CN112999330B

CN112999330B - Preparation method of estradiol benzoate and oxytocin long-acting suspension injection

Info

Publication number: CN112999330B
Application number: CN202110290222.1A
Authority: CN
Inventors: 郭晓强; 何顺东; 康泰然; 宋芹; 姚倩; 李小红
Original assignee: Chengdu University
Current assignee: Chengdu University
Priority date: 2021-03-18
Filing date: 2021-03-18
Publication date: 2023-04-18
Anticipated expiration: 2041-03-18
Also published as: CN112999330A

Abstract

The invention provides a preparation method of estradiol benzoate and oxytocin long-acting suspension injection, which comprises the following steps: poloxamer 188 is used as a wetting agent, sodium carboxymethylcellulose is used as a suspending agent, the mixture is mixed with estradiol benzoate in water, the mixture is homogenized by adopting an ultrasonic cell disruption mode, then the mixture is mixed with oxytocin solution, and the pH value is adjusted by acetic acid to prepare estradiol benzoate and oxytocin suspension; the mass concentration of the wetting agent in the estradiol benzoate and oxytocin suspension is 0.1-0.9%, and the mass concentration of the suspending agent is 0.1-0.5%. Experiments show that the mixture of the two medicines has better effect on postpartum hemostasis when being used as veterinary medicines; the intramuscular injection of the suspension can facilitate the administration and is beneficial to improving the curative effect, the compliance and the like. The preparation method is simple to operate, stable and reliable in quality, and can provide effective guidance for enterprise scale-up production.

Description

Preparation method of estradiol benzoate and oxytocin long-acting suspension injection

Technical Field

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a preparation method of estradiol benzoate and oxytocin long-acting suspension injection.

Background

Oxytocin is a cyclic nonapeptide neuropeptide, can selectively excite uterine smooth muscle to cause uterine contraction, can be upwards pulled by cervix, and can stimulate endometrium to release prostaglandin to soften cervix, so that the oxytocin becomes an important means for treating postpartum hemorrhage. Oxytocin can be degraded by oxytocin in uterus, and estrogen has the effects of inhibiting oxytocin, regulating oxytocin receptors, regulating uterine blood flow and the like. The common estrogen drug is estradiol benzoate, the chemical name of which is 3-hydroxyestra-1,3,5 (10) -triene-17 beta-alcohol-3-benzoate, which is an estrogen prodrug, is converted into estradiol after being metabolized in vivo to proliferate endometrium and enhance contraction of uterine smooth muscle, and can act synergistically with oxytocin.

Estradiol benzoate and oxytocin are main medicines for inducing parturition and stopping bleeding in the postpartum period of animals. Oxytocin has excellent water solubility, is sensitive to temperature and pH, is basically ineffective when being orally taken, is generally administered sublingually or by injection, and has very little dosage; estradiol benzoate is poorly water soluble. At present, the two medicines are separately used when being used by animals, and the estradiol benzoate is generally oil-soluble injection which mainly has the defects of strong pain feeling, poor compliance, short action time and the like when in use; oxytocin is applied as water soluble injection. When the animal directly joins the two, the operation is complex, and the drug administration is not convenient and rapid. However, no related studies on the mixed dosage forms of the two drugs have been reported.

Disclosure of Invention

In view of the above, the present application provides a method for preparing estradiol benzoate and oxytocin long-acting suspension injection, and the present invention adopts suspension injection to mix the two to achieve the purpose of combined administration, which makes the administration for animals more convenient, etc.

The invention provides a preparation method of estradiol benzoate and oxytocin long-acting suspension injection, which comprises the following steps:

mixing poloxamer 188 as a wetting agent and sodium carboxymethylcellulose as a suspending agent with estradiol benzoate in water, homogenizing by ultrasonic cell disruption, mixing with oxytocin solution, and adjusting pH with acetic acid to obtain estradiol benzoate and oxytocin suspension;

the mass concentration of the wetting agent in the estradiol benzoate and oxytocin suspension is 0.1-0.9%, and the mass concentration of the suspending agent is 0.1-0.5%.

