WO2012034360A1

WO2012034360A1 - Insulin crystal microsphere, suspension and preparation method thereof

Info

Publication number: WO2012034360A1
Application number: PCT/CN2011/001560
Authority: WO
Inventors: 梁伟; 任武贤; 魏秀莉; 禹玉洪; 于继兵; 许振江
Original assignee: 中国科学院生物物理研究所; 北京亚宝生物药业有限公司
Priority date: 2010-09-15
Filing date: 2011-09-14
Publication date: 2012-03-22
Also published as: CN102397535B; CN102397535A

Abstract

An insulin crystal microsphere, suspension and preparation method thereof are disclosed. The insulin crystal microsphere is prepared by method of balling and crystallizing in liquid, which utilizes the isoelectric point of insulin. The insulin crystal microsphere has an average diameter of 1-10μm. The insulin crystal microsphere suspension is formed by suspending the freeze-dried insulin crystal microsphere in protective agents such as PEG and phospholipids etc.

Description

Insulin crystalline microspheres, suspensions thereof, and preparation methods

The invention belongs to the field of medicine and relates to an insulin crystalline microsphere suspension and a preparation method thereof. More specifically, it relates to an insulin crystalline microsphere suspension prepared by a liquid-phase crystallization technique and a preparation method thereof. Background technique

At present, the incidence of diabetes is increasing year by year, becoming the third most deadly disease after cardiovascular disease and cancer. Since its first use in the treatment of diabetes in 1922, insulin has been the drug of choice for insulin-dependent diabetes patients to date due to its unique hypoglycemic effect. As a protein and peptide drug, insulin is easily degraded by proteolytic enzymes of the gastrointestinal tract when it is taken orally, and its molecular weight is not easy to pass through biofilms. The bioavailability during oral administration is very low, and it cannot exert its proper hypoglycemic effect. Currently, all of the other insulin preparations are administered by injection, except that Generex's buccal film product, Omlin-LynTM, has been marketed in India and Ecuador. This not only imposes a large economic burden on patients, but also affects the quality of life of patients and their families. At the same time, frequent long-term injections can also cause many adverse reactions such as: insulin edema, hypoglycemia, hypertrophic fat malnutrition and subcutaneous fat. Shrinking and so on. - Current non-injectable administrations of insulin include transdermal administration, pulmonary inhalation administration, nasal administration, oral and buccal mucosal administration. Pulmonary inhaled insulin suffers from long-term application safety and long-term efficacy. The Exubera® automatic withdrawal market has, to a certain extent, cast a shadow on the development of inhaled insulin. The oral administration route of insulin can simulate physiological insulin secretion, and the ratio of portal insulin concentration to peripheral circulating insulin concentration in the physiological state is approximately 1:5. Therefore, the oral delivery system is the most ideal non-injection route for insulin, avoiding The pain and inconvenience of the injection, the patient's compliance is strong. The key problem to be solved by the insulin oral delivery system is how to prevent the insulin from being damaged by the enzyme in the gastrointestinal tract. The currently reported preparation methods for insulin oral delivery systems are as follows: (1) addition of absorption enhancers: absorption enhancers include protease inhibitors and penetration enhancers, commonly used absorption enhancers include cholates, surfactants, Bacitracin and amino acid derivatives, and the like. However, long-term use of absorption enhancers can damage the gastrointestinal epithelium, affect the absorption of nutrients, and can also lead to the entry of bacterial toxins, which may cause systemic infections and toxic reactions; (2) Protection of insulin stability in the gastrointestinal tract by formulation techniques and promotion of its absorption: These formulation techniques include encapsulation of insulin in liposomes, solid microspheres/nanoparticles, micelles, hydrogels, Emulsions, etc. However, the above preparation techniques all have certain limitations: poor stability of liposomes; organic solvents, high temperature/high pressure are often used in the preparation of nanoparticles/microspheres, and these undesirable conditions may cause insulin loss activity, and some polymer materials are in vivo. Can cause inflammatory reactions, etc.; (3) chemical modification of insulin or synthesis of prodrugs: chemical modification of insulin with fatty acids can enhance its lipophilicity and increase its absorption in the intestine, but change the structure of insulin or before synthesis The body drug is very complex, and if the structural modification changes its binding to the receptor binding site, it may affect its biological activity.

