CN109106688B

CN109106688B - Preparation method of octreotide acetate microspheres

Info

Publication number: CN109106688B
Application number: CN201810958532.4A
Authority: CN
Inventors: 蔡晨辰; 徐朋; 蒋刚锋; 何超; 梁慧君; 蒋永康; 江晓漫
Original assignee: Livzon Pharmaceutical Group Inc
Current assignee: Livzon Pharmaceutical Group Inc
Priority date: 2018-08-22
Filing date: 2018-08-22
Publication date: 2021-08-17
Anticipated expiration: 2038-08-22
Also published as: CN109106688A

Abstract

A method of preparing an octreotide acetate microsphere formulation, comprising: (1) dissolving octreotide acetate in methanol to form a solution A; (2) dissolving PLGA in dichloromethane to form a solution B; (3) mixing the solution A and the solution B under stirring to form a uniform solution C; (4) adding silicone oil into the solution C at a constant speed under stirring to form a condensate; (5) adding the obtained aggregate into a curing liquid to form microspheres; (6) and (5) filtering the microspheres obtained in the step (5), cleaning, adding a proper amount of mannitol solution, vacuum drying and sieving. The method has simple operation, high quality of the prepared product, rapid and uniform distribution of the spheres and no aggregation when in use. The microspheres prepared by the method of the invention require fewer suspension steps, are simple to operate, have no special requirements on formulators, do not need special training, and can obviously improve the convenience of terminal hospitals and patients when using the medicine.

Description

Preparation method of octreotide acetate microspheres

Technical Field

The invention relates to the technical field of microsphere medicinal preparations, in particular to a method for preparing octreotide acetate microspheres by a phase separation method.

Background

Octreotide is an artificially synthesized octapeptide derivative of natural somatostatin, which was first synthesized by the chemist Wilfriend Bauer in 1979, retains the same pharmacological action as growth hormone, and can inhibit the secretion of many hormones including gastrin, cholecystokinin, glucagon, Growth Hormone (GH), insulin, pancreatic polypeptide, thyrotropin, vasoactive intestinal peptide, and the like. The traditional Chinese medicine composition is mainly used for clinically treating acromegaly and gastroenteropancreatic endocrine tumors (carcinoid, growth hormone releasing factor adenoma, pancreatic hyperglycosemia, gastrinoma/Zollinger-Ellison syndrome, insulinoma and vasoactive intestinal peptide tumor), and is widely applied clinically.

There are two types of octreotide acetate preparations approved by FDA to be marketed, one is injection

Subcutaneous injections, 3 times a day; the other is a reservoir type injection microsphere

Depot, intramuscular injection, once a month.

The clinical results show that the compound can effectively control the level of growth hormone and insulin-like growth factor 1 for a long time and can effectively reduce tumors. However, the marketed octreotide acetate microspheres need to use a special glucose-PLGA star polymer (GLU-PLGA) as a matrix.

At present, many scholars research on the preparation of octreotide acetate microspheres by using a multiple emulsion-solvent evaporation method. There are now generally two ways of preparing multiple emulsions:

(1) one-time preparation: the volume ratio of the primary emulsion to the external water phase is generally 1:10-1:40, and the solvent is directly volatilized in a container after the multiple emulsion is prepared. For example, as disclosed in patent CN1927906A entitled "biodegradable star-structure poly (lactide-co-glycolide) drug-carrying microsphere and preparation method thereof", colostrum is injected into an external water phase under stirring, the volume ratio of the colostrum to the external water phase is 4-10:100, W/O/W emulsion is formed by emulsification, and stirring is carried out until dichloromethane is completely volatilized, so that the solidification is carried out to form a sphere ". Also disclosed in patent application CN1330921A entitled "method for producing sustained-release microspheres by multiple emulsion method" is a "stage of preparing microparticles encapsulating a drug such as a drug by dispersing the produced 1-time emulsion phase or emulsion in an aqueous phase and removing an organic solvent from the microspheres in which the drug is dispersed". An "oil-in-water-in-oil-in-water emulsion" as disclosed in US patent 7846479, also entitled "microsphere composition and method of preparation", is prepared by the following steps: water-in-oil emulsion is prepared by mixing an aqueous phase with an organic solvent containing a polymer, and water-in-oil-in-water emulsion is prepared by mixing the prepared emulsion with an aqueous phase containing a surfactant.