The invention prepares the estradiol benzoate and oxytocin long-acting suspension injection (also called estradiol benzoate and oxytocin suspension injection, etc.) by a process mode of an ultrasonic cell disruption method; the ultrasonic cell disruption method is to use ultrasonic cell disruption equipment to carry out ultrasonic treatment for a certain time so as to uniformly mix and disperse.

Enterprises generally adopt a high-pressure homogenizer when preparing a large amount of single pharmaceutical preparations, but the preparation amount is large, and the high-pressure homogenizer is not suitable for laboratory experiments. The applicant researches and discovers that the preparation process such as ultrasonic anti-solvent method, vortex oscillation anti-solvent method and the like usually needs to add ethanol, cannot completely remove the ethanol, and easily causes poor suspension and over-low yield. The invention adopts an ultrasonic cell disruption method to realize the preparation of the estradiol benzoate and oxytocin long-acting suspension injection without adding ethanol; the preparation amount can be properly increased; the suspension is better and the particle size is small.

In addition, the invention mainly researches the types, the dosage and other factors of the suspending agent and the wetting agent. In the embodiment of the invention, a proper amount of sodium carboxymethylcellulose (CMC-Na) serving as a suspending agent and a proper amount of poloxamer 188 serving as a wetting agent are weighed, preferably, the sodium carboxymethylcellulose and the poloxamer 188 are heated and dissolved by ultrapure water, and the mixture is filtered when the mixture is hot and is subjected to constant volume to prepare a blank solution. Then, in the embodiment of the invention, a certain amount of estradiol benzoate is precisely weighed in the blank liquid, the blank liquid is homogenized by an ultrasonic crusher after being properly shaken, oxytocin solution is added and uniformly mixed at normal temperature, the pH value is adjusted by acetic acid, and the volume is fixed by ultrapure water.

In the invention, poloxamer 188 is used as a wetting agent, and sodium carboxymethylcellulose is used as a suspending agent, so that the synergistic effects of wetting, thickening and the like are good. The poloxamer is polyoxyethylene polyoxypropylene ether block copolymer; sodium carboxymethylcellulose is a cellulose derivative having a degree of polymerization of glucose of 100 to 2000, and has various applications in the pharmaceutical industry. In the estradiol benzoate and oxytocin suspension, the mass concentration of the wetting agent poloxamer 188 is 0.1-0.9%, and the preferable dosage range is 0.25-0.35% with better effect; the mass concentration of the suspending agent sodium carboxymethyl cellulose is 0.1-0.5%, the dosage range is preferably 0.15-0.25%, and the effect is better.

The ultrasonic time for the ultrasonic cell disruption is preferably 5 to 15min, and more preferably 7 to 13min. In a preferred embodiment of the invention, the specific mixture ratio is as follows: poloxamer 188.25% as wetting agent, and carboxymethylcellulose sodium 0.20% as suspending agent; the ultrasonic treatment time is 7min.

Estradiol benzoate and oxytocin adopted by the application are commercially available medicines (the purity is more than 99%); the proportion of the two main drugs is determined according to the main single drug dosage in the current market, and the proportion can be properly adjusted according to different use objects (the proportion range is in the range specified by the pharmacopoeia).

In some embodiments of the present application, the optimal prescription is: 100mg of estradiol benzoate, 500 units of oxytocin, 0.20 percent of sodium carboxymethylcellulose, 0.25 percent of poloxamer 188 and a proper amount of acetic acid, and can be prepared into 50mL of estradiol benzoate and oxytocin suspension.

The pH value of the estradiol benzoate and oxytocin suspension is generally 3.0-4.5, preferably 3.5-4.4, and more preferably 3.9-4.1. And the particle diameter of the estradiol benzoate and oxytocin suspension is less than 8 mu m.

