WO2012034360A1 - Insulin crystal microsphere, suspension and preparation method thereof - Google Patents

Insulin crystal microsphere, suspension and preparation method thereof Download PDF

Info

Publication number
WO2012034360A1
WO2012034360A1 PCT/CN2011/001560 CN2011001560W WO2012034360A1 WO 2012034360 A1 WO2012034360 A1 WO 2012034360A1 CN 2011001560 W CN2011001560 W CN 2011001560W WO 2012034360 A1 WO2012034360 A1 WO 2012034360A1
Authority
WO
WIPO (PCT)
Prior art keywords
insulin
crystalline
polyethylene glycol
microspheres
solution
Prior art date
Application number
PCT/CN2011/001560
Other languages
French (fr)
Chinese (zh)
Inventor
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 (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院生物物理研究所, 北京亚宝生物药业有限公司 filed Critical 中国科学院生物物理研究所
Publication of WO2012034360A1 publication Critical patent/WO2012034360A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/28—Insulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • Insulin crystalline microspheres 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
  • 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.
  • 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;
  • Protection of insulin stability in the gastrointestinal tract by formulation techniques and promotion of its absorption include encapsulation of insulin in liposomes, solid microspheres/nanoparticles, micelles, hydrogels, Emulsions, etc.
  • formulation techniques include encapsulation of insulin in liposomes, solid microspheres/nanoparticles, micelles, hydrogels, Emulsions, etc.
  • 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.
  • the main problem is the high dose of oral administration, low bioavailability, and difficulty in clinical application.
  • 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 are also important.
  • 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.
  • the mucus layer is also a potential barrier to drug absorption.
  • the drug 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.
  • the thickness of the mucus layer can significantly affect the rate at which macromolecules and charged molecular drugs enter the basal tissue.
  • 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.
  • 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.
  • a protective agent such as PEG or phospholipid
  • 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;
  • step (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.
  • the salt solution is preferably a NaCl solution
  • the polyethylene glycol is polyethylene glycol 2000 or polyethylene glycol 4000.
  • the heating step has a temperature of 55 to 65 °C.
  • 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.
  • the polar organic solvent is a polyethylene glycol.
  • the polyethylene glycol has a molecular weight of from 200 to 600, such as PEG 200, PEG 400.
  • the lipophilic component is a phospholipid.
  • the phospholipid is phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol or phosphatidylserine.
  • 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.
  • 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 ⁇ ⁇ or less, preferably 5 ⁇ m or less.
  • the invention has the advantages that: the degeneration of insulin caused by ⁇ 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.
  • the insulin drug microsphere suspension of the present invention can be used for oral administration.