(2) The preparation of the multiple emulsion and the volatilization of the solvent are respectively carried out: the colostrum is mixed with a continuous medium to prepare a multiple emulsion, and the multiple emulsion is added into an effective amount of an extraction medium to extract an organic solvent to form microspheres. Mixing said dispersion (i.e. colostrum) with an effective amount of a continuous working medium to form an emulsion containing said working medium and microdroplets comprising said octreotide acetate, said solvent and said linear poly (lactide-co-glycolide), as disclosed in the invention patent CN17110073A entitled "pharmaceutical composition comprising octreotide microparticles"; immediately after forming said emulsion, adding all of said emulsion to an effective amount of an extraction medium to extract said solvent from said microdroplets to form said microparticles. The extraction medium is preferably 10 times or more in volume ".

However, the above method has many disadvantages. Specifically, when the production of microspheres is carried out, when the first multiple-emulsion production method is employed, a multiple-emulsion tank of large capacity must be provided in order to satisfy the production amount of microspheres and the volume requirement of the external aqueous phase. The requirements of the double emulsion tank for microsphere production are relatively high, and emulsification stirring, namely, rapid stirring (rotating speed of 500-2000 rpm for preparing double emulsion) and slow stirring (rotating speed of 200-500 rpm for solvent volatilization process) must be provided usually. For a large-capacity compound emulsion tank, it is difficult to equip a high-efficiency, stable and high-quality rapid stirring device. And the external water phase has too large volume, and the balling property of the microspheres is difficult to control properly. When the second multiple emulsion production method is adopted, the capacity of a multiple emulsion tank can be properly reduced, but the emulsification effect is often poor in the production of large-batch microspheres, and the microspheres are easy to adhere when the production amount is large, so that the quality of the final microspheres is affected. And the capacity of the re-emulsifying tank limits the re-emulsifying, the maximum production is relatively fixed, and the production cannot be further increased.

Patent US5538739 discloses a method for preparing octreotide microspheres, which is simple in process and suitable for industrial production. However, the product obtained by the method has uneven microsphere distribution, and is very easy to aggregate even if the product is prepared at present, thereby seriously hindering the clinical use of the medicine.

Therefore, it is necessary to research and develop a simple preparation process meeting the production requirements, and the prepared microspheres are uniformly distributed and easy to prepare and use, thereby bringing convenience to clinical use.

Disclosure of Invention

The invention overcomes the defects in the prior art, and provides a brand new method for preparing octreotide acetate microspheres by using a phase separation technology.

In one aspect, the invention provides a method for preparing octreotide acetate microspheres, which comprises the following steps:

(1) dissolving octreotide acetate in methanol to form a solution A;

(2) dissolving PLGA in dichloromethane to form a solution B;

(3) mixing the solution A and the solution B under stirring to form a uniform solution C;

(4) adding silicone oil into the solution C at a constant speed under stirring to form a condensate;

(5) adding the obtained aggregate into a curing liquid to form microspheres;

(6) and (5) filtering the microspheres formed in the step (5), cleaning, adding a proper amount of mannitol solution, drying in vacuum, and sieving.

Preferably, the mass ratio of octreotide acetate to methanol in the step (1) is 1:5-1: 36; more preferably, the ratio is 1: 20. Researches show that the use amount of the methanol has certain influence on experimental results, and the strict control needs to be carried out: when the amount of the methanol is too small, the solution C is easily milky and turbid, and the medicine is crystallized.

