CN112972383A

CN112972383A - Tildipirosin nanoemulsion, preparation method and application thereof in prevention and treatment of calf colibacillosis diarrhea

Info

Publication number: CN112972383A
Application number: CN202110417832.3A
Authority: CN
Inventors: 高海慧; 段孝谋; 乔波; 刘苗苗; 曹晓伟; 李磊; 李乐; 李正; 徐滔; 李�真; 陈文东; 刘宸铄
Original assignee: Institute Of Animal Science Ningxia Academy Of Agricultural And Forestry Sciences
Current assignee: Institute Of Animal Science Ningxia Academy Of Agricultural And Forestry Sciences
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2021-06-18
Anticipated expiration: 2041-04-19
Also published as: CN112972383B

Abstract

A tildipirosin nanoemulsion, wherein the weight of each 100g of the nanoemulsion is as follows: 0.01-6.0 g of tildipirosin, 2.0-10.0 g of dodecanal, 0.1-8.0 g of isopropyl myristate, 5.0-15.0 g of polyoxyethylene lauryl ether, 15.0-25.0 g of polyoxyethylene (60) hydrogenated castor oil, 1.0-8.0 g of glycerol and the balance of deionized water. The tildipirosin nanoemulsion is an oil-in-water emulsion, is solid when the temperature is less than or equal to 35 ℃, is liquid when the temperature is more than 35 ℃, and has the particle size of drug emulsion drops between 10 and 60 nm. The invention solves the problem that the Tildipirosin is insoluble in water, can be clinically administered by drinking water, is more convenient to use, has improved bioavailability and good safety, and can be used for preventing and treating diarrhea caused by calf escherichia coli infection. The invention further discloses a preparation method of the veterinary drug, the process has strong operability, the production can be carried out by conventional veterinary drug production equipment, the technology is easy to convert, and the market prospect is wide.

Description

Tildipirosin nanoemulsion, preparation method and application thereof in prevention and treatment of calf colibacillosis diarrhea

Technical Field

The invention belongs to the technical field of veterinary drugs, and particularly relates to a tildipirosin nanoemulsion, a preparation method and application thereof in preventing and treating calf colibacillosis.

Background

Colibacillosis of calves is caused by infection of pathogenic escherichia coli, pathogenic bacteria destroy intestinal mucosa through endotoxin, exotoxin, invasion factors, cytotoxin and the like, the calves have incomplete development due to immune function and a rumen microecological system, the infection resistance to external escherichia coli is poor, once infection occurs, diarrhea is easy to occur, and the calves can be seriously dehydrated and die. At present, colibacillosis diarrhea has become one of important diseases which hinder the development of cattle raising industry in China, in the prior art, medicines such as gentamycin, streptomycin, amikacin, ampicillin, florfenicol and the like are usually used for treating the disease, but after long-term use of the medicines, serious drug resistance is generated in many areas, the effect is increasingly poor, and a new antibiotic for animals is urgently needed to replace in the market, so that the development of the cattle raising industry is promoted.

The tildipirosin is a novel macrolide antibiotic special for animals, and the in vitro drug sensitivity evaluation result in a laboratory shows that the minimum inhibitory concentration of the tildipirosin is lower than that of other clinically common antibiotics, such as gentamicin, streptomycin, amikacin, ampicillin and florfenicol. The Tildipirosin has short time to market, basically has no drug resistance problem at present, and is a veterinary drug with good market prospect. However, the water solubility of the tildipirosin is poor, the drug cannot be administered in a drinking water mode, for calves, the daily ration structure of the calves mainly adopts breast milk, water can be drunk at ordinary times, feed is only slowly contacted with the calves to feed in the later stage of lactation, if escherichia coli infection occurs and diarrhea symptoms occur, the mode of mixing the feed and administering the drug is undoubtedly not as convenient as the drinking water, but the defect of poor water solubility of the tildipirosin causes great obstruction in the popularization of the drug.

