytical balance (AUW120D, Shimadzu Corporation Japan); multi-purpose electric oven (Beijing Yongguangming Medical Instrument Factory); constant temperature and humidity test chamber (Chongqing Sida Testing Equipment Co., Ltd.); acidity meter (PHS-3C, Shanghai Yueci Electronic Technology Co., Ltd.); UV-visible spectrophotometer (GoldS53, Shanghai Huayan Instrument Equipment Co., Ltd.).

[0050] 2. Reagents

[0051] Isopropyl myristate (chemically pure, Sinopharm Chemical Reagent Co., Ltd.); Tween 80 (AR, Tianjin Damao Chemical Reagent Factory); Span 80, anhydrous ethanol (AR, Sinopharm Chemical Reagent Co., Ltd.); Polyvinylpyrrolidone K 30 (Boai Xinkaiyuan Pharmaceutical Co., Ltd.); PBS (PH = 7.0); Mequidoxine (injection grade, Hubei Zhongmu Anda Pharmaceutical Co., Ltd.); methanol, dimethylformamide (AR, Tianjin Damao Chemical Reagent Co., Ltd.); hydrochloric acid (AR, Shenzhen Tenglongyuan Industrial Co., Ltd.).

[0052] Example 1

[0053] A method for preparing a mequidoxine injectable sustained-release nanoemulsion preparation comprises the following steps:

[0054] (1) Weigh 60 mL of Tween 80, 12 mL of Span 80, and 24 mL of isopropyl myristate and mix them evenly to obtain a composite emulsion; then weigh 10 g of mequindox and add it to the composite emulsion, continue heating to 20° C., and dissolve it to obtain liquid A;

[0055] (2) Weigh 2 g of polyvinyl pyrrolidone K 30 Add 40 mL of anhydrous ethanol, heat to 20°C, and then add liquid A as the oil phase;

[0056] (3) Take about 880 mL of purified water as the aqueous phase;

[0057] (4) placing the oil phase on a constant temperature magnetic stirrer at 18-22° C., and dripping the aqueous phase into the oil phase in a constant temperature stirring state under the drainage of a constant flow pump (controlling the dripping amount of the aqueous phase so that the total amount after the oil phase and the aqueous phase are mixed is 1000 mL), until the solution changes from clear to turbid, and then from turbid to clear and transparent, to obtain a nanoemulsion;

[0058] (5) The obtained nanoemulsion was filtered through a filter membrane with a pore size of 1 μm, nitrogen was introduced into the filter, and the mixture was sterilized at 119° C. and high temperature and high pressure for 20 min to obtain a mequidoxine injectable sustained-release nanoemulsion preparation.

[0059] Example 2

[0060] A method for preparing a mequidoxine injectable sustained-release nanoemulsion preparation comprises the following steps:

[0061] (1) Weigh 180 mL of Tween 80, 36 mL of Span 80, and 72 mL of isopropyl myristate and mix them evenly to obtain a composite emulsion, weigh 30 g of mequindox into the composite emulsion, continue heating to 80° C., and dissolve to obtain Solution A;

[0062] (2) Weigh 6 g of polyvinyl pyrrolidone K 30 Add to 120 mL of anhydrous ethanol, heat to 80°C, and add liquid A as the oil phase;

[0063] (3) Take about 660 mL of purified water as the aqueous phase;

[0064] (4) placing the oil phase on a constant temperature magnetic stirrer at 78-82° C., and dripping the aqueous phase into the oil phase in a constant temperature stirring state under the drainage of a constant flow pump (controlling the dripping amount of the aqueous phase so that the total amount after the oil phase and the aqueous phase are mixed is 1000 mL), until the solution changes from clear to turbid, and then from turbid to clear and transparent, to obtain a nanoemulsion;

[0065] (5) The obtained nanoemulsion was filtered through a filter membrane with a pore size of 0.5 μm, nitrogen was introduced into the filter, and the mixture was sterilized at 123° C. and high temperature and high pressure for 10 min to obtain a mequidoxine injectable sustained-release nanoemulsion preparation.