Preferably, the mass ratio of PLGA to dichloromethane in step (2) is 1:25-1: 50; more preferably, the ratio is from 1:34.5 to 1: 50; most preferably, the ratio is from 1:34.5 to 1: 40. Researches show that the using amount of dichloromethane has certain influence on experimental results, and needs to be strictly controlled: when the PLGA concentration is increased, namely the addition amount of dichloromethane is reduced, the in vitro release is slowed down, and when the PLGA concentration is increased to a certain concentration, the balling property is obviously poor, and a large amount of deformed spheres can be formed in the preparation process; when the concentration of PLGA is reduced, namely the addition amount of dichloromethane is large, the particle size and the release are not obviously influenced, but the aggregation of spheres in the curing process is easily caused, and the difficulty in removing the organic solvent of the final product is increased.

Preferably, the mass ratio of methanol in step (1) to dichloromethane in step (2) is from 1:11 to 1:86, more preferably, the ratio is from 1:11 to 1: 45; further preferably, the ratio is from 1:11 to 1: 36; most preferably, the ratio is 1: 26.7. Researches show that the use amount of the methanol has certain influence on experimental results, and the strict control needs to be carried out: when the using amount of the methanol is reduced, the particle size of the microsphere is in a growth trend, and the in vitro release is reduced; when the amount of methanol is increased, the particle size of the microspheres tends to decrease, but when the ratio is reduced to a certain degree, the particle size does not change significantly any more, and the burst release is increased significantly.

Preferably, the viscosity of the silicone oil in step (4) is 350-600cs (centistokes), more preferably 350 cs. Researches show that the viscosity of the silicone oil has certain influence on experimental results, and the strict control needs to be carried out: the increase in viscosity of the silicone oil results in a decrease in the average particle size of the microspheres; meanwhile, the higher viscosity of the silicone oil increases the requirement on the input power of mechanical stirring in the condensation stage, and increases the difficulty of sterilization and filtration.

Preferably, the mass/volume ratio of dichloromethane in step (2) to silicone oil in step (4) is from 1g:1ml to 1g:2ml, more preferably from 1g:1ml to 1g:1.6ml, most preferably 1g:1.6 ml. Researches show that the ratio of dichloromethane to silicone oil has certain influence on experimental results, and needs to be strictly controlled: when the ratio of dichloromethane to silicone oil is large, the balling property is remarkably reduced, and when the ratio is reduced to about 1g:1.6ml, the balling property and the particle size tend to be stable. Too large or too small a ratio of dichloromethane to silicone oil leads to a slow in vitro release.

Preferably, the addition time of the silicone oil in the step (4) is 10min to 40min, more preferably 10min to 20 min. The silicone oil is added for too short time, namely the silicone oil is added too fast, so that the particle size uniformity is reduced partially; however, when the silicone oil is added for a long time, emulsion droplets aggregate to form large particles, the particle size uniformity of the microspheres is reduced, and the surface pores become large.

Preferably, in step (4), the system temperature is controlled to be 8 ℃ to 10 ℃. The condensation temperature affects the release behavior, with slower release at higher temperatures and a pronounced burst effect at lower temperatures.

Preferably, the curing liquid in step (5) may be: a mixture of n-heptane, water and Span80, or a mixture of n-heptane, water, Span80 and silicone oil; more preferably, the volume ratio of n-heptane to water is from 70:30 to 95: 5; further preferably, the ratio of the total volume of n-heptane and water to the volume of Span80 is from 120:1 to 360:1, preferably 180: 1; further preferably, the volume ratio of n-heptane to silicone oil added in step (4) is from 3.5:1 to 7: 1. Different curing fluid compositions have a certain influence on the release behaviour and at the same time on the temperature during curing. When the volume ratio of the n-heptane to the water is too low, the solution is viscous in the curing process, and spheres are difficult to disperse; when the volume ratio of n-heptane to water is too high, a burst phenomenon occurs.

Preferably, in step (5), the temperature of the system is controlled to be 0 ℃ to 20 ℃, more preferably 2 ℃ to 10 ℃. When the temperature is higher than 20 ℃, aggregation and agglomeration among spheres can occur, which is not beneficial to collection and washing.