In summary, although there are many research reports and patents on insulin oral delivery systems, the final clinical application is minimal. The main problem is the high dose of oral administration, low bioavailability, and difficulty in clinical application. . In the related research of insulin oral delivery system, the main focus is on how to overcome the enzyme barrier and intestinal epithelial barrier in the gastrointestinal tract, increase the permeability of the intestinal epithelium and open tight junctions, while ignoring the mucus layer on the surface of the gastrointestinal tract. The effects of drug absorption. The human intestinal mucus layer consists of mucin, water, electrolytes, exfoliated epithelial cells, lipids, salts, enzymes, microorganisms and their products, up to several hundred microns thick. Phospholipids make the mucus layer hydrophobic, and the mucin and water content determine the viscoelastic properties of the mucus layer. A small change in water content can make a very significant change in the rheological properties of the mucus. The mucus layer contains a large amount of esterase and protein digestive enzyme, which is the main site of insulin degradation by enzymes.

Many studies have shown that in addition to protecting the basement membrane from risk factors in the intestinal contents, the mucus layer is also a potential barrier to drug absorption. For oral delivery systems, regardless of the physicochemical properties of the drug, the drug must first pass through the mucus layer before it is absorbed through the epithelium. In addition to neutral small molecule drugs, the thickness of the mucus layer can significantly affect the rate at which macromolecules and charged molecular drugs enter the basal tissue. For example, the mucus layer can significantly delay the diffusion of hydrogen ions compared to water of equivalent thickness. Studies have shown that when the particles are larger than 60 nm, the mucus barrier cannot be worn. However, it has been found that PEG can prevent large particles (particle size > 500 nm) from adhering to the surface of the mucus layer, and the particles can quickly cross the mucus layer, while the PEG unmodified particles adsorb. On the surface of the mucus layer and gathered together, can not enter the mucus layer. Summary of the invention The object of the present invention is to prepare a novel insulin oral delivery system, namely an insulin drug crystalline microsphere suspension, under the dual conditions of ensuring the effectiveness and safety of the insulin oral delivery system.

The invention provides an insulin drug crystalline microsphere suspension and a preparation method thereof, wherein the insulin drug crystal microspheres of a certain density and size are prepared by a liquid-phase crystallization technique. After lyophilizing the insulin crystalline microspheres to obtain insulin crystalline microspheres lyophilized powder, the suspension is suspended in a protective agent such as PEG or phospholipid to obtain an insulin microsphere suspension. The preparation of the microspheres does not rely on any polymer and organic solvent, thus ensuring the safety of the delivery system. PEG' and pity are beneficial for the drug microspheres to rapidly pass through the intestinal mucus layer, avoid degradation by proteases, increase contact with intestinal lymphoid tissue, and promote drug absorption.

The rate at which the insulin crystalline microsphere suspension prepared by the present invention rapidly passes through the intestinal mucus layer can be achieved by the chain length of the PEG, the type of phospholipid, and the ratio between the PEG and the phospholipid.

The invention provides a preparation method of insulin crystalline microspheres, which comprises the following steps: (1) adding insulin to a salt solution, adding glacial acetic acid dropwise and stirring until the insulin is completely dissolved, adding an appropriate amount of polyethylene glycol, stirring Dissolved; the weight ratio of insulin to polyethylene glycol is 1:20-200;

(2) adjusting the pH of the solution in step (1) to 3.5-4.5 to precipitate the precipitate; (3) heating and stirring the solution in step (2) until the precipitate is completely dissolved; (4) placing the solution at room temperature and adjusting the pH to 5.0-6.0, placed at 1-10 ° C (preferably 4 ° C), precipitates again; and (5) centrifuge, wash, and separate the precipitate to obtain insulin crystalline microspheres.

The method of the present invention further comprises the following steps: (6) The obtained insulin crystalline microspheres are suspended in a polyethylene glycol solution and freeze-dried to obtain a lyophilized powder form of the insulin crystalline microspheres. The polyethylene glycol solution is polyethylene glycol 2000 or polyethylene glycol 4000.

In the step (1), the salt solution is preferably a NaCl solution, and the polyethylene glycol is polyethylene glycol 2000 or polyethylene glycol 4000.