Aiming at the characteristics of the Tildipirosin medicine and the defects and deficiencies in the popularization process, the Tildipirosin nano-emulsion is prepared, the emulsion is of an oil-in-water type, the main component is Tildipirosin, the particle size of the medicine is 10-60 nm through the preparation process disclosed by the invention, the problem of poor water solubility of the medicine is solved, the specific surface area of the medicine is increased after the medicine is reduced, the bioavailability is obviously improved, the bitter taste of the medicine is covered to a certain extent, the problem of poor palatability when drinking water is used is avoided, the drinking water amount is not influenced, the Tildipirosin nano-emulsion also has the advantages of high safety, good stability, no separation, no layering, no demulsification, no degradation and the like of the medicine after being diluted by water, and the market prospect is wide.

Disclosure of Invention

The invention aims to provide a tildipirosin nanoemulsion, and also aims to provide a second invention aim of a preparation method and a third invention aim of the invention.

Based on the purpose, the invention adopts the following technical scheme: a tildipirosin nanoemulsion, wherein the weight of each 100g of the nanoemulsion is as follows: 0.01-6.0 g of tildipirosin, 2.0-10.0 g of dodecanal, 0.1-8.0 g of isopropyl myristate, 5.0-15.0 g of polyoxyethylene lauryl ether, 15.0-25.0 g of polyoxyethylene (60) hydrogenated castor oil, 1.0-8.0 g of glycerol and the balance of deionized water.

Preferably, the nanoemulsion consists of, per 100g by weight: 1.0-5.0 g of tildipirosin, 3.0-9.0 g of dodecanal, 1.0-7.0 g of isopropyl myristate, 6.0-14.0 g of polyoxyethylene lauryl ether, 16.0-24.0 g of polyoxyethylene (60) hydrogenated castor oil, 2.0-7.0 g of glycerol and the balance of deionized water.

Further preferably, the nanoemulsion consists of, per 100g by weight: 3.0g of tildipirosin, 6.0g of dodecanal, 4.0g of isopropyl myristate, 10.0g of polyoxyethylene lauryl ether, 20.0g of polyoxyethylene (60) hydrogenated castor oil, 4.5g of glycerol and 52.5g of deionized water.

Preferably, the deionized water is sterilized deionized water.

Preferably, the formulation is of the oil-in-water type (as in figure 1).

The tylonolide nanoemulsion is solid when the temperature is less than or equal to 35 ℃, and is liquid when the temperature is more than 35 ℃.

The preparation method of the tildipirosin nanoemulsion comprises the following steps: (a) uniformly mixing dodecanal and isopropyl myristate, heating to 40-50 ℃, adding the tildipirosin, and stirring until the mixture is completely dissolved to obtain an oil phase; (b) adding polyoxyethylene lauryl ether and polyoxyethylene (60) hydrogenated castor oil into the oil phase, uniformly mixing, and maintaining the system temperature at 40-50 ℃ to obtain a mixture 1; (c) mixing glycerol and deionized water uniformly to obtain a mixture 2; (d) and adding the mixture 2 into the mixture 1 for emulsification until the system becomes a uniform colorless or light yellow system, stopping emulsification, cooling to room temperature, and solidifying the system to obtain the nano-emulsion.

The storage conditions of the tildipirosin nanoemulsion of the invention are as follows: and (5) storing in a shady, ventilated and dry place in a sealed way.

The formula of the invention is reasonable, the prepared nano-emulsion is oil-in-water type, and the emulsion droplet particles are spherical or nearly spherical and have the diameter of 10-60 nm (as shown in figure 2) through the magnified observation of an electron microscope. The nano-scale drug particle size is beneficial to increasing the specific surface area and improving the bioavailability of the drug through the small-size effect. The emulsion of the invention is safe in clinical use, and has very good prevention and treatment effects on diarrhea caused by common escherichia coli infection of calves. The drug is strong in stability after being diluted by water, the problems of drug precipitation, layering, emulsion breaking, degradation and the like do not exist, the palatability is good, the water intake of calves is not influenced, the preparation process is strong in operability, easy to convert and produce, low in manufacturing cost and good in market popularization prospect.