[0066] Example 3

[0067] A method for preparing a mequidoxine injectable sustained-release nanoemulsion preparation comprises the following steps:

[0068] (1) Weigh 90 mL of Tween 80, 18 mL of Span 80, and 36 mL of isopropyl myristate, mix them evenly to obtain a composite emulsion, weigh 15 g of mequindox into the composite emulsion, continue heating to 40° C., and dissolve to obtain Solution A;

[0069] (2) Weigh 3 g of polyvinyl pyrrolidone K 30 Add to 60 mL of anhydrous ethanol, heat to 40°C, and add liquid A as the oil phase;

[0070] (3) Take about 820 mL of purified water as the aqueous phase;

[0071] (4) placing the oil phase on a constant temperature magnetic stirrer at 38-42° C., and dripping the aqueous phase into the oil phase in a constant temperature stirring state under the drainage of a constant flow pump (controlling the dripping amount of the aqueous phase so that the total amount after the oil phase and the aqueous phase are mixed is 1000 mL), until the solution changes from clear to turbid, and then from turbid to clear and transparent, to obtain a nanoemulsion;

[0072] (5) The obtained nanoemulsion was filtered through a filter membrane with a pore size of 0.4 μm, nitrogen was introduced into the filter, and the mixture was sterilized at 120° C. and high temperature and high pressure for 18 min to obtain a mequindox injectable sustained-release nanoemulsion preparation.

[0073] Example 4

[0074] A method for preparing a mequidoxine injectable sustained-release nanoemulsion preparation comprises the following steps:

[0075] (1) Weigh 150 mL of Tween 80, 30 mL of Span 80, and 60 mL of isopropyl myristate and mix them evenly to obtain a composite emulsion, weigh 25 g of mequindox into the composite emulsion, continue heating to 60° C., and dissolve to obtain Solution A;

[0076] (2) Weigh 5 g of polyvinyl pyrrolidone K 30 Add 100 mL of anhydrous ethanol, heat to 60°C, and then add liquid A as the oil phase;

[0077] (3) Take about 680 mL of purified water as the aqueous phase;

[0078] (4) placing the oil phase on a constant temperature magnetic stirrer at 58-62° C., and dripping the aqueous phase into the oil phase in a constant temperature stirring state under the drainage of a constant flow pump (controlling the dripping amount of the aqueous phase so that the total amount after the oil phase and the aqueous phase are mixed is 1000 mL), until the solution changes from clear to turbid, and then from turbid to clear and transparent, to obtain a nanoemulsion;

[0079] (5) The obtained nanoemulsion was filtered through a filter membrane with a pore size of 0.3 μm, nitrogen was introduced into the filter, and the mixture was sterilized at 122° C. and high temperature and high pressure for 12 min to obtain a mequidoxine injectable sustained-release nanoemulsion preparation.

[0080] Example 5

[0081] A method for preparing a mequidoxine injectable sustained-release nanoemulsion preparation comprises the following steps:

[0082] (1) Weigh 120 mL of Tween 80, 24 mL of Span 80, and 48 mL of isopropyl myristate and mix them evenly to obtain a composite emulsion, weigh 20 g of mequindox into the composite emulsion, continue heating to 55° C., and dissolve to obtain Solution A;

[0083] (2) Weigh 4 g of polyvinyl pyrrolidone K 30Add 80 mL of anhydrous ethanol, heat to 55°C, and then add liquid A as the oil phase;

[0084] (3) Take about 750 mL of purified water as the aqueous phase;

[0085] (4) placing the oil phase on a constant temperature magnetic stirrer at 53-57° C., and dripping the water phase into the oil phase in a constant temperature stirring state under the drainage of a constant flow pump (controlling the dripping amount of the water phase so that the total amount after the oil phase and the water phase are mixed is 1000 mL) to obtain a nanoemulsion;

[0086] (5) The obtained nanoemulsion was filtered through a filter membrane with a pore size of 0.2 μm, nitrogen was introduced into the filter, and the mixture was sterilized at 121° C. and high temperature and high pressure for 15 min to obtain a mequindox injectable sustained-release nanoemulsion preparation.

[0087] 1. Nanoemulsion identification test:

[0088] Centrifuge at 2000r / min for 10min. If it is a uniform yellow clear liquid with Tyndall optical phenomenon, it is a nanoemulsion. Divide the prepared nanoemulsion into two parts in a small beaker and drop two drops of Sudan red dye and two drops of methylene blue dye into each part. Observe the diffusion rate of the dye in the nanoemulsion. If the diffusion rate of methylene blue dye in the nanoemulsion is faster than that of Sudan red dye, it is an oil-in-water nanoemulsion (O / W), otherwise it is an oil-in-water nanoemulsion (W / O).