In the step (3), the heating step has a temperature of 55 to 65 °C.

In the step (5), the washing process was washed 3 times with 0.5% NaCl, and then washed 3 times with a 0.5% polyethylene glycol 2000 solution.

The present invention also relates to an insulin crystalline microsphere obtained by the above method.

The invention also relates to an insulin crystalline microsphere suspension, which is microcrystallized from the above insulin The ball, a polar organic solvent and a lipophilic component.

The insulin crystalline microsphere suspension of the present invention contains less than 10% water.

In the insulin crystalline microsphere suspension of the present invention, the polar organic solvent is a polyethylene glycol. Preferably, the polyethylene glycol has a molecular weight of from 200 to 600, such as PEG 200, PEG 400. In the insulin crystalline microsphere suspension of the present invention, the lipophilic component is a phospholipid. Preferably, the phospholipid is phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol or phosphatidylserine.

In the insulin crystalline microsphere suspension of the present invention, the weight/volume ratio (g/ml) of the insulin crystalline microspheres to polyethylene glycol and phospholipid is 1:1-100, in phospholipids and polyethylene glycols. The weight/volume ratio (g/ml) is 0.001-0.05:1.

The insulin crystalline microsphere suspension of the present invention can be encapsulated in a soft capsule or a hard capsule. Preferably, the soft and hard capsules are enteric capsules or enteric coated.

The invention further relates to a method for preparing an insulin drug crystalline microsphere suspension, which comprises suspending the above insulin drug crystalline microspheres in a phospholipid-containing PEG solution to obtain an insulin microsphere suspension.

The present invention also relates to the use of the above-mentioned insulin drug crystalline microspheres or the above-mentioned insulin drug crystal microsphere suspension in the preparation of an oral hypoglycemic agent.

The size of the drug-crystallized microsphere of the present invention is ΙΟ μηι or less, preferably 5 μm or less. The invention has the advantages that: the degeneration of insulin caused by ρΗ and temperature change during microcrystallization can be reduced, and the activity is lost. The freeze-drying step has no effect on the biological activity of insulin. The dose of oral administration of insulin drug crystal microspheres is appropriate. When the dose of oral administration of 50 IU/kg is normal, the percentage of blood glucose lowering is 25%. It has broad clinical application prospects and can avoid the series of long-term injection of diabetes patients. side effect.

Therefore, the insulin drug microsphere suspension of the present invention can be used for oral administration.

Other objects and advantages of the present invention will be described in further detail in conjunction with the embodiments. DRAWINGS

Figure 1 Scanning electron micrograph of insulin crystalline microsphere freeze-dried powder

Figure 2 Determination of the particle size analyzer of insulin crystalline microsphere suspension Figure 3: Changes in blood glucose levels after duodenal administration in normal rats with fasting anesthesia

Figure 4: Changes in blood glucose levels after duodenal administration in normal rats with free diet

Figure 5 In vitro protective effect of PEG chain length and dosage on insulin drug crystalline microspheres

Figure 6 Different doses of insulin crystalline microsphere suspension in the duodenum of normal rats

Post-glycemic level change chart

Figure 7 Changes in blood glucose levels after administration of insulin-crystallized microsphere suspensions containing different phospholipids in the duodenum of normal rats

Fig. 8 Variation of blood glucose level after administration of insulin crystallization microsphere suspension containing different PEG chain length in duodenum of normal rats

Example 1

20 ml of 0.5% NaCl aqueous solution, add 50 mg of insulin, add glacial acetic acid, stir and dissolve, add 3 g of PEG 2000, stir to dissolve, adjust the pH to 3.9 with 1 M aqueous sodium acetate solution, heat to 65 ° C under magnetic stirring, dissolve, place After room temperature, the pH was adjusted to 5.6 with a 1 M aqueous sodium acetate solution. Place at 4 ° C for 12 h, lOOO r / min (TDZ5-WS multi-tube rack automatic balance centrifuge, rotor-2), centrifuge for 10 min, the resulting precipitate was washed 3 times with 0.5% NaCl aqueous solution, then 0.5% PEG 2000 The aqueous solution was washed 3 times, and the resulting precipitate was suspended in 1 ml of a 0.5% aqueous solution of PEG 2000, and lyophilized to obtain crystalline microspheres of insulin. The structure of the insulin crystalline microspheres (Fig. 1) and the particle diameter distribution (Fig. 2) were determined by a low temperature scanning electron microscope and a particle size analyzer based on centrifugation and light transmission of solid particles. The insulin content of the microspheres was determined by HLPC, and the insulin microspheres were obtained by grinding and suspending the insulin crystal microspheres in a PEG 200 solution containing 2.0% soybean phospholipid according to the requirements of the animal test. Example 2