In the formula of the invention, the components have the following effects:

1) the selected tildipirosin is a novel macrolide antibiotic for veterinary use, does not have the problem of drug resistance at present, has good prevention and treatment effects on diarrhea caused by common pathogenic escherichia coli infection of calves, can replace the existing antibiotic which is clinically used for years at present to be used, and has more ideal effects;

2) the dodecanal and the isopropyl myristate are selected as the mixed oil, and the mixed system of the dodecanal and the isopropyl myristate has a good dissolving effect on the tildipirosin, so that the emulsion drop drug loading is favorably improved, and the oil is very suitable for serving as an oil phase;

3) lauryl alcohol polyoxyethylene ether and polyoxyethylene (60) hydrogenated castor oil are used as the components of the invention, because the mixed system of the two surfactants has good emulsification effect on the oil phase consisting of dodecanal and isopropyl myristate, the prepared system is not easy to demulsify, has strong stability and is beneficial to storage and transportation;

4) the glycerol is used as the component of the invention, firstly, the glycerol can be used as the co-emulsifier to enhance the stability of the system, shorten the emulsification time in the production process and be beneficial to improving the production efficiency. And secondly, the glycerol also has a dissolving effect on the tildipirosin, so that the drug loading capacity is further improved and the drug stability is enhanced.

The invention is an oil-in-water emulsion (as figure 1), when in clinical use, the medicine can be dissolved and diluted by water for the calf to drink, the liquid medicine is uniformly distributed in the water, which is beneficial to uniform administration, and the original bitter taste of the medicine is covered due to the coating effect of the emulsion drops on the medicine, so that the palatability and the drinking water amount are not influenced.

Compared with the prior art, the invention achieves the technical effects that:

(1) the tildipirosin nanoemulsion can dissolve tildipirosin with poor water solubility in water, improves the drug solubility, and can meet the clinical drinking water administration requirement;

(2) according to the nano-emulsion prepared by the invention, emulsion droplets are spherical or nearly spherical (as shown in figure 2), the diameter is 10-60 nm, the specific surface area of the medicine is large, the relative bioavailability is high, and the full play of the medicine effect is facilitated;

(3) the emulsion medicine prepared by the invention is coated, the original bitter taste of the medicine is covered, the palatability problem is not generated, and the influence on the drinking amount is not caused during clinical use;

(4) the emulsion prepared by the invention is solid when the temperature is less than or equal to 35 ℃, and is converted into liquid when the temperature is more than 35 ℃, and the characteristic ensures that the medicine exists in a solid form when being stored or transported at normal temperature, and the stability is stronger than that of the common liquid emulsion, thereby being more beneficial to long-distance transportation and having longer quality guarantee period.

(5) The product of the invention has stable property and strong operability of the preparation process, and is beneficial to production conversion.

Drawings

FIG. 1 is a photograph showing the type discrimination of the nanoemulsion of the present invention, in which the left side shows a state where methylene blue is dropped and then left to stand for 30min, and the right side shows a state where Sudan III is dropped and then left to stand for 30 min.

Fig. 2 is an electron micrograph of the nanoemulsion prepared by the invention, wherein the black near-spherical particles are drug emulsion droplets.

Detailed Description

The invention will be further illustrated by the following specific examples, which are not intended to limit the scope of the invention in any way.

Examples 1 to 10

For the sake of brevity of the description, the weight composition of the tylonolide nanoemulsion of the present invention described in examples 1-10 is given below in the form of a table, which is shown in table 1.