Preferably, the vacuum drying in the step (6) adopts a temperature programmed vacuum drying method, wherein the temperature of the first stage vacuum drying is 15-25 ℃, and preferably 20 ℃; the second stage vacuum drying temperature is 35-50 deg.C, preferably 40-45 deg.C; the temperature of vacuum drying in the third stage is 45-50 deg.C, preferably 50 deg.C. By setting the vacuum drying program (time, temperature), the methylene chloride level can be effectively reduced to below 0.1%.

In another aspect, the present invention provides octreotide acetate microspheres prepared according to the above method.

In a further aspect, the present invention provides the use of octreotide acetate microspheres prepared according to the above method for the preparation of a medicament for the treatment of acromegaly and gastroenteropancreatic endocrine tumors such as carcinoid, growth hormone releasing factor adenoma, pancreatic hyperglycosemia, gastrinoma/zollinger-ellison syndrome, insulinoma, vasoactive intestinal peptidoma.

The invention has the following advantages and positive effects:

1. the method has simple steps and is suitable for production and use.

2. The microsphere prepared by the method has high quality, can be prepared on site, and has the advantages of rapid and uniform distribution of spheres and no aggregation.

3. When the microspheres prepared by the method are used, fewer suspension steps are needed, and the operation is simple.

4. When the microspheres prepared by the method are prepared, no special requirements are required for preparation personnel, no special training is required, and the convenience of terminal hospitals and patients in using the medicine can be obviously improved.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1 shows an SEM (scanning Electron microscope) image of the microspheres obtained in example 1;

FIG. 2 shows an SEM image of the microspheres obtained in example 1 at a higher magnification;

FIG. 3 shows an SEM image of the microspheres obtained in example 2;

FIG. 4 shows an SEM image of the microspheres obtained in example 2 at a higher magnification;

FIG. 5 shows an SEM image of microspheres obtained in example 3;

FIG. 6 shows an SEM image of the microspheres obtained in example 3 at a higher magnification;

FIG. 7 shows a schematic representation of the suspension results of example 5;

FIG. 8 is a graph showing the results of experimental group A of example 7;

FIG. 9 is a graph showing the results of experimental group B of example 7;

FIG. 10 is a graph showing the results of experimental group C of example 7;

FIG. 11 is a graph showing the results of experimental group D of example 7;

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

Example 1

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving 10g of PLGA in 400g of dichloromethane; stirring and mixing the two solutions, and forming a uniform solution at a rated rotation speed of 120 rpm; adding 640ml silicone oil (viscosity 350cs) at constant speed within 13min, stirring for 5min, and controlling temperature at 8-10 deg.C to form condensate; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying, and sieving to obtain the final product. SEM images are shown in figures 1 and 2.

The accelerated release results for the resulting product were as follows:

TABLE 1-1

Note: the results are cumulative accelerated Release degrees of accelerated Release 1h, 4h and 24h

The particle size distribution of the resulting product was as follows:

tables 1 to 2

Batch number	D₁₀	D₅₀	D₉₀	Span
					W03-171107-3-1-1	35.79	50.87	72.39	0.719

Note: d₁₀Is a physical parameter of the particle size distribution, which means that 10% of the particles are smaller than the particle size, and so on;

span is used to characterize the uniformity of particle size distribution Span ═ D₉₀-D₁₀/D₅₀。

Example 2

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving 10g of PLGA in 345g of dichloromethane; stirring and mixing the two solutions, and forming a uniform solution at a rated rotation speed of 120 rpm; adding 640ml silicone oil (viscosity 350cs) at constant speed within 13min, stirring for 5min, and controlling temperature at 8-10 deg.C to form condensate; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying for 20 hr, and sieving to obtain the final product. SEM images are shown in figures 3 and 4.