  • 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 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 2 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 3 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 Example 3
  • Example 4 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 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
  • Example 5 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 Example 5
  • Example 6 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 Example 6
  • 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.
  • 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.
  • 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 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).
  • 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.
  • the effect of the amount of phospholipid on the biofilm effect of insulin crystalline microspheres 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.
  • PEG 4000 refers to 25% PEG4000 in PEG 400
  • insulin crystalline microspheres 200 IU.kg-l
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Diabetes (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Endocrinology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Obesity (AREA)
  • Emergency Medicine (AREA)
  • Dispersion Chemistry (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

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

胰岛素结晶微球、 其混悬剂、 以及制备方法 技术领域  Insulin crystalline microspheres, suspensions thereof, and preparation methods
本发明属于医药领域， 涉及一种胰岛素结晶微球混悬剂及其制备方 法。 更具体而言， 涉及一种采用液间成球结晶技术制备的胰岛素结晶微球 混悬剂及其制备方法。 背景技术  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
目前， 糖尿病的发病率正在逐年增加， 成为仅次于心血管疾病和癌症 的第三大致死性疾病。 胰岛素自 1922年首次用于治疗糖尿病后， 由于其 独特的降血糖疗效， 迄今为止是胰岛素依赖性糖尿病患者的首选药。 胰岛 素作为蛋白质与多肽药物， 口服时易被胃肠道的蛋白水解酶降解， 且分子 量较大不易透过生物膜等因素， 口服时的生物利用度很低， 不能发挥应有 的降血糖作用。 目前， 除了 Generex公司的颊贴膜产品 Omlin-Lyn™已在 印度与列厄瓜多尔上市外， 其它所有的胰岛素制剂都通过注射给药。 这不 仅给患者带来很大的经济负担， 影响了患者及其家属的生活质量， 同时频 繁长期注射用药还会产生诸多不良反应如： 胰岛素浮肿、 低血糖反应、 肥 大性脂肪营养不良及皮下脂肪萎縮等。 - 目前有关胰岛素的非注射给药包括透皮给药、 肺部吸入给药、 鼻腔给 药、 口服及颊粘膜给药等。 肺部吸入型胰岛素受到长期应用安全性和远期 疗效的困扰， Exubera®自动撤出市场在一定程度上对吸入型胰岛素的研制 带来了阴影。 胰岛素的口服给药途径可模拟生理性胰岛素分泌， 重建生理 状态下门静脉胰岛素浓度和外周循环胰岛素浓度近似 1 :5的比例， 因此， 口 服输送***是胰岛素最理想的非注射给药途径， 避免了注射疼痛及不便， 患者的依从性强。胰岛素口服输送***要解决的关键问题是如何防止胰岛 素在胃肠消化道内不被酶破坏的难题。 目前报道的有关胰岛素口服输送系 统的制备方法有以下几类： （1 )加入吸收促进剂： 吸收促进剂包括蛋白酶 抑制剂与渗透促进剂， 常用的吸收促进剂包括胆酸盐、 表面活性剂、 杆菌 肽与氨基酸衍生物等。 但长期使用吸收促进剂可损伤胃肠道上皮， 影响营 养物质吸收，还可导致细菌毒素的进入，有可能引起全身感染及毒性反应； (2) 通过制剂技术保护胰岛素在胃肠道的稳定性以及促.进其吸收： 这些 制剂技术包括将胰岛素包载于脂质体、 固体微球 /纳米粒、 胶束'、 水凝胶、 乳剂等。 但上述制剂技术均存在一定的局限性： 脂质体的稳定性差； 制备 纳米粒 /微球时常使用到有机溶剂、 高温 /高压， 这些不良条件有可能胰岛 素丧失活性， 并且有些聚合物材料在体内可引起炎症反应等； （3 )对胰岛 素进行化学修饰或合成前体药物： 采用脂肪酸对胰岛素进行化学修饰， 可 以强其亲脂性， 提高其在肠道的吸收， 但改变胰岛素的结构或合成前体药 物非常复杂， 如果结构修饰改变了其与受体结合位点的结合， 可能影响其 生物活性。 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.
综上所述， 虽然有关胰岛素口服输送***的研究报道、 专利很多， 但 最终实现临床应用的却微乎其微， 主要问题是口服给药剂量偏高， 生物利 用度较低， 难于在临床上实际推广应用。 在胰岛素口服输送***的相关研 究中， 主要集中于如何克服胃肠道中的酶屏障及肠上皮屏障， 增加肠上皮 的通透性及打开紧密连接等， 而忽略了胃肠道表面的粘液层对药物吸收的 影响。人类的肠道粘液层由粘蛋白、水、 电解质、脱落的上皮细胞、 脂类、 盐、 酶、 微生物及其产物组成， 厚度达数百微米。 磷脂使粘液层呈现疏水 特性， 粘蛋白与含水量决定了粘液层的粘弹性质。 含水量微小的变化可使 粘液的流变性质产生非常显著的变化。 粘液层中含有大量的酯酶、 蛋白消 化酶， 是胰岛素被酶降解的主要场所。  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.
许多研究表明粘液层除了保护基底膜免受肠内容物中危险因素的伤 害外， 还是影响药物吸收的潜在屏障。 对于口服输送***而言， 不管药物 的理化性质如何， 药物通过上皮吸收之前, 首先必须通过粘液层。 除中性 小分子药物外， 粘液层的厚度可显著影响大分子与带电荷分子药物进入基 底组织的速率。 例如， 与当量厚度的水相比， 粘液层可显著延迟氢离子的 扩散。研究表明当粒子大于 60 nm时即无法穿粘液屏障， 但研究发现 PEG 可防止大粒子（粒径 >500nm)附著于粘液层表面，粒子可快速穿越粘液层， 而 PEG未修饰的粒子则会吸附在粘液层表面并聚集成一团， 无法进入粘 液层中。 发明内容 本发明的目的是在确保胰岛素口服输送***有效性与安全性的双重 条件下， 制备一种新型胰岛素口服输送***， 即胰岛素药物结晶微球混悬 剂。 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.