40 ml of 0.8% NaCl solution, add 100 mg of insulin, add glacial acetic acid, stir and dissolve, add 6 g of PEG 2000, stir to dissolve, adjust the pH to 4.2 with 1 M sodium acetate aqueous solution, heat to 60 ° C under magnetic stirring, dissolve, place After room temperature, the pH was adjusted to 5.5 with a 1 M aqueous solution of sodium acetate. Place at 4 ° C for 16 h, 1000 r / min (TDZ5-WS multi-tube rack automatic balance centrifuge, rotor-2), centrifuge for 10 min, the resulting precipitate was washed 3 times with 0.8% NaCl aqueous solution, then 0.5% PEG The 2000 aqueous solution was washed 3 times, and the resulting precipitate was suspended in 1.5 ml of 0.5% PEG 2000 solution, and lyophilized to obtain insulin crystalline microspheres (Fig. 1, Fig. 2). The insulin content of the microspheres was determined by HLPC, and the insulin crystalline microspheres were ground and suspended in a PEG 400 solution containing 1.0% soybean phospholipid according to the needs of the animal test to obtain an insulin crystalline microsphere suspension. Example 3

20 ml of 0.9% NaCl aqueous solution, add 50 mg of insulin, add glacial acetic acid dropwise, dissolve and dissolve, add 3 g of PEG 4000, stir to dissolve, adjust the pH to 4.1 with 2 M sodium acetate aqueous solution, heat to 60 ° C under magnetic stirring, dissolve, place room temperature The pH was adjusted to 5.7 with a 2 M aqueous sodium acetate solution. Place at 4 ° C overnight, 1000 r / min (TDZ5-WS multi-tube rack automatic balance centrifuge, rotor-2), centrifuge for 10 min, the resulting precipitate was washed 3 times with 0.5% NaCl aqueous solution, then 0.5% PEG 4000 aqueous solution After washing 3 times, the resulting precipitate was suspended in 1 ml of 0.5% PEG 4000 aqueous solution, and lyophilized to obtain crystalline microspheres of insulin (Fig. 1, Fig. 2). The content of insulin in the microspheres was measured by HLPC, and the insulin microspheres were obtained by grinding and suspending the insulin crystal microspheres in a PEG 200 solution containing 2.0% soybean phosphoester according to the needs of the animal test. Example 4

80 ml of 0.7% NaCl aqueous solution, add 200 mg of insulin, add glacial acetic acid dropwise, dissolve and dissolve, add 9 g of PEG 2000, stir to dissolve, adjust the pH to 4.0 with 2 M sodium acetate aqueous solution, heat to 60 ° C under magnetic stirring, dissolve, place room temperature The pH was adjusted to 5.7 with a 2 M aqueous sodium acetate solution. Place at 4 ° C overnight, 1000 r / min (TDZ5-WS multi-tube rack automatic balance centrifuge, rotor-2), centrifuge for 10 min, the resulting precipitate was washed 3 times with pH 5.7 0.5% NaCl aqueous solution, then 0.5% PEG The 4000 aqueous solution was washed 3 times, and the resulting precipitate was suspended in 2.5 ml of 0.5% PEG 4000 aqueous solution, and lyophilized to obtain crystalline microspheres of insulin (Fig. 1, Fig. 2). The insulin content of the microspheres was determined by HLPC, and the insulin crystal microspheres were ground and suspended in a PEG 400 solution containing 1.0% soybean phospholipid according to the needs of the animal test to obtain an insulin microsphere suspension. Example 5