The preparation method comprises the steps of preparing a long-acting suspension of estradiol benzoate and oxytocin by adopting an ultrasonic cell disruption method, and optimizing a prescription, wherein the CMCNa with the mass concentration of 0.20% is used as a suspending agent, the poloxamer 188 with the mass concentration of 0.25% is used as a wetting agent, and the prepared suspension has good sedimentation volume ratio and redispersion property. Experiments show that the mixture of the two medicines has better effect on postpartum hemostasis when being used as veterinary medicines; the intramuscular injection of the suspension can facilitate the administration and is beneficial to improving the curative effect, the compliance and the like. The preparation method is simple to operate, stable and reliable in quality, and can provide effective guidance for enterprise scale-up production.

Drawings

FIG. 1 is a graph showing the sedimentation volume ratio and the weight dispersibility for different CMC-Na concentrations in examples of the present invention;

FIG. 2 is a graph showing the sedimentation volume ratio and the weight dispersibility for different concentrations of poloxamer 188 in the examples of the present invention;

FIG. 3 is a linear relationship of estradiol benzoate in accordance with examples of the present invention;

FIG. 4 is a linear relation of oxytocin in an embodiment of the present invention;

FIG. 5 shows the hemostasis of the incomplete abortion of rats with the suspension of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

For further understanding of the present application, the preparation method of estradiol benzoate and oxytocin long-acting suspension injection provided by the present application is specifically described below with reference to the examples.

In the following examples, the drugs and main reagents used were: estradiol benzoate (batch number, 20190921, purity not less than 99%), chengdu gold pharmaceutical Co., ltd; oxytocin (540 units/mg) (batch No. 20191217, purity not less than 99%), chengdu gold pharmaceutical Co., ltd; sodium carboxymethylcellulose (lot number, 2015040901), medeto chemical reagent plant; poloxamer 188 (lot number, T24J10Z 80481), sourced lobe biology ltd; acetic acid (lot number, RH 137206), rayne reagent;

the main instruments used were: agilent-1260 type high performance liquid chromatograph (Agilent technologies, inc.); JY92-II N type ultrasonic cell disruptor (Ningbo Xinzhi Biotechnology GmbH); BSA-124S type electronic analytical balance (Sadoris scientific instruments, inc.); HH-4 model digital display thermostat water bath (Australian instruments, inc., changzhou); a pH meter model FE28 (mettler-toledo instruments ltd); biomicroscopes model PH100-2B41L-IPL (Phoenix optics, inc.); CT14D model desk-top high speed centrifuge (shanghai tianmei biochemical instruments engineering ltd); model RE-2000A rotary evaporator (shanghai monokoku instruments ltd).

Example 1

1. Sodium carboxymethylcellulose, sodium alginate and methylcellulose are respectively used as suspending agents and are mixed with the two main drugs (the estradiol benzoate content is 2mg/mL, the oxytocin content is 10 u/mL), and the following screening results are obtained by observation:

table 1 screening of suspending agents

2. Sodium dodecyl sulfate, tween 80, polyethylene glycol 400, polyethylene glycol 4000 and poloxamer 188 are respectively used as wetting agents, sodium carboxymethylcellulose (0.5%) is used as a suspending agent, the two main medicines are mixed (the content of estradiol benzoate is 2mg/mL, and the content of oxytocin is 10 u/mL), and the following screening results are obtained by observation:

TABLE 2 screening of wetting Agents

Example 2

Preparing a blank liquid: weighing appropriate amount of carboxymethylcellulose sodium as suspending agent and appropriate amount of poloxamer 188 as wetting agent, dissolving with ultrapure water under heating, filtering while hot, and metering volume.

Preparing a sample: 100mg of estradiol benzoate was precisely weighed in 50mL of the blank solution, and homogenized by an ultrasonic cell disrupter after shaking appropriately. Adding oxytocin solution at normal temperature, mixing uniformly, adjusting pH with acetic acid, and then using ultrapure water to fix the volume to 50mL.

Wherein the concentration of CMC-Na is 0.1%; poloxamer 188 concentration was 0.3%; the ultrasonic treatment time is 10min.

Examples 3 to 6

The preparation process of example 2 is followed with the only difference that the CMC-Na concentrations are, in order: 0.2%, 0.3%, 0.4%, 0.5%.