本发明提供了一种胰岛素药物结晶微球混悬剂及其制备方法， 其中一 定密度与尺寸的胰岛素药物结晶微球采用液间成球结晶技术制备。冻干所 述胰岛素结晶微球获得胰岛素结晶微球冻干粉后， 将其混悬在 PEG、磷脂 等保护剂中即得胰岛素微球混悬剂。微球的制备不借助于任何聚合物与有 机溶剂， 从而保证了输送***的安全性。 PEG'与憐脂有利于药物微球迅速 通过肠粘液层， 避免被蛋白酶降解， 增加与肠淋巴组织的接触， 促进药物 吸收。  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.
本发明所制备的胰岛素结晶微球混悬剂迅速通过肠粘液层的速度可 以通过 PEG的链长、 磷脂的种类及改变 PEG与磷脂之间的比例实现。  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.
本发明提供了一种胰岛素结晶微球的制备方法， 其特征在于包括如下 步骤： （1 ) 胰岛素加入盐溶液中， 滴加冰醋酸并搅拌至胰岛素完全溶解， 加入适量的聚乙二醇，搅拌溶解；胰岛素与聚乙二醇的重量比为 1:20-200; 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) 调节步骤 （1 ) 中的溶液 pH至 3.5-4.5， 析出沉淀； （3 ) 加热并搅拌 步骤 （2 ) 中的溶液至沉淀完全溶解； （4 ) 将溶液放置窒温后调 pH 至 5.0-6.0， 1-10°C (优选 4°C )下放置， 再次析出沉淀； 和 （5 ) 离心、 洗涤、 分离沉淀， 得胰岛素结晶微球。 (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.
本发明的方法还进一步包含如下步骤： （6)将所得胰岛素结晶微球混 悬于聚乙二醇溶液中， 冷冻干燥， 得到胰岛素结晶微球的冻干粉形态。 所 述的聚乙二醇溶液为聚乙二醇 2000或聚乙二醇 4000。  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.
在步骤 （1 ) 中， 所述的盐溶液优选为 NaCl溶液， 所述的聚乙二醇为 聚乙二醇 2000或聚乙二醇 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.
在步骤 （3 ) 中， 所述的加热步骤的温度为 55-65°C。  In the step (3), the heating step has a temperature of 55 to 65 °C.
在步骤 （5 ) 中， 所述的洗涤过程为用 0.5% NaCl洗涤 3次， 然后采 用 0.5% 聚乙二醇 2000溶液洗涤 3次。  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.
本发明还涉及一种胰岛素结晶微球， 所述胰岛素药物结晶微球由上述 方法制备获得。  The present invention also relates to an insulin crystalline microsphere obtained by the above method.
本发明还涉及一种胰岛素结晶微球混悬剂， 由上述的胰岛素结晶微 球， 极性有机溶媒与亲脂性成份组成。 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.
本发明的胰岛素结晶微球混悬剂中含有少于 10%的水。  The insulin crystalline microsphere suspension of the present invention contains less than 10% water.
在本发明的胰岛素结晶微球混悬剂中， 所述的极性有机溶媒为聚乙二 醇类。优选地，所述聚乙二醇的分子量为 200-600，例如 PEG200, PEG400, 在本发明的胰岛素结晶微球混悬剂中， 所述的亲脂性成份为磷脂。 优 选地， 所述磷脂为磷脂酰胆碱、 磷脂酰乙醇胺、 磷脂酰肌醇或磷脂酰丝 氨酸。  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.
在本发明的胰岛素结晶微球混悬剂中， 所述胰岛素结晶微球与聚乙二 醇、 磷脂的重量 /体积比 (g/ml)为 1:1-100， 磷脂与聚乙二醇中的重量 /体积 比 (g/ml)为 0.001-0.05:1。  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.
本发明的胰岛素结晶微球混悬剂可灌封于软胶囊或硬胶囊中。 优选 地， 所述的软、 硬胶囊为肠溶胶囊或进行肠溶包衣。  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.
本发明还涉及胰岛素药物结晶微球混悬剂的制备方法， 将上述胰岛素 药物结晶微球混悬于含磷脂的 PEG溶液中， 得到胰岛素微球混悬剂。  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.