The accelerated release results for the resulting product were as follows:

TABLE 2-1

	Drug loading	1h	4h	24h
					W03-171031-1-1-1	4.60％	2.06％	32.27％	56.65％

The particle size distribution of the resulting product was as follows:

tables 2 to 2

Batch number	D₁₀	D₅₀	D₉₀	Span
					W03-171031-1-1-1	36.225	49.63	67.89	0.638

Example 3

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving 10g of PLGA in 400g of dichloromethane; stirring and mixing the two solutions, and forming a uniform solution at a rated rotation speed of 120 rpm; adding 640ml silicone oil (viscosity 600cs) at constant speed within 13min, stirring for 5min, and controlling temperature at 8-10 deg.C to form condensate; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying for 20 hr, and sieving to obtain the final product. SEM images are shown in figures 5 and 6.

The accelerated release results for the resulting product were as follows:

TABLE 3-1

	Drug loading	1	4	24
					W03-171113-2-0-1	4.72％	2.51％	28.55％	54.97％

The particle size distribution of the resulting product was as follows:

TABLE 3-2

Batch number	D₁₀	D₅₀	D₉₀	Span
					W03-171113-2-0-1	36.9	51.55	71.49	0.67

Examples4Effect of vacuum drying on solvent residue

Preparing a microsphere intermediate by adopting the preparation process of the embodiment 1, and performing vacuum drying under different conditions to improve the solvent residue;

the first condition is as follows: 24h at 20 ℃, 24h → 40 ℃ 24h → 45 ℃ 24 h;

and a second condition: 24h at 20 ℃ → 24h at 40 ℃;

and (3) carrying out a third condition: 24h at 20 ℃, 24h → 45 ℃ 24h → 45 ℃ 24 h;

and a fourth condition: 24h at 20 ℃ → 24h at 40 ℃ → 24h at 50 ℃;

the experimental results are as follows:

table 4 results for material and solvent residues:

batch number	DCM residual solvent	Residue of n-heptane
			W03-170817-1 (initial level)	2.28％	2.15％
Condition one	0.30％	2.37％
			Condition two	0.53％	2.44％
Condition three	0.19％	2.14％
			Condition four	0.08％	1.83％

Depending on the impurities and solvent residue limits requirements: DCM residue should not be higher than 0.5%, and n-heptane residue should not exceed 2.5%. In view of the classification of ICH solvents, DCM belongs to the second class of solvents and has carcinogenicity, the maximum daily intake is 60ppm, and the concentration of DCM is not more than 0.18% according to the sustained-release administration time of 30 days; and n-heptane belongs to three types of solvents, namely low-toxicity solvents, and the daily limit is 5000 ppm.

As can be seen from table 4, the requirements of octreotide impurities and solvent residue limit can be met under the conditions of one, three and four; but the conditions three and four are safer for human body; under the fourth condition, the DCM solvent residue and the n-heptane solvent residue are the lowest, and the safety is the best.

Example 5Comparative experiment with the formulation prepared in patent US 5538739:

(1) experimental products:

the preparation of patent US5538739 was set a in experimental group a, and the microsphere product was prepared by the above preparation process of example 1, and set B in experimental group B.

(2) The experimental scheme is as follows:

the product of experiment A, B group was taken out from the refrigerated environment at 4 deg.C, left at room temperature for 5min, added with 2.5ml of the same suspending agent (the suspending agent was a solution of sodium carboxymethylcellulose, mannitol and deionized water, wherein the concentration of mannitol was 6mg/ml and the concentration of sodium carboxymethylcellulose was 5mg/ml), shaken gently (vigorous shaking was not allowed), and the suspension results were observed.

(3) The experimental results are as follows: experiment A group has aggregation, suspension failure and no administration; experiment group B was rapidly and evenly distributed and suspended successfully.

The results are shown in FIG. 7.

Example 6PLGA/DCM dosageInfluence of the comparison on the results

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving a certain amount of PLGA in a certain amount of dichloromethane; stirring and mixing the two solutions, wherein the rated rotation speed is 120rpm, and the temperature is controlled to be 10 ℃ to form a uniform solution; adding 640ml silicone oil (viscosity 350cs) at constant speed within 13min, and stirring for 5min to form condensate; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying for 20 hr, and sieving to obtain the final product. Different PLGA/dichloromethane dosage ratios were investigated, experimental phenomena were observed and in vitro release was studied, the results are shown in the table below.