TABLE 1 compositions by weight per 100g of the invention in examples 1-10

The method for preparing the tylonolide nanoemulsion according to embodiments 1-10, comprising the steps of:

(a) uniformly mixing dodecanal and isopropyl myristate, heating to 45 ℃, adding the tildipirosin, and stirring until the mixture is completely dissolved to obtain an oil phase;

(b) adding polyoxyethylene lauryl ether and polyoxyethylene (60) hydrogenated castor oil into the oil phase, uniformly mixing, and maintaining the system temperature at 45 ℃ to obtain a mixture 1;

(c) mixing glycerol and deionized water uniformly to obtain a mixture 2;

(d) and adding the mixture 2 into the mixture 1 for emulsification until the system becomes a uniform colorless or light yellow system, stopping emulsification, and cooling to room temperature to gradually solidify the system to obtain the nano-emulsion.

In other embodiments, the heating temperature in step (a) and step (b) is 40-50 ℃.

Electron microscope tests on the nano-emulsion obtained by the invention show that the emulsion droplet particle size distribution of the emulsion prepared by the invention is between 10 and 60 nm. The invention takes example 1 as an example to give an electron microscope photo of the nanoemulsion, in the figure, a black near-spherical shape is a medicament emulsion droplet, and the particle size of the emulsion droplet is distributed between 10 nm and 60 nm.

Test examples 1 to 4:

test example 1

In order to identify the type of the nano-emulsion, the characteristics of methylene blue as a water-soluble dye and Sudan red III as a fat-soluble dye are utilized, a sample (taking the sample prepared in example 1 as an example) of the nano-emulsion is placed at a constant temperature of 40 ℃, after the sample is converted into liquid, the methylene blue and the Sudan red III are respectively dripped into the taken sample, the sample is slightly shaken and is kept still for 30min for observation, and the result is shown in figure 1.

And (4) judging the standard:

if the system is oil-in-water type, the external phase is water phase, the internal phase is oil phase, methylene blue shows diffusion, Sudan red III does not diffuse, and floats on the liquid level;

if the system is water-in-oil type, the external phase is oil phase, the internal phase is water phase, methylene blue is not diffused and floats on the liquid surface, and Sudan red III is diffused;

if the system is an oil-water emulsion, the diffusion of methylene blue and Sudan red is shown.

As can be seen from fig. 1, the tylonolide nanoemulsion of the present invention exhibited methylene blue diffusion, and sudan iii did not appear to diffuse, but floated on the surface of the system.

The results show that: the tildipirosin nano emulsion is oil-in-water type.

Test example 2

Properties and stability tests of the products of the invention

The samples of the examples 1-10 of the present invention were taken, and high temperature, normal temperature, cold storage and freezing stability tests were performed, and the samples were placed in the environment of 40 ℃, 25 ℃, 4 ℃ and-20 ℃ respectively, and were sampled and observed at 1d, 15d, 1 month, 3 months, 6 months, 12 months and 24 months, respectively, and as a result, it was found that the products obtained in the examples were free from instability such as precipitation, delamination, discoloration and drug precipitation, and had good drug stability.

Test example 3

Animal safety test

16 calves of 2 months old are selected in the same cattle farm and randomly divided into 4 groups of a control group, a low-dose drug group, a medium-dose drug group and a high-dose drug group, wherein 4 calves in the control group drink water normally, and the low-dose drug group, the medium-dose drug group and the high-dose drug group respectively drink 10ml, 20ml and 40ml of the nanoemulsion (taking the nanoemulsion prepared in example 1 as an example in the test) of the invention intensively from each cow respectively, the drugs are taken for 1 time every day, during which, the test calves in each group are raised in the same environment and managed by the same breeder, the test period is 15d, and the behavior and physiological reaction of each group of calves are observed and recorded for 1 time every 3d, so that whether the behavior and physiological reaction of each group of calves are normal or not is observed. If the cattle is poisoned and died, the cattle is dissected in situ, and the pathological changes of the organs are observed and recorded.