[0089] 2. Nanoemulsion morphology observation and particle size measurement:

[0090] Take a quantitative amount of mequidoxine nanoemulsion, dilute it, and apply a small amount on a copper mesh. After drying, observe its morphological distribution under a transmission electron microscope, and complete the measurement of the nanoemulsion particle size under a transmission electron microscope.

[0091] 3. Content determination:

[0092] (1) Selection of measurement wavelength:

[0093] An appropriate amount of mequindox raw material reference substance (i.e. pure mequindox) was added with 10 mL of dimethylformamide and 30 mL of 0.1 mol / L hydrochloric acid solution to dissolve the solution, and then methanol was added to prepare a 10 μg / mL solution. Methanol was used as a blank and the solution was scanned in the wavelength range of 200 to 400 nm. The absorption spectrum was recorded and the maximum absorption was found at a wavelength of 380 nm. The solvent used for mequindox was tested and there was no absorption at this wavelength, so 380 nm was used as the measurement wavelength.

[0094] (2) Determination of the absorbance of the reference solution (standard curve):

[0095] Accurately weigh 50 mg of mequindoxime reference substance, place it in a 250 mL brown volumetric flask, add 10 mL of dimethylformamide and 30 mL of hydrochloric acid solution (0.1 mol / L), shake to dissolve, add water to the scale, shake well, accurately measure 0.5, 1, 2, 3, 5, and 10 mL respectively, place it in a 100 mL brown volumetric flask, dilute to the scale with methanol, shake well. Use methanol as blank and measure the absorbance at a wavelength of 380 nm.

[0096] (3) Determination of Nanoemulsion Content:

[0097] Accurately measure 2 mL of mequindox nanoemulsion, place it in a 250 mL brown volumetric flask, add 10 mL of dimethylformamide and 30 mL of hydrochloric acid solution (0.1 mol / mL). Shake well, add water to the scale, shake well, accurately measure 5 mL, place it in a 100 mL brown volumetric flask, dilute to the scale with methanol, shake well, and measure at a wavelength of 380 nm according to the spectrophotometric method.

[0098] (4) Accelerated stability study of nanoemulsion preparations:

[0099] The stability test was conducted according to the provisions of the drug accelerated test in the Technical Specifications for Stability Test of Veterinary Drugs. Three batches of mequindox nanoemulsion for injection were placed in a constant temperature and humidity chamber at 30°C and a relative humidity of 65%. Samples were taken out regularly to examine the appearance, particle size, pH value and drug content changes of the nanoemulsion.

[0100] Experimental results:

[0101] 1. Absorbance and regression equation of reference solution:

[0102] The specific results are shown in Table 2. The regression equation of absorbance (y) to concentration (x, μg / mL) is: y = 0.055x + 0.0091, R2 = 0.999. The test results show that within the concentration range of 1-20 μg / mL, the absorbance and concentration have a good linear relationship.

[0103] Table 2 Absorbance concentration of reference solution

[0104]

[0105] 2. Determination of properties of the injectable sustained-release nanoemulsion preparation of mequidoxine obtained in the present invention:

[0106] The results are shown in Table 3. It can be seen that the mequidoxine injectable sustained-release nanoemulsion preparation obtained by the present invention has high transparency, high drug content, small particle size of nanoemulsion particles, and stable state.

[0107] Table 3 Performance data of the injectable sustained-release nanoemulsion preparation of mequindox obtained in the present invention

[0108]

[0109] 3. Identification test of the injectable sustained-release nanoemulsion preparation of mequidoxine obtained in the present invention:

[0110] The results show that the mequindox injectable sustained-release nanoemulsion preparations (Examples 1-5) obtained by the present invention are uniform yellow clear liquids, and have the Tyndall optical phenomenon, and are nanoemulsions. The diffusion experiment results show that the diffusion rate of the methylene blue dye in the mequindox injectable sustained-release nanoemulsion preparations (Examples 1-5) obtained by the present invention is faster than that of the Sudan red dye, and is an oil-in-water nanoemulsion (O / W).

[0111] 4. Morphological observation and particle size determination of the injectable sustained-release nanoemulsion preparation of mequidoxine obtained in Example 5 of the present invention:

[0112] The results are as follows Figure 2 The mequindox injectable sustained-release nanoemulsion particles obtained in Example 5 of the present invention are relatively regular spherical, evenly distributed, and have an average particle size of 50 nm (eg Figure 3 shown).

[0113] 5. Accelerated stability test of the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention:

[0114] The results are shown in Table 4. The injectable sustained-release nanoemulsion preparation of mequidoxine obtained in Example 5 of the present invention has good stability, and all indicators meet the requirements.