本发明还涉及上述的胰岛素药物结晶微球或上述的胰岛素药物结晶 微球混悬剂在制备口服降血糖药物中的应用。  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.
本发明的药物结晶微球的大小为 ΙΟ μηι以下， 优选为或 5 μιη以下。 本发明的优点是： 可以减少胰岛素在微晶化过程中因 ρΗ与温度变化 导致的变性， 活性丧失。 冷冻干燥步骤对胰岛素的生物活性无影响。 胰岛 素药物结晶微球的口服给药剂量适当， 正常大鼠口服给药剂量为 50IU/kg 时， 降血糖百分率达 25%, 具有广泛的临床应用前景， 可以避免糖尿病患 者长期注射给药产生的系列副作用。  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.
因此， 本发明的胰岛素药物微球混悬剂可用于口服给药。  Therefore, the insulin drug microsphere suspension of the present invention can be used for oral administration.
结合实施方案对本发明的其他目的和优点做进一步详细的描述， 使其 更为清楚。 附图说明  Other objects and advantages of the present invention will be described in further detail in conjunction with the embodiments. DRAWINGS
图 1 胰岛素结晶微球冻干粉的扫描电镜照片 Figure 1 Scanning electron micrograph of insulin crystalline microsphere freeze-dried powder
图 2 胰岛素结晶微球混悬剂的粒度分析仪测定图 图 3 空腹麻醉正常大鼠十二指肠给药后的血糖水平变化图 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
图 4 自由饮食清醒正常大鼠十二指肠给药后的血糖水平变化图 Figure 4: Changes in blood glucose levels after duodenal administration in normal rats with free diet
图 5 PEG链长与用量对胰岛素药物结晶微球的体外保护作用 Figure 5 In vitro protective effect of PEG chain length and dosage on insulin drug crystalline microspheres
图 6 正常大鼠十二指肠给予不同剂量的胰岛素结晶微球混悬剂 Figure 6 Different doses of insulin crystalline microsphere suspension in the duodenum of normal rats
后的血糖水平变化图 Post-glycemic level change chart
图 7 正常大鼠十二指肠给予含不同磷脂含量的胰岛素结晶微球混 悬剂后的血糖水平变化图 Figure 7 Changes in blood glucose levels after administration of insulin-crystallized microsphere suspensions containing different phospholipids in the duodenum of normal rats
图 8 正常大鼠十二指肠给予含不同 PEG链长的胰岛素结晶微球混 悬剂后的血糖水平变化图 具体实施方式 Fig. 8 Variation of blood glucose level after administration of insulin crystallization microsphere suspension containing different PEG chain length in duodenum of normal rats
实施例 1 Example 1