Quality evaluations were performed on the suspension samples prepared in examples 2-6 by the following methods:

1. examination of characters

By observation, the suspension appears to be a milky, particle-free liquid and the system remains stable for a certain period of time. After long-term standing and storage, slight layering occurs, and the suspension is recovered after slight shaking.

2. Volume ratio of sedimentation

The prepared suspension was 50mL, stoppered, measured with a stoppered cylinder and the initial height H of the suspension before settling was recorded ₀ After shaking vigorously for 1min and standing for 3H, the height H of the suspension after settling was recorded. By the formula: sedimentation volume ratio (F) = H/H ₀ . The closer the F value to 1, the more stable the suspension, the more than 0.9 the F value indicates compliance (according to the pharmacopoeia of the people's republic of china (fourth part 2020).

3. Test for weight dispersibility

Simulating redispersion test after full precipitation, placing the sample in a centrifuge tube, and subjecting to 10000 r.min ^-1 And (3) generating a precipitate after centrifuging for 10min, repeatedly inverting the precipitate, and recording the reciprocal of the inversion frequency of complete uniform dispersion, wherein the larger the numerical value is, the better the redispersibility of the test sample is.

4. Determination of pH

The pH of oxytocin should be stable between 3.0 and 4.5, as measured with a precision pH meter, with the most stable pH between 3.5 and 4.4.

The sedimentation volume ratio and redispersion results of different CMC-Na concentrations are shown in figure 1, and figure 1 shows that the effect of the suspending agent sodium carboxymethyl cellulose is better when the dosage is between 0.15% and 0.25%.

Example 7

The procedure of example 2 was followed except that the CMC-Na concentration was 0.2%; poloxamer 188 was present at a concentration of 0.1%.

Examples 8 to 11

The preparation process of example 7 was followed, except that the concentrations of poloxamer 188 were 0.3%, 0.5%, 0.7%, 0.9% in this order.

The results of the sedimentation volume ratio and redispersion of different concentrations of poloxamer 188 are shown in fig. 2, and fig. 2 shows that the wetting agent poloxamer 188 has a better effect when the dosage is in the range of 0.25% -0.35%.

Example 12

The procedure of example 2 was followed except that the CMC-Na concentration was 0.25%; poloxamer 188 concentration was 0.15%; the ultrasonic treatment time is 7min.

Sedimentation volume ratio: 0.98 of; redispersion times: 19.

example 13

The procedure of example 2 was followed except that the CMC-Na concentration was 0.25%; poloxamer 188 concentration was 0.20%; the ultrasonic treatment time is 10min.

Sedimentation volume ratio: 1.00; the redispersion times are as follows: 23.

example 14

The preparation process of example 2 was followed except that the CMC-Na concentration was 0.25%; poloxamer 188 concentration was 0.25%; the ultrasonic treatment time is 13min.

Sedimentation volume ratio: 1.00; the redispersion times are as follows: 48.

example 15

The preparation process of example 2 was followed except that the CMC-Na concentration was 0.30%; poloxamer 188 concentration was 0.15%; the ultrasonic treatment time is 10min.

Sedimentation volume ratio: 0.99; the redispersion times are as follows: 24.

example 16

The preparation process of example 2 was followed except that the CMC-Na concentration was 0.30%; poloxamer 188 concentration was 0.20%; the ultrasonic treatment time is 13min.

Sedimentation volume ratio: 0.99; the redispersion times are as follows: 34.

example 17

The procedure of example 2 was followed except that the CMC-Na concentration was 0.30%; poloxamer 188 concentration was 0.25%; the ultrasonic treatment time is 7min.

Sedimentation volume ratio: 1.00; the redispersion times are as follows: 32.

example 18

The procedure of example 2 was followed except that the CMC-Na concentration was 0.35%; poloxamer 188 concentration was 0.15%; the ultrasonic treatment time is 13min.

Sedimentation volume ratio: 0.98 of; the redispersion times are as follows: 25.

example 19

The preparation process of example 2 was followed except that the CMC-Na concentration was 0.35%; poloxamer 188 concentration was 0.20%; the ultrasonic treatment time is 7min.