50 ml of 0.5% NaCl aqueous solution, add 100 mg of insulin, add glacial acetic acid, stir and dissolve, add 7 g of PEG 2000, stir to dissolve, adjust the pH to 3.9 with 1 M sodium hydroxide solution, magnetic The mixture was heated to 58 ° C with stirring, and allowed to stand at room temperature, and then adjusted to pH 5.6 with a 1 M aqueous sodium hydroxide solution. Place at 4 ° C overnight, 1000 r / min (TDZ5-WS multi-tube rack automatic balance centrifuge, rotor-2), centrifuge for 10 min, the resulting precipitate with pH 5.6 0.5% NaCl aqueous solution 3 times, then use 0.5% PEG 4000 The aqueous solution was washed 3 times, and the resulting precipitate was suspended in 2 ml of 0.5% PEG 4000 aqueous solution, and lyophilized to obtain crystalline microspheres of insulin (Fig. 1, Fig. 2). The content of insulin in the microspheres was determined by HLPC, and the insulin microspheres were obtained by grinding and suspending the insulin crystal microspheres in a PEG 400 solution containing 1.0% soybean phosphonite according to the needs of the animal test. Example 6

Fifteen healthy male Sprague-Dawley rats, weighing 180-200 g, were randomly divided into 3 groups. The first group was given 0.5 ml PBS as a control, the second group was given insulin solution (50 IU.kg), and the third group was given insulin drug microspheres. Suspension (SO IU.k^ fasting for 16 h before the experiment, free to drink water. Rats were anesthetized with intraperitoneal injection of pentobarbital sodium, the rat abdomen was fixed on the plate, and the abdominal cavity was opened along the midline of the rat. Duodenal administration, the specific operation is as follows: Injection is about 5 cm away from the pylorus. In order to avoid solution spillage, a small needle should be used as far as possible, and the drug should be injected downward along the intestinal tract. Barbital sodium, ensuring that the rats were always anesthetized during the test. Blood was taken from the tail vein of rats at 0, 0.5, 1, 2, 3, 4, 6, 8, 10 h, and blood glucose levels were measured with a blood glucose meter. (Fig. 3) It can be seen from the figure that the blood glucose levels of the three groups increased during the 30 minutes after administration, and then decreased. The blood glucose changes in the control group and the insulin solution group were basically the same, insulin drug microsphere suspension group The blood glucose level was significantly lower than that of the control group and the insulin solution group, and the blood glucose level showed a downward trend within 4-8 h. At 8 h, the blood glucose level dropped to the lowest level, about 30% of the initial value, and then the blood glucose began to rise. It is indicated that the anesthetized fasting rat has a significant hypoglycemic effect after administration of the insulin drug microsphere suspension in the duodenum.

Fifteen healthy male Sprague-Dawley rats, weighing 180-200 g, were randomly divided into 3 groups. The first group was given 0.5 ml PBS as a control, the second group was given insulin solution (SO IU.kg- ¹ ), and the third group was given insulin drugs. Microsphere suspension (50 IU.kg^ free diet before the experiment. Rats were anesthetized with intraperitoneal injection of pentobarbital sodium. The rats were fixed on the abdomen face up, and the abdominal cavity was opened along the midline of the rat. Duodenum For administration, the specific operation is as follows: Injection is administered about 5 cm away from the pylorus. For solution spillage, a small needle should be used as far as possible and administered downward in the direction of the intestine. Subsequently, the abdomen of the rats was surgically sutured, and the rats were completely awake at about 1.5 h, and blood was taken from the tail veins of the rats at 3, 4, 6, 8, and 10 h, respectively, and blood glucose levels were measured by a blood glucose meter (Fig. 4). It can be seen from the figure that the blood glucose changes in the control group and the insulin solution group are basically the same, and the blood glucose level in the insulin drug microsphere suspension group is significantly lower than that in the control group and the insulin solution. In group, blood glucose dropped to the lowest level at 4 h, about 25% of the initial value, and then blood sugar began to rise.