0.5%NaCl水溶液 20 ml， 加入 50mg胰岛素， 滴加冰醋酸搅拌溶解后 加入 3 g PEG 2000， 搅泮溶解， 用 1 M醋酸钠水溶液调 pH至 3.9， 磁力搅 拌下加热至 65°C溶解， 放置室温后用 1 M醋酸钠水溶液调 pH至 5.6。 置 4°C放置 12 h， lOOO r/min (TDZ5-WS多管架自动平衡离心机， rotor-2)， 离心 10 min,所得沉淀用 0.5% NaCl水溶液洗涤 3次，然后采用 0.5% PEG 2000水溶液洗涤 3次， 将所得沉淀混悬在 1ml 0.5% PEG 2000水溶液中， 冷冻干燥得胰岛素的结晶微球。采用低温扫描电镜及基于固体颗粒的离心 沉降和光透射原理的粒度仪测定胰岛素结晶微球的结构形态 （图 1 ) 及粒 径分布（图 2)。采用 HLPC测定微球中胰岛素的含量， 依据动物试验的需 要将胰岛素结晶微球通过研磨、 混悬在含 2.0%大豆磷酯的 PEG 200溶液 中即得胰岛素微球混悬液。 实施例 2  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
0.8% NaCl水溶液 40 ml， 加入 100 mg胰岛素， 滴加冰醋酸搅拌溶解 后加入 6 g PEG 2000， 搅拌溶解， 用 1 M醋酸钠水溶液调 pH至 4.2， 磁力 搅拌下加热至 60°C溶解， 放置室温后用 1 M醋酸钠水溶液调 pH至 5.5。 置 4°C放置 16 h ， 1000 r/min (TDZ5-WS多管架自动平衡离心机， rotor-2), 离心 10 min,所得沉淀用 0.8% NaCl水溶液洗涤 3次，然后采用 0.5% PEG 2000水溶液洗涤 3次， 将所得沉淀混悬在 1.5 ml 0.5% PEG 2000溶液中， 冷冻干燥得胰岛素结晶微球（图 1， 图 2)。 采用 HLPC测定微球中胰岛素 的含量，依据动物试验的需要将胰岛素结晶微球通过研磨、混悬在含 1.0% 大豆磷酯的 PEG 400溶液中即得胰岛素结晶微球混悬液。 实施例 3 · 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
0.9%NaCl水溶液 20 ml， 加入 50mg胰岛素， 滴加冰醋酸搅拌溶解后 加入 3 g PEG 4000， 搅拌溶解， 用 2 M醋酸钠水溶液调 pH至 4.1， 磁力搅 拌下加热至 60°C溶解， 放置室温后用 2 M醋酸钠水溶液调 pH至 5.7。 置 4°C放置过夜， 1000 r/min (TDZ5-WS多管架自动平衡离心机， rotor-2) , 离心 10 min，所得沉淀用 0.5% NaCl水溶液洗涤 3次，然后采用 0.5% PEG 4000水溶液洗涤 3次， 将所得沉淀混悬在 1ml 0.5% PEG 4000水溶液中， 冷冻干燥得胰岛素的结晶微球（图 1， 图 2)。 采用 HLPC测定微球中胰岛 素的含量， 依据动物试验的需要将胰岛素结晶微球通过研磨、 混悬在含 2.0%大豆磷酯的 PEG 200溶液中即得胰岛素微球混悬液。 实施例 4  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
0.7%NaCl水溶液 80 ml，加入 200mg胰岛素， 滴加冰醋酸搅拌溶解后 加入 9 g PEG 2000， 搅拌溶解， 用 2 M醋酸钠水溶液调 pH至 4.0， 磁力搅 拌下加热至 60°C溶解， 放置室温后用 2 M醋酸钠水溶液调 pH至 5.7。 置 4°C放置过夜， 1000 r/min (TDZ5-WS多管架自动平衡离心机， rotor-2), 离心 10 min,所得沉淀用 pH 5.7 0.5% NaCl水溶液洗涤 3次，然后采用 0.5% PEG 4000水溶液洗涤 3次，将所得沉淀混悬在 2.5 ml 0.5% PEG 4000水溶 液中， 冷冻干燥得胰岛素的结晶微球（图 1， 图 2)。 采用 HLPC测定微球 中胰岛素的含量， 依据动物试验的需要将胰岛素结晶微球通过研磨、 混悬 在含 1.0%大豆磷酯的 PEG 400溶液中即得胰岛素微球混悬液。 实施例 5  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
0.5%NaCl水溶液 50 ml，加入 lOOmg胰岛素， 滴加冰醋酸搅拌溶解后 加入 7 g PEG 2000， 搅拌溶解， 用 1 M氢氧化钠水溶液调 pH至 3.9， 磁力 搅拌下加热至 58°C溶解，放置室温后用 1 M氢氧化钠水溶液调 pH至 5.6。 置 4°C放置过夜， 1000 r/min (TDZ5-WS多管架自动平衡离心机， rotor-2)， 离心 10 min，所得沉淀用 pH 5.6 0.5% NaCl水溶液 3次，然后采用 0.5% PEG 4000水溶液洗涤 3次， 将所得沉淀混悬在 2 ml 0.5% PEG 4000水溶液中， 冷冻干燥得胰岛素的结晶微球（图 1， 图 2)。 采用 HLPC测定微球中胰岛 素的含量， 依据动物试验的需要将胰岛素结晶微球通过研磨、 混悬在含 1.0%大豆磷酯的 PEG 400溶液中即得胰岛素微球混悬液。 实施例 6 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