TABLE 5-1 influence of PLGA and Dichloromethane dosage ratio on the results

In vitro release result comparison:

TABLE 5-2

As can be seen from tables 5-1 and 5-2, when the dosage ratio of PLGA/DCM is below 1:34.5, the release shows a slow trend, and the release behavior is better between 1:34.5 and 1: 50; however, as the amount of DCM increases, the likelihood of sphere agglomeration during curing increases, resulting in cure failure. In general, the ratio of PLGA to DCM is in the range of 1:25 to 1:50, preferably in the range of 1:34.5 to 1: 50; most preferably in the range of 1:34.5 to 1: 40.

Example 7Influence of the ratio of the amount of dichloromethane to the amount of silicone oil on the experimental results

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving 10g of PLGA in 400g of dichloromethane; stirring and mixing the two solutions, wherein the rated rotation speed is 120rpm, and the temperature is controlled to be 10 ℃ to form a uniform solution; adding a certain amount of silicone oil (viscosity 350cs) at constant speed within 13min, and stirring for 5min to form condensate; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying for 20 hr, and sieving to obtain the final product. Different dichloromethane/silicone oil dosage ratios were investigated, experimental phenomena were observed and in vitro release was studied, the results are shown in the table below.

TABLE 6-1 influence of the ratio of the amounts of methylene chloride and silicone oil on the results of the experiment

TABLE 6-2

And (4) conclusion: from the data in the table above and the scanning electron micrographs (FIGS. 8-11), it can be seen that the release is slowed when the silicone oil/dichloromethane ratio is lower than 1: 1.6; when the ratio is less than 1:1, the balling property is obviously reduced, and the drug loading capacity is reduced; when the ratio of silicone oil to dichloromethane is increased, the release behavior has no significant influence, but spheres are easy to gather in the curing process, and the silicone oil on the spheres is easy to clean in the later period. Comprehensively considering, when the mass ratio of the dichloromethane to the silicone oil is 1:1-1:2, the experiment can be smoothly finished; when the ratio is 1:1-1:1.6, the experiment is better; the experimental results were best when the ratio was 1:1.6 (group B).

Example 8Effect of selecting different silicone oil viscosities on the results of the experiment

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving 10g of PLGA in 400g of dichloromethane; stirring and mixing the two solutions, wherein the rated rotation speed is 120rpm, and the temperature is controlled to be 10 ℃ to form a uniform solution; adding 640ml silicone oil (with different viscosities) at constant speed within 13min, and stirring for 5min to obtain condensate; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying for hr, and sieving to obtain the final product. The influence of different silicone oil viscosities on the preparation is investigated, experimental phenomena are observed, and the results are shown in the following table.

TABLE 7 influence of different silicone oil viscosities on the experimental results

Experimental group	Different viscosity of silicone oil	Description of experimental results or phenomena
			A	250cs	Can be filtered and sterilized at normal temperature, but the spheres are easy to aggregate during solidification
B	350cs	Normal temperature filtering and sterilizing
			C	600cs	The filtration and sterilization at normal temperature are difficult
D	1000cs	Difficult to filter and sterilize at normal temperature

And (4) conclusion: for aseptic injection, the difficulty of sterilization and filtration needs to be considered during production. Therefore, the experimental results in the table show that the silicone oil with 350cs-600cs viscosity is selected, which is beneficial to the subsequent filtering sterilization process; the experiment effect is best when the viscosity is 350 cs.

Example 9Influence of the differences in the Silicone oil addition times on the test results

Microspheres were prepared according to example 1 except that the time of addition of the silicone oil was varied. The addition times of different silicone oils were investigated and the particle size distribution was investigated, the results are shown in the table below.