The results are given in the following table:

TABLE 2 safety test results of the tylonolide nanoemulsion of the present invention

First day

No. 3d

6d th

9d th

12d th

Th 15d

Group 1

Is normal

Group 2

Is normal

Group 3

Is normal

Group 4

Is normal

The results show that: the cattle and the control group of each test group have no death, and the behavioral expression and the physiological reaction have no difference and are normal, which indicates that the tildipirosin nanoemulsion of the invention has good safety.

Test example 4

Clinical efficacy test

20 diseased calves which are similar in disease degree of 2 months old and cause diarrhea due to escherichia coli infection are selected, the clinical manifestations of diarrhea are achieved, excrement is not formed, sticky excrement is frequently generated at the tail root part, the milk sucking amount is reduced, the spirit is not vibrating, people do not like to lie on the ground, and the calves are not sensitive to external stimulation. 20 sick cattle are randomly divided into 4 groups, each group comprises 5 cattle, wherein the 1 st group is a blank control group and does not use any medicine, the 2 nd group is a clinical common medicine gentamicin sulfate treatment group, each cattle is dissolved in water for centralized drinking according to the dose of 10mg/kg body weight, the 3 rd group is a raw material medicine group of tildipirosin, each cattle is added into physiological saline according to the dose of 10mg/kg body weight, the mixture is shaken up and suspended and then drenched, the 4 th group is a medicine group of the nanoemulsion (taking the nano emulsion prepared in example 1 in the test as an example) of the invention, and the medicines are dissolved in water for centralized drinking according to the dose of 0.333g/kg body weight (the actual amount of the tildipirosin medicine is 10 mg/kg). Each group of cattle was administered 1 time per day for 3 days. During the period, all the cows are raised in the same environment, the feed, the drinking water, the illumination, the temperature and the humidity in the house, the breeders, the management conditions and the like are all the same, the testers make records of the ingestion, the drinking water, the behaviors, the death conditions and the like, and the drug effect is evaluated after the test is finished.

Evaluation criteria of drug efficacy:

death: the calf died during the test, score-1;

and (4) invalidation: after the medicine is applied, although the calf does not die, the calf still has diarrhea, the general symptom expression is not obviously improved, and the score is 0;

turning to: after the prescription is used, the symptoms of the calf are obviously relieved, although some symptoms exist compared with the completely healthy cattle, the body is not completely recovered, but the symptoms are obviously improved by 1 minute compared with the symptoms before the prescription is used;

and (3) curing: after the test is finished, the diarrhea of the calf is completely disappeared, the tail root is clean, the excrement is formed, and the behaviors of feeding, drinking and the like are not different from those of healthy calves of the same age by 2 minutes;

the cure rate is as follows: the ratio of the number of the cured animals to the total number of the test animals in the group is multiplied by 100 percent;

the total effective rate is as follows: the ratio of the total number of animals returned and cured after the drug administration to the total number of the test animals in the group is multiplied by 100%.

TABLE 3 clinical efficacy test of the tylonolide nanoemulsion of the present invention

Group of	Death (/ only)	Invalid (/ only)	Returning (/ only)	Cure (/ only)	Score of
						Group 1	1	4	0	0	-1
Group 2	0	2	2	1	4
						Group 3	0	1	2	2	6
Group 4	0	0	1	4	9

The test results are as follows:

on the premise that the group 1 does not use any medicine for treatment, diseases are allowed to naturally develop, 1 death finally occurs, 4 deaths do not occur, and the cure rate and the total effective rate are 0 to obtain 1 min;

group 2 treated with gentamicin sulfate concentrated drinking water, 0 died, 2 failed, 2 returned, 1 cured, the cure rate was 20%, the total effective rate was 60%, and the score was 4;

the group 3 is treated by oral administration of the Tildipirosin raw material medicine, 0 patient dies, 1 patient fails, 2 patients return, and 2 patients are cured, the cure rate is 40 percent, the total effective rate is 80 percent, and the score is 6;

group 4 treated with the tylonolide nanoemulsion of the invention, 0 died, 0 failed, 1 returned and 4 cured, the cure rate is 80%, the total effective rate is 100%, and 9 points are obtained.