Sedimentation volume ratio: 0.99; the redispersion times are as follows: 18.

example 20

The procedure of example 2 was followed except that the CMC-Na concentration was 0.35%; poloxamer 188 concentration of 0.25%; the ultrasonic treatment time is 10min.

Sedimentation volume ratio: 1.00; redispersion times: 61.

the optimal scheme is as follows: 0.25% of wetting agent poloxamer 188, 0.20% of suspending agent sodium carboxymethyl cellulose and 7min of ultrasound treatment time.

For the optimal scheme, the quality evaluation content is specifically as follows:

the sedimentation volume ratio of the prepared suspension is 99.19 percent by measurement and calculation, and the prepared suspension conforms to the regulation of Chinese pharmacopoeia (2020 edition).

The prepared suspension is processed at 10000 r.min ^-1 And after centrifugation for 10min, precipitates are generated, and the suspension is repeatedly inverted and redispersed, so that the suspension has good redispersibility.

The measured pH value was 4.0; the prepared suspension has a particle size range of less than 8 μm measured by a microscope.

5mL of the optimally prepared suspension was withdrawn, 0.45 needle elapsed time 42 seconds; 0.6 the needle takes 15 seconds; 0.7 the needle is used for 9 seconds; the needle penetration was good when the needle was used for 0.8 seconds and 6 seconds.

The above needle penetration test: the shaken suspension was withdrawn using a 10mL syringe using 0.45, 0.6, 0.7, 0.8 needle, respectively, and the time taken to withdraw 5mL was recorded.

And (3) particle size measurement: the suspension with proper dilution is put on a glass slide, then a cover glass is covered on the glass slide to lightly press the glass slide, the whole visual field of the glass slide is immediately detected under a microscope with 50 to 100 times of marked scales, no agglomeration phenomenon exists, and particles with 50 mu m cannot be detected. The particle size was then randomly inspected under a 400-fold microscope.

The quality evaluation research shows that the suspension prepared by the invention has the advantages of high weight dispersibility, good needle penetration, and the quality indexes of particle size, sedimentation volume ratio and the like which all accord with the relevant requirements of pharmacopoeia.

In addition, an HPLC method is established in the application to determine the content of the main drug of the suspension.

HPLC chromatographic conditions

Estradiol benzoate high performance liquid phase conditions: elution was carried out using a Sinochrom OSD-BP (4.6 mm. Times.150mm, 5 μm) column, mobile phase A being methanol and mobile phase B being water, according to 85; the sample volume was 20. Mu.L, and the flow rate was 1.0 mL. Min ^-1 The detection wavelength is 230nm.

Oxytocin high performance liquid phase conditions: using a SinochromOSD-BP (4.6 mm. Times.150mm, 5 μm) chromatographic column, mobile phase A being acetonitrile, mobile phase B being water, mobile phase C being 15.6 g.L ^-1 Sodium dihydrogen phosphate solution, gradient elution: 0-40 min, 12-20% of A, 12-20% of B, 76-40% of C, sample size of 20 μ L, flow rate of 1.0 mL/min ^-1 The detection wavelength is 220nm.

Linear relation

Accurately weighing 100mg estradiol benzoate standard substance, dissolving with appropriate amount of methanol under ultrasonic condition, and making into 2mg/mL with methanol ^-1 The estradiol benzoate stock solution of (1). Then 2,4,6, 8 and 10mL of stock solutions are respectively put into a 25mL volumetric flask, and the volume is determined by methanol.

Precisely weighing oxytocin standard substance 10mg, dissolving with appropriate amount of ultrapure water under ultrasonic condition, and preparing into 0.4 mg/mL with ultrapure water ^-1 The oxytocin stock solution. Then 0.5, 1,2,3 and 4mL of the stock solutions were respectively put into a 10mL volumetric flask, and the volume was fixed with ultra pure water.