The results showed that there was a significant hypoglycemic effect after the administration of the insulin drug microsphere suspension in the duodenum of the free-fed rats. It can be seen from Fig. 3 and Fig. 4 that the hypoglycemic effect of the insulin microsphere suspension is closely related to the state of the experimental animal, and there is a significant difference in the hypoglycemic effect between the fasting anesthetized rats and the free diet rats. Example 8

Five healthy male Sprague-Dawley rats, weighing 180-200 g, were scraped off the ice immediately after anesthesia, and the PEG chain length (Fig. 5.A) and dosage (Fig. 5.B) were observed at 37 °C in vitro. Protective effect on insulin in the intestinal mucus layer. As can be seen from the figure: When insulin PBS solution contains 60% PEG (PEG/PEG 200 and PEG 400 is V/V, PEG4000 is W/V), the protective effect on insulin increases with the increase of PEG chain length, but PEG 400 There was no significant difference in the effect of PEG 4000 at different time points (Fig. 5. A); the protective effect of PEG 400 on insulin was positively correlated with its dosage. When the amount of PEG 400 was increased to 60%, the intestinal mucus layer was significantly inhibited. Degradation of insulin by protein digestive enzymes (Fig. 5. B). When the solution does not contain PEG, insulin is rapidly degraded by the protein digestive enzyme in the mucus layer, and no insulin content is detected at 30 min. Example 9

The relationship between the amount of insulin crystalline microspheres and the hypoglycemic effect in vivo was investigated in normal rats. 15 healthy male Sprague-Dawley rats weighing 180-200 g were randomly divided into 3 groups. The first group was given insulin drug microsphere suspension (50 IU.kg-l), and the second group was given insulin drug microsphere suspension. (200 IU.kg-l), the third group was given insulin drug microsphere suspension (500 IU.kg-l). Fasting for 16 h before the experiment, free to drink water. The rats were anesthetized with intraperitoneal injection of pentobarbital sodium. The rats were fixed on the abdomen with the abdomen facing up. The abdominal cavity was opened along the midline of the rat and the duodenum was administered. Bottom: Injected at a distance of about 5 cm from the pylorus. In order to avoid spillage of the solution, a small needle should be used as far as possible and administered downward in the direction of the intestine. Subsequently, the abdomen of the rats was surgically sutured, and blood was taken from the tail vein of the rats at 0.5, 1, 2, 3, 4, 5, 6, and 8 h, respectively, and blood glucose levels were measured by a blood glucose meter (Fig. 6). It can be seen from the figure that the blood glucose lowering amplitude of the insulin crystalline microspheres is dose-dependent, and the decrease of the blood glucose level of the animal increases with the increase of the administered dose, suggesting that the absorption of the drug increases correspondingly with the increase of the administered dose. Example 10

The effect of the amount of phospholipid on the biofilm effect of insulin crystalline microspheres (200 IU.kg-l) was investigated in normal rats. Twenty healthy male Sprague-Dawley rats, weighing 180-200 g, were randomly divided into 4 groups. The first group was given a phospholipid-free insulin drug microsphere suspension, and the second group was given an insulin drug microsphere suspension containing 1% phospholipid. In the third group, an insulin drug microsphere suspension containing 5% phospholipid was administered, and the fourth group was administered an insulin drug microsphere suspension containing 10% phospholipid. Fasting for 16 h before the experiment, free to drink. Rats were anesthetized with intraperitoneal injection of pentobarbital sodium. The rats were fixed on the plate with the abdomen facing up. The abdominal cavity was opened along the midline of the rat and the duodenum was administered. The specific operation was as follows: Injection at a distance of about 5 cm from the pylorus medicine. In order to avoid spillage of the solution, a small needle should be used as far as possible and administered downward in the direction of the intestine. Subsequently, the abdomen of the rats was surgically sutured, and blood was taken from the tail vein of the rats at 0.5, 1, 2, 3, 4, 5, 6, and 8 h, respectively, and blood glucose levels were measured by a blood glucose meter (Fig. 7). As can be seen from the figure, the presence or absence of phospholipids in the insulin microsphere suspension of insulin is essential. When no phospholipid is added to the system, the insulin drug crystal microspheres show almost no hypoglycemic activity. The amount of phospholipid in the system has little effect on the biological effects of the crystalline microspheres of insulin drugs, and when the content is increased to 10%, the hypoglycemic activity tends to decrease. Example 11