The results show that:

1) compared with the group 2 and the group 3, the group 3 has the cure rate and the total effective rate which are both 20 percent higher than those of the group 2 and the score is 2 percent higher by using the medicine with the same dosage, which shows that the effect of the tildipirosin on the treatment of calf colibacillary diarrhea is better than that of gentamycin sulfate.

2) Compared with the group 3 and the group 4, when the same dose of the tildipirosin is used, the cure rate and the total effective rate of the group 4 are respectively 40% higher and 20% higher than those of the group 3, and the score is 3 points higher, which indicates that the bioavailability of the medicine is improved after the tildipirosin is prepared into the nano-emulsion of the invention.

And (4) test conclusion: tildipirosin can be used for treating diarrhea diseases caused by calf Escherichia coli infection. After the tildipirosin is prepared into the nano emulsion, the bioavailability of the medicine is improved, and the treatment effect is remarkably improved.

Claims

1. The tildipirosin nanoemulsion is characterized in that the weight of each 100g of the nanoemulsion is as follows: 0.01-6.0 g of tildipirosin, 2.0-10.0 g of dodecanal, 0.1-8.0 g of isopropyl myristate, 5.0-15.0 g of polyoxyethylene lauryl ether, 15.0-25.0 g of polyoxyethylene (60) hydrogenated castor oil, 1.0-8.0 g of glycerol and the balance of deionized water.

2. The tildipirosin nanoemulsion according to claim 1, wherein the composition per 100g of the nanoemulsion is: 1.0-5.0 g of tildipirosin, 3.0-9.0 g of dodecanal, 1.0-7.0 g of isopropyl myristate, 6.0-14.0 g of polyoxyethylene lauryl ether, 16.0-24.0 g of polyoxyethylene (60) hydrogenated castor oil, 2.0-7.0 g of glycerol and the balance of deionized water.

3. The tildipirosin nanoemulsion according to claim 2, wherein the composition per 100g of the nanoemulsion is: 3.0g of tildipirosin, 6.0g of dodecanal, 4.0g of isopropyl myristate, 10.0g of polyoxyethylene lauryl ether, 20.0g of polyoxyethylene (60) hydrogenated castor oil, 4.5g of glycerol and 52.5g of deionized water.

4. The tildipirosin nanoemulsion according to claim 1, 2 or 3, wherein said deionized water is sterilized deionized water.

5. The tylonolide nanoemulsion of claim 4, wherein the nanoemulsion formulation is of the oil-in-water type.

6. The tildipirosin nanoemulsion of claim 5, which is solid at a temperature of 35 ℃ or lower and liquid at a temperature of 35 ℃ or higher.

7. A method for preparing a tildipirosin nanoemulsion according to claim 1, 2 or 3, comprising the steps of: (a) uniformly mixing dodecanal and isopropyl myristate, heating to 40-50 ℃, adding the tildipirosin, and stirring until the mixture is completely dissolved to obtain an oil phase; (b) adding polyoxyethylene lauryl ether and polyoxyethylene (60) hydrogenated castor oil into the oil phase, uniformly mixing, and maintaining the system temperature at 40-50 ℃ to obtain a mixture 1; (c) mixing glycerol and deionized water uniformly to obtain a mixture 2; (d) and adding the mixture 2 into the mixture 1 for emulsification until the system becomes a uniform colorless or light yellow system, stopping emulsification, cooling to room temperature, and solidifying the system to obtain the nano-emulsion.

8. Use of the tildipirosin nanoemulsion of claim 1, 2 or 3 for the prevention and treatment of colibacillosis in calves.