[0115] Table 4 Stability test results of the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 (x±s, n=3)

[0116]

[0117] 6. In vitro release test of the injectable sustained-release nanoemulsion preparation of mequidoxine obtained in Example 5 of the present invention:

[0118] (1) Materials: Six-cup drug dissolution apparatus (Wuhan Espe Scientific Instrument Co., Ltd.), mequidoxine injection (Sichuan Qianxing Animal Pharmaceutical Co., Ltd.), mequidoxine injectable sustained-release nanoemulsion preparation obtained in Example 5, and UV-visible spectrophotometer (GoldS53, Shanghai Huayan Instrument Equipment Co., Ltd.).

[0119] (2) Methods:

[0120] The selection of the measurement wavelength and the measurement of the absorbance of the reference solution (standard curve) refer to the selection of the measurement wavelength and the measurement of the absorbance of the reference solution in the above-mentioned content determination.

[0121] Release determination method: accurately measure equal amounts (calculated according to the content of mequindox) of mequindox nanoemulsion and mequindox injection and place them in dialysis bags, place the dialysis bags in hanging baskets, and place the hanging baskets in dissolution cups filled with 350mL release medium (PBS); control the dissolution apparatus at (37±0.5)℃ and 100r / min, and regularly take 3.5mL samples from the cup with a syringe, and replenish the release medium at the same time. Dilute the sample to 7mL with PBS, filter it through a filter membrane after high-speed centrifugation, and measure the absorbance value under ultraviolet spectrophotometer, and use the formula to calculate its cumulative release percentage.

[0122] Cumulative release percentage = (C n × dilution factor + ∑C n-1 ×3.5) / G×100%

[0123] In the formula, C n represents the drug concentration in the release medium at each sampling;

[0124] C n-1 represents the drug concentration in the release medium of the previous sampling;

[0125] G is the drug content in the nanoparticles; G = nanoparticle mass × drug loading.

[0126] Experimental results:

[0127] The in vitro release test results of the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention are as follows Figure 2 As shown, the release curve of the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention is smoother than that of the common injection, and 70% is released after 80 hours in vitro, while the release rate of the common mequindox injection is close to 100% after 40 hours in vitro. Therefore, the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention has a significant sustained-release function compared with the common mequindox injection.

[0128] The mequidoxine injectable sustained-release nanoemulsion preparation obtained by the present invention is prepared by an oil-in-water emulsification method, and the drug is wrapped in microspheres, so that the release of the drug needs to break through a layer of membrane outside the microspheres first, thereby playing a role in delaying the release of the drug.

[0129] Application Example 1

[0130] Clinical treatment test of mequindox injectable sustained-release nanoemulsion injection preparation on yellow and white diarrhea in piglets obtained in Example 5 of the present invention:

[0131] (1) Materials:

[0132] Reagents and drugs: mequidoxine ordinary injection (Sichuan Qianxing Animal Pharmaceutical Co., Ltd.), mequidoxine injectable sustained-release nanoformulation obtained in Example 5 of the present invention (injection administration, homemade in the laboratory), syringe (9-gauge needle);

[0133] Animals: 120 piglets with yellow and white diarrhea were selected from a large pig farm in Hunan Province. The diagnostic criteria are as follows:

[0134] Symptoms: The sick piglets are depressed, thirsty, unwilling to feed, and excrete yellow or grayish-yellow watery feces containing small pieces of curd and bubbles, or milky white, grayish-white slurry or paste-like feces with a fishy smell. They are depressed, eat less milk, become emaciated, and are dehydrated.

[0135] (2) Methods:

[0136] Efficacy criteria:

[0137] Recovery: After taking the medicine, the clinical symptoms of the sick pigs disappeared, their spirits became normal, their bowel movements became normal, and their appetites recovered.

[0138] Improvement: Clinical symptoms begin to disappear and stool gradually becomes thicker than normal.

[0139] Ineffectiveness: Clinical symptoms do not improve or the disease becomes increasingly severe due to the bacteria developing resistance to the preparation, with persistent diarrhea and severe dehydration.

[0140] Total effective rate = recovery + improvement.

[0141] Experimental Design:

[0142] Piglets with yellow and white diarrhea (the pathogen is Escherichia coli) were randomly divided into 2 groups, including 40 piglets in the test group and 40 piglets in the control group. The piglets in the test group were intramuscularly injected with the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention once a day for three consecutive days. The control group was injected with the above-mentioned ordinary mequindox injection twice a day for three consecutive days.