And (4) adopting the corresponding HPLC conditions for sample injection, and performing linear fitting on the absorption peak area and the corresponding concentration to obtain a standard curve regression equation.

According to the given method and conditions, the retention time of estradiol benzoate is 9.48min, and the standard curve equation is: y =68875.44x +184.17 ² =0.9998, and the linear relationship thereof is shown in fig. 3. From this, it was found that 0.16 to 0.80 mg/mL of estradiol benzoate is present at 230nm ^-1 In the concentration range of (a), the absorbance (y) has a good linear relationship with the concentration of estradiol benzoate.

The retention time of oxytocin is 18.35min, and the standard curve equation is as follows: y =19780.91x-3.28，R ² =0.9990, the linear relationship of which is shown in fig. 4. Therefore, oxytocin is 0.02-0.16 mg/mL at 220nm ^-1 Within the concentration range of (a), the absorbance (y) has a good linear relationship with the corresponding concentration of oxytocin.

Determination of precision

The preparation was 0.32,0.48, 0.64mg. Multidot.mL ^-1 0.04,0.08, 0.12mg.ml of estradiol benzoate methanol solution ^-1 Every mass concentration of the oxytocin aqueous solution is measured once every 3 hours on the same day, 4 times of measurement are carried out, and the precision in the day is calculated; the 3 mass concentrations of the sample were measured 1 time per day and 4 times per day, and the day precision was calculated. Precision is expressed as Relative Standard Deviation (RSD), estradiol benzoate and oxytocin should both be less than 5% precision in the day and less than 10% precision in the daytime.

As can be seen from Table 3, the diurnal precisions of estradiol benzoate and oxytocin were less than 10% and the intra-day precisions were less than 5%. The Relative Standard Deviations (RSD) of the mean intra-day precision and mean inter-day precision of estradiol benzoate were 0.3291% and 1.8503%, respectively; the Relative Standard Deviations (RSDs) of the mean inter-day and intra-day precision of oxytocin were 2.0575% and 2.0555%, respectively. The precision of the instrument is good, and the measuring method is stable and reliable.

TABLE 3 determination of the precision of estradiol benzoate and oxytocin

Determination of recovery

200mL of the prepared suspension is taken for freeze-drying, dissolved by using a proper amount of methanol through ultrasonic waves to be 100mL, and directly sampled for determining the concentration of the oxytocin in the sample. 20mL of oxytocin solution to be tested was taken, a part of methanol was removed by rotary evaporation at low temperature under reduced pressure, and 0.4 mg. Multidot.mL of oxytocin solution was added ^-1 Each of the oxytocin solutions is 1,2,3mL, the methanol constant volume is 20mL, namely the addition concentration of the oxytocin solution to be tested is 0.02,0.04, 0.06mg.mL ^-1 。

Freeze-drying 200mL of prepared suspension, and ultrasonically dissolving with appropriate amount of methanol to constant volumeWhen the concentration of the estradiol benzoate solution reaches 100mL, 8mL of the solution is taken and is made into a volume of 100mL by using methanol, and a sample is taken for measuring the concentration of the estradiol benzoate. 20mL of the test estradiol benzoate solution was taken, and a portion of methanol was removed by rotary evaporation at low temperature under reduced pressure, and 1.6 mg. Multidot.mL of the solution was added ^-1 2,4,6mL of each estradiol benzoate, and 20mL of methanol constant volume, namely the addition concentration of the estradiol benzoate solution to be tested is 0.16,0.32 and 0.48mg/mL ^-1 。

The test was repeated 5 times for each concentration, and the recovery was calculated by substituting the regression equation with the measured concentration and the added concentration.

TABLE 4 recovery determination of estradiol benzoate and oxytocin

As can be seen from table 4, the average recovery rate of estradiol benzoate was 97.48%, the average recovery rate of oxytocin was 95.62%, and the coefficients of variation were 0.8023 and 1.0417, respectively. The method is stable and accurate, and can be used for measuring the content of the estradiol benzoate and oxytocin long-acting suspension injection.