TABLE 8 influence of the Silicone oil addition time on the test results

Experimental group	Time of addition of silicone oil	Description of experimental results or phenomena
			A	30s	Does not form balls
B	2.5min	D₅₀About 55um, the particle size distribution span is increased
			C	10min	D₅₀About 50um, particle sizeIs distributed more uniformly
D	13min	D₅₀About 50um, uniform particle size distribution
			E	20min	D₅₀About 50um, uniform particle size distribution
F	40min	D₅₀About 50um, uniform particle size distribution
			G	100min	D₅₀About 80um, the particle size distribution span is increased

And (4) conclusion: when the adding speed of the silicone oil is too high (30s), the DCM extraction speed is too high, so that emulsion droplets are not formed yet, PLGA is separated out, and the preparation is not spherical; when the adding time of the silicone oil is reduced to 2.5min, the average particle size has no significant change, and the particle size span is increased; when the adding time of the silicone oil is prolonged to 100min, the average particle size is remarkably increased, and the particle size span is remarkably reduced; when the silicone oil is added for 10-40min, the balling property is better, the formed preparation particles are uniform, and the particle size span value is smaller; considering the preparation process time, the optimal selection is 10min-20 min.

Example 10Effect of temperature in the condensation stage on the results of the experiment

Weighing 0.75g of octreotide acetate and dissolving in 15g of methanol; dissolving 10g of PLGA in 400g of dichloromethane; stirring and mixing the two solutions, wherein the rated rotation speed is 120rpm, and the temperature is controlled to be 10 ℃ to form a uniform solution; 640ml of silicone oil (350cs) is added at a constant speed within 13min, the mixture is continuously stirred for 5min, and the temperature is controlled to be certain, so that condensate is formed; adding curing liquid composed of n-heptane (3200ml), water (400ml), Span80(20ml) and silicone oil (100ml), curing for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying, and sieving to obtain the final product.

TABLE 9 Effect of temperature during the agglomeration phase on the results

Experimental group	Temperature range (. degree.C.)	Description of experimental results or phenomena
			A	3-4	The release is accelerated, and the burst release is obviously increased
B	8-10	Qualified release rate
			C	14-15	Slow release
D	25	Release is significantly slowed down

And (4) conclusion: as can be seen from the results in Table 9, the experimental agglomerates should be released acceptably at 8-10 ℃. The release of the product is affected when the temperature is higher or lower than 8-10 ℃.

Example 11Influence of different kinds of curing liquids on experimental results

Using the procedure of example 1, agglomerates were formed; adding into different solidifying liquid, solidifying at 10 deg.C for 2 h; solidifying to form microsphere, filtering, collecting, washing with n-heptane for 3-4 times, adding appropriate amount of mannitol solution, vacuum drying for 20 hr, and sieving to obtain the final product.

TABLE 10 Effect of different curing solutions on the results of the experiment

And (4) conclusion: the proportion of the selected n-heptane to the water is 70:30-95:5, the balling property is good, and no burst release behavior exists.

Example 12Influence of the proportion of n-heptane to silicone oil in the coagulation stage on the experimental results

According to example 1, different amounts of silicone oil and n-heptane were used in the coacervation stage and the curing stage, respectively, and the effect of different ratios of n-heptane to coacervate stage silicone oil on the formulation was examined.

TABLE 11 influence of different n-heptane to coacervation stage silicone oil ratios on the experimental results

And (4) conclusion: it was finally determined that significant agglomeration of spheres would occur when the n-heptane to coacervate stage silicone oil ratio was less than 3: 1.

Example 13Effect of curing temperature on test results

The effect of different curing temperatures on the formulations was examined according to example 1.