[0143] The experimental results are shown in Table 5.

[0144] Table 5 Clinical efficacy of different treatment groups in clinical treatment trials

[0145]

[0146] As can be seen from Table 5, the total effective rate of the experimental group for the treatment of yellow and white diarrhea in piglets reached 97.5%, and the cure rate was significantly higher than that of other groups. The total effective rate of the control group for the treatment of yellow and white diarrhea in piglets was 90.0%. While reducing the number of manual administrations and the dosage, the effect of the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention was significantly better than that of the control group. Under the same number of administrations, the effect of the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention was significantly better than that of the control group. It can be seen that the injectable sustained-release nanoemulsion preparation of mequindox obtained in Example 5 of the present invention has a unique sustained-release and synergistic effect compared with the ordinary mequindox injection.

[0147] Embodiment 6-17

[0148] Example 5 was repeated except for the differences described in Table 6.

[0149] Table 6

[0150]

[0151]

[0152] The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

Claims

1. A mequidoxine nano sustained-release injection, characterized in that: Including Tween 80, Span 80, isopropyl myristate, anhydrous ethanol, polyvinyl pyrrolidone K 30 , mequindox and water; in each 1000mL of mequindox nano sustained-release injection, the content of Tween 80 is 60-180mL, the content of Span 80 is 12-36mL, the content of isopropyl myristate is 24-72mL, the content of anhydrous ethanol is 40-120mL, and the content of polyvinylpyrrolidone K is 24-72mL. 30 The content of is 2-6g, and the content of mequidoxine is 10-30g.

2. The mequidoxine nano sustained-release injection according to claim 1, characterized in that: In each 1000mL of mequindox nano sustained-release injection, the content of Tween 80 is 80-150mL, the content of Span 80 is 18-30mL, the content of isopropyl myristate is 36-60mL, the content of anhydrous ethanol is 60-100mL, and the content of polyvinylpyrrolidone K is 100mL. 30 The content of is 3-5g, and the content of mequidoxine is 15-25g.

3. The mequidoxine nano sustained-release injection according to claim 1, characterized in that: In the mequidoxine nano sustained-release injection, the volume ratio of Tween 80, Span 80 and isopropyl myristate is 4-6:1:1-3.

4. The mequidoxine nano sustained-release injection according to claim 1, characterized in that: In the mequidoxine nano sustained-release injection, anhydrous ethanol and polyvinyl pyrrolidone K 30 The ratio is 18-22mL:1g.

5. The mequidoxine nano sustained-release injection according to any one of claims 1 to 4, characterized in that: The mequidoxine nano sustained-release injection is an oil-in-water nanoemulsion preparation.

6. The method for preparing the mequidoxine nano sustained-release injection according to any one of claims 1 to 5, characterized in that: The steps include: S1. Mix Tween 80, Span 80, isopropyl myristate and mequidoxine in a certain ratio to obtain solution A. According to the ratio of anhydrous ethanol, polyvinyl pyrrolidone K 30 Mix well to obtain liquid B; S2, mixing the liquid A and the liquid B evenly to obtain an oil phase; S3. Add water dropwise into the oil phase according to the ratio, stirring while adding water, so that the solution changes from clear to turbid and then from turbid to clear, to obtain a finished product of mequidox nano sustained-release injection.

7. The preparation method according to claim 6, characterized in that: In S1, Tween 80, Span 80 and isopropyl myristate are firstly mixed evenly to obtain a composite emulsion; then mequidoxine is mixed with the composite emulsion and heated to 20-80° C. to fully dissolve mequidoxine to obtain liquid A.

8. The preparation method according to claim 6, characterized in that: In S2, the liquid B is preheated to 20-80° C. and then mixed evenly with the liquid A to obtain an oil phase.

9. The preparation method according to claim 6, characterized in that: In S3, during the dropping process, the temperature of the oil phase is maintained at 20-80°C, preferably 40-60°C, and more preferably 45-55°C.

10. The preparation method according to claim 6, characterized in that: The method further comprises S4, i.e. filtering the finished product of mequindox nano sustained-release injection through a filter membrane, introducing an inert gas for sealing, and sterilizing at 119-123° C. for 10-20 min; preferably, the pore size of the filter membrane is less than 1 μm, preferably less than 0.5 μm, and more preferably less than 0.2 μm.

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