In the invention, HPLC method is adopted to respectively determine the correlation coefficient R of the standard curve of the contents of estradiol benzoate and oxytocin ² 0.9998 and 0.9990 respectively, good linear relation, and strong specificity of precision and recovery rate.

Determination of content of principal drug in sample

The sample is prepared into powder by adopting vacuum freeze drying, then the powder is dissolved in a small amount of methanol, and after the volume is fixed to a certain volume, the corresponding method is adopted for measurement.

The content of estradiol benzoate in the main drug of the sample is 1.9957 mg.mL ^-1 (ii) a The oxytocin content is 0.0184 mg/mL ^-1 (9.9352 units/mL).

According to the invention, a set of detection method suitable for the suspension is formulated through the quality control research; the research result can provide effective guidance for the amplification production of enterprises.

The invention carries out animal experiments on the suspension for animals, and specifically comprises the following steps:

1. establishment of rat uterine incomplete abortion hemorrhage model

Rats were assigned to male and female 2:1 proportion, and carrying out vaginal smear examination the next morning to find that the sperm is the 1 st d of pregnancy. At 7d, mifepristone (8, 8.3 mg/kg) and misoprostol (18, 00, 100 μ g/kg) were separately instilled with one metered dose of sterile cotton ball (85-90 mg) intravaginally, the following day at 8:00 and 18:00 taking out the cotton ball, placing into a plastic bag, sealing, refrigerating, preserving, replacing a new cotton ball in vagina, observing vaginal bleeding, continuously until d14, killing animals, and measuring bleeding amount of the collected cotton ball in vagina of each mouse ^[1-5] 。

2. Grouping and administration of drugs

Pregnant rats were randomly divided into 6 groups of 8 per group, i.e.:

blank group: injecting normal saline into muscle on 8 th and 11 th days of gestation;

negative control group: molding on day 7 of pregnancy (intragastric mifepristone (8, 00,8.3 mg/kg) and misoprostol (18, 00, 100 μ g/kg), respectively), and intramuscular injecting physiological saline on days 8 and 11 of pregnancy;

small dose suspension group: molding on 7 th day of pregnancy, and performing intramuscular injection of 2mg/mL estradiol benzoate and 10 units/mL oxytocin suspension at 0.09mL/Kg on 8 th and 11 th days of pregnancy;

bulk suspension group: molding on 7 th day of pregnancy, and performing intramuscular injection of 2mg/mL estradiol benzoate and 10 units/mL oxytocin suspension at 0.18mL/Kg on 8 th and 11 th days of pregnancy;

oxytocin group: the model is made on 7 th day of pregnancy, and oxytocin is injected intramuscularly on 8 th and 11 th days of pregnancy at a ratio of 0.9 unit/Kg ^[5] ；

Estradiol benzoate group: the model is built on the 7 th day of pregnancy, and 0.18mg/Kg of estradiol benzoate is injected intramuscularly on the 8 th and 11 th days of pregnancy. Sacrificed after 14d of gestation.

3. Blood volume determination

Uterine bleedingAnd (3) blood volume measurement: 0.02mL of blood is collected from the tail vein of the rat by a hemoglobin straw, and is added into 4mL of 50g/L NaOH solution to be uniformly mixed for later use. The collected cotton balls with uterine bleeding every day of each mouse are respectively placed in a beaker, a proper amount of 50g/L NaOH solution is added according to the condition of the amount of the bleeding to soak, squeeze and scrub the cotton balls to soak the blood, and the soaked solution is poured into another beaker for storage. Then adding a proper amount of 50g/L NaOH solution to soak the cotton balls, squeezing and scrubbing bloodstains, and pouring the bloodstains into a preservation container. And (4) determining whether to soak and wash for 1-2 times according to the blood stain elution condition of the cotton balls. Mixing all leaching solutions, recording total amount of 50g/L NaOH solution, filtering 5mL leaching solution, respectively filtering (1) 4mL filtered leaching solution, (2) 4mL NaOH solution of 50g/L rat tail venous blood, and (3) recording absorbance (A) value at 546nm wavelength with 50g/L NaOH solution as blank control ^[1-5] 。

Calculating the formula: uterine bleeding volume (mL) = tail vein blood volume (0.02 mL) × intrauterine leaching solution A value × V ₂ V (tail vein A blood value. Times.V) ₁ )；

V ₁ = amount of 50g/L Na0H solution used to dilute tail vein blood (4 mL);

V ₂ = amount of NaOH solution used for extraction of uterine blood 50g/L (mL).