TABLE 12 Effect of different curing temperatures on the results of the experiment

And (4) conclusion: the curing temperature has no obvious influence on the release, but at the higher temperature of 20 ℃, the aggregation and caking among spheres can occur, and the difficulty of the next step of collection and washing is increased.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. A method for preparing octreotide acetate microspheres comprises the following steps:

(1) dissolving octreotide acetate in methanol to form a solution A, wherein the mass ratio of octreotide acetate to methanol is 1:5-1: 36;

(2) dissolving PLGA in dichloromethane to form a solution B, wherein the mass ratio of PLGA to dichloromethane is 1:34.5-1: 50;

(4) adding silicone oil into the solution C at a constant speed under stirring to form a condensate, wherein the viscosity of the silicone oil is 350-600cs, the adding time of the silicone oil is 10-20min, and the system temperature is controlled at 8-10 ℃; wherein the mass/volume ratio of the dichloromethane in the step (2) to the silicone oil in the step (4) is 1g:1ml-1g:2 ml;

(5) adding the obtained aggregate into a curing liquid to form microspheres, wherein the temperature of the system is controlled to be 0-20 ℃, and the curing liquid is a mixture of n-heptane, water and Span80, or a mixture of n-heptane, water, Span80 and silicone oil; wherein the volume ratio of the n-heptane to the water is 70:30-95: 5; wherein the volume ratio of the n-heptane to the silicone oil added in the step (4) is 3.5:1-7: 1;

(6) and (5) filtering the microspheres obtained in the step (5), cleaning, adding a proper amount of mannitol solution, vacuum drying and sieving.

2. The method according to claim 1, wherein the mass ratio of octreotide acetate to methanol in step (1) is 1: 20.

3. The method according to claim 1, wherein the mass ratio of PLGA to methylene chloride in step (2) is 1:34.5 to 1: 40.

4. The method according to claim 1, characterized in that the viscosity of the silicone oil in step (4) is 350 cs.

5. The process according to claim 1, characterized in that the mass ratio of methanol in step (1) to dichloromethane in step (2) is from 1:11 to 1: 86.

6. The process according to claim 5, wherein the mass ratio of methanol in step (1) to dichloromethane in step (2) is from 1:11 to 1: 45.

7. The process according to claim 6, wherein the mass ratio of methanol in step (1) to dichloromethane in step (2) is from 1:11 to 1: 36.

8. The process according to claim 7, characterized in that the mass ratio of methanol in step (1) to dichloromethane in step (2) is 1: 26.7.

9. The process according to claim 1, characterized in that the mass/volume ratio of dichloromethane in step (2) to silicone oil in step (4) is from 1g:1ml to 1g:1.6 ml.

10. The process according to claim 9, characterized in that the mass/volume ratio of dichloromethane in step (2) to silicone oil in step (4) is 1g:1.6 ml.

11. The process of claim 1, wherein the ratio of the total volume of n-heptane and water to the volume of Span80 is from 120:1 to 360: 1.

12. The process of claim 11, wherein the ratio of the total volume of n-heptane and water to the volume of Span80 is 180: 1.

13. The method according to any one of claims 1 to 12, wherein in the step (5), the temperature of the system is controlled to be 2 ℃ to 10 ℃.

14. The method according to any one of claims 1 to 12, wherein the vacuum drying in step (6) is performed by a temperature programmed vacuum drying method, wherein the first stage vacuum drying temperature is 15 to 25 ℃; the second stage vacuum drying temperature is 35-50 ℃; the third stage vacuum drying temperature is 45-50 deg.C.

15. The method of claim 14, wherein the first stage vacuum drying temperature is 20 ℃.

16. The method of claim 14, wherein the second stage vacuum drying temperature is 40-45 ℃.

17. The method of claim 14, wherein the third stage vacuum drying temperature is 50 ℃.

18. Octreotide acetate microspheres prepared according to the method of any one of claims 1 to 17.

19. Use of octreotide acetate microspheres according to claim 18 in the manufacture of a medicament for the treatment of acromegaly and gastro-pancreatic endocrine tumors.

20. The use according to claim 19, characterized in that the gastroenteropancreatic endocrine tumor is carcinoid, growth hormone releasing factor adenoma, pancreatic hyperglycosemia, gastrinoma, insulinoma or vipoma.