The effect of PEG chain length (PEG 4000 refers to 25% PEG4000 in PEG 400) on the biological effects of insulin crystalline microspheres (200 IU.kg-l) was investigated using normal rats. Fifteen healthy male SD rats weighing 180-200 g were randomly divided into 3 groups. Fasting for 16 h before the experiment, free to drink water. Rats were anesthetized with intraperitoneal injection of pentobarbital sodium. The rats were fixed on the plate with the abdomen facing up, and the abdominal cavity was opened along the midline of the rat's abdomen. The duodenum was administered as follows: Injection at a distance of about 5 cm from the pylorus medicine. In order to avoid solution spillage, try to use a small needle and follow the intestine The drug is administered in a downward direction. Subsequently, the abdomen of the rats was surgically sutured, and blood was taken from the tail vein of the rats at 0.5, 1, 2, 3, 4, 5, 6, and 8 h, respectively, and the blood glucose level was measured by a blood glucose meter (Fig. 8). It can be seen from the figure that the PEG chain length has no significant effect on the hypoglycemic activity of the insulin drug crystalline microspheres to some extent.

Claims

Rights request

A method for preparing insulin crystalline microspheres, comprising the steps of:

(1) Insulin is added to the salt solution, glacial acetic acid is added dropwise and stirred until the insulin is completely dissolved, and an appropriate amount of polyethylene glycol is added and stirred to dissolve; the weight ratio of insulin to polyethylene glycol is 1:20-200;

(2) adjusting the pH of the solution in step (1) to 3.5-4.5, and precipitating the precipitate;

(3) heating and stirring the solution in step (2) until the precipitate is completely dissolved;

(4) After the solution is placed at room temperature, the pH is adjusted to 5.0-6.0, and placed at 1-10 ° C, and the precipitate is precipitated again;

(5) Centrifugation, washing, and separation of the precipitate to obtain insulin crystal microspheres.

2. The method for preparing insulin crystalline microspheres according to claim 1, further comprising the steps of:

(6) The obtained insulin crystalline microspheres were suspended in a polyethylene glycol solution, and lyophilized to obtain a lyophilized powder form of the insulin crystalline microspheres.

The method for preparing insulin crystalline microspheres according to claim 1, wherein the salt solution in the step (1) is a NaCl solution.

The method for preparing a crystalline insulin microparticle according to claim 1, wherein the polyethylene glycol according to the step (1) is polyethylene glycol 2000 or polyethylene glycol 4000.

The method for producing insulin crystalline microspheres according to claim 1, wherein the temperature of the heating step in the step (3) is 55 to 65 °C.

The method for preparing insulin crystalline microspheres according to claim 1, wherein the washing process in the step (5) is washing three times with 0.5% NaCl, and then washing with a 0.5% polyethylene glycol 2000 solution. Times.

The method for producing insulin crystalline microspheres according to claim 2, wherein the polyethylene glycol solution is polyethylene glycol 2000 or polyethylene glycol 4000.

An insulin crystalline microsphere, characterized in that the insulin drug crystalline microsphere is obtained by the method according to any one of claims 1-7.

An insulin crystalline microsphere suspension comprising the insulin crystalline microsphere of claim 8, a polar organic solvent and a lipophilic component.

10. The insulin crystalline microsphere suspension according to claim 9, characterized by comprising Less than 10% water.

The insulin crystalline microsphere suspension according to claim 9, wherein the polar organic solvent is a polyethylene glycol.

The insulin crystalline microsphere suspension according to claim 9, wherein the lipophilic component is a phospholipid.

The insulin crystalline microsphere suspension according to claim 9, wherein the weight ratio of the insulin crystalline microspheres to polyethylene glycol and phospholipids (g/ml) is 1:1 to 100, The weight/volume ratio (g/ml) in the phospholipid to the polyethylene glycol is from 0.001 to 0.05:1.

The insulin crystalline microsphere suspension according to claim 9, wherein the polyethylene glycol has a molecular weight of from 200 to 600.

The insulin crystalline microsphere suspension according to claim 9, wherein the phospholipid is phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol or phosphatidylserine.

16. The insulin crystalline microsphere suspension of claim 9, wherein the suspension is potable in a soft or hard capsule.

The method for preparing an insulin drug crystalline microsphere suspension according to any one of claims 9 to 16, characterized in that the insulin drug crystalline microsphere of claim 8 is suspended in a phospholipid-containing PEG solution. In the middle, an insulin microsphere suspension is obtained.

18. Use of the insulin drug crystalline microsphere of claim 8 or the insulin drug crystalline microsphere suspension of any of claims 9-16 for the preparation of an oral hypoglycemic agent.