Reference to the literature

[1] Wang Xiaodong. Establishment of model of uterine bleeding in early pregnancy induced by drugs [ J ]. Chinese pharmacological advisory, 1999, 15 (2): 182.

[2] jin Zhichun, study of the action and mechanism of action of YALIAN granule in preventing and treating vaginal bleeding after drug abortion [ D ] Huazhong university of science and technology, 2005.

[3] Liu Minghui influence and mechanism research of Haimaichong bleeding tablets on bleeding after drug abortion in early pregnant rats [ D ]. Hebei university of medical science, 2009.

[4] Kang Xuliang, zhou Aixiang, li Xiaoqin, et al, influence of YIMUANGONG Capsule on drug induced abortion in early pregnant rats [ J ] Chinese J.EXPERIMENT FORMULATION, 2006,12 (007): 48-49.

[5] Liu Gongmei, experimental study of biochemical hemostatic drink for preventing and treating bleeding after drug abortion in rats [ D ], heilongjiang university of traditional Chinese medicine, 2006.

4. Results of the experiment

The experimental results are shown in table 5 and fig. 5, and it can be seen from fig. 5 that the mixed use of the two medicines has a better effect on postpartum hemostasis. The intramuscular injection of the suspension of the invention can facilitate the administration.

TABLE 5 hemostasis of incomplete abortion in rats with suspensions

From the above examples, it can be seen that, in the examples of the present application, the ultrasonic cell disruption method is adopted to prepare the long-acting suspension of estradiol benzoate and oxytocin, and by optimizing the prescription, the prepared suspension has good sedimentation volume ratio and redispersion by using 0.20% by mass of CMC-Na as a suspending agent and 0.25% by mass of poloxamer 188 as a wetting agent. Experiments show that the mixture of the two medicines has better effect on postpartum hemostasis when being used as veterinary medicines; the intramuscular injection of the suspension can facilitate the administration and is beneficial to improving the curative effect, the compliance and the like. The preparation method is simple to operate, stable and reliable in quality, and can provide effective guidance for enterprise scale-up production.

Claims

1. A preparation method of estradiol benzoate and oxytocin long-acting suspension injection comprises the following steps:

poloxamer 188 is used as a wetting agent, sodium carboxymethylcellulose is used as a suspending agent, the mixture is mixed with estradiol benzoate in water, the mixture is homogenized by adopting an ultrasonic cell disruption mode, then the mixture is mixed with oxytocin solution, and the pH value is adjusted by acetic acid to prepare estradiol benzoate and oxytocin suspension;

the mass concentration of the wetting agent in the estradiol benzoate and oxytocin suspension is 0.1-0.9%, and the mass concentration of the suspending agent is 0.1-0.5%; the pH value of the estradiol benzoate and oxytocin suspension is 3.0-4.5.

2. The preparation method according to claim 1, wherein the mass concentration of the wetting agent in the estradiol benzoate and oxytocin suspension is 0.25-0.35%, and the mass concentration of the suspending agent is 0.15-0.25%.

3. The method according to claim 1, wherein the ultrasonic time for the ultrasonic cell disruption is 5 to 15min.

4. The method according to claim 3, wherein the sonication time for the sonication of the cell disruption solution is 7 to 13min.

5. The method according to any one of claims 1 to 4, wherein the estradiol benzoate and oxytocin suspension has a particle size of less than 8 μm.

6. The preparation method according to any one of claims 1 to 4, wherein the estradiol benzoate and oxytocin suspension comprises 100mg of estradiol benzoate, 500 units of oxytocin, 0.20% of sodium carboxymethylcellulose and 0.25% of poloxamer 188.