健康雄性 SD大鼠 15只， 体重 180-200 g， 随机分成 3组， 第一组给 予 0.5 ml PBS作为对照， 第二组给予胰岛素溶液 （50 IU.kg )， 第三组给 予胰岛素药物微球混悬剂 （SO IU.k^ 实验前禁食 16 h， 可自由饮水。 腹腔注射戊巴比妥钠麻醉大鼠， 将大鼠腹部朝上固定在平板上， 沿大鼠腹 中线打开腹腔， 十二指肠给药， 具体操作如下： 在距幽门约 5 cm处注射 给药。 为了避免溶液外溢， 应尽量采用小号针头， 且顺着肠道方向向下注 射给药。 定时补充注射戊巴比妥钠， 保证大鼠在试验过程中一直处于麻醉 状态。 分别于 0、 0.5、 1、 2、 3、 4、 6、 8、 10 h大鼠尾静脉取血， 用血糖 仪测定血糖值（图 3 )。 由图可知， 三组在给药后 30min时， 因手术刺激血 糖均呈上升趋势， 随后下降。 对照组与胰岛素溶液组的血糖变化趋势基本 一致， 胰岛素药物微球混悬剂组的血糖水平明显低于对照组与胰岛素溶液 组， 且血糖水平在 4-8 h内呈下降趋势， 8 h时血糖降至最低水平， 约为初 始值的 30%左右， 随后血糖开始上升。 结果表明， 麻醉空腹大鼠十二指肠 给予胰岛素药物微球混悬剂后， 有明显的降血糖作用。 实施例 7  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.
健康雄性 SD大鼠 15只， 体重 180-200 g， 随机分成 3组， 第一组给 予 0.5 ml PBS作为对照， 第二组给予胰岛素溶液 （SO IU.kg—1 ), 第三组给 予胰岛素药物微球混悬剂 （50 IU.kg^ 实验前自由饮食。 腹腔注射戊巴 比妥钠麻醉大鼠，将大鼠腹部朝上固定在平板上，沿大鼠腹中线打开腹腔， 十二指肠给药， 具体操作如下： 在距幽门约 5 cm处注射给药。 为了避免 溶液外溢， 应尽量采用小号针头， 且顺着肠道方向向下注射给药。 随后将 大鼠腹部进行手术缝合， 大鼠约于 1.5 h左右完全清醒， 分别于 3、 4、 6、 8、 10 h大鼠尾静脉取血， 用血糖仪测定血糖值 （图 4)。 由图可知， 对照 组与胰岛素溶液组的血糖变化趋势基本一致， 与给药前的血糖水平相比变 化不大， 而胰岛素药物微球混悬剂组的血糖水平明显低于对照组与胰岛素 溶液组， 4 h时血糖降至最低水平， 约为初始值的 25%左右， 随后血糖开 始上升。 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.
结果表明， 自由饮食清清醒大鼠十二指肠给予胰岛素药物微球混悬剂 后， 有明显的降血糖作用。 从图 3与图 4中可以看出， 胰岛素微球混悬剂 的降血糖效果与与实验动物的状态密切相关， 对空腹麻醉大鼠及自由饮食 大鼠的降血糖作用存在明显差别。 实施例 8  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
健康雄性 SD大鼠 5只， 体重 180-200 g， 麻醉处死后立即在冰上刮取 肠粘液层， 体外 37°C孵育考察 PEG链长 （图 5. A) 与用量 （图 5. B ) 对 肠粘液层中胰岛素的保护作用。 由图可知： 当胰岛素 PBS溶液中含 60% 的 PEG (PEG 200与 PEG 400为 V/V， PEG4000为 W/V) 时， 随 PEG链 长的增加， 对胰岛素的保护作用增强， 但 PEG 400与 PEG 4000在不同时 间点的作用无明显差别 （图 5. A); PEG 400对胰岛素的保护作用与其用 量呈正相关性， 当 PEG 400的用量增加至 60%时， 可显著抑制肠粘液层中 蛋白消化酶对胰岛素的降解作用 （图 5. B)。 当溶液中不含 PEG时， 胰岛 素在粘液层中快速被蛋白消化酶降解， 30 min时已检测不出胰岛素含量。 实施例 9  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
采用正常大鼠考察胰岛素结晶微球的用量与其体内降血糖效应的关 系。 健康雄性 SD大鼠 15只， 体重 180-200 g， 随机分成 3组， 第一组给 予胰岛素药物微球混悬剂 （50 IU.kg-l )， 第二组给予胰岛素药物微球混悬 剂 （200 IU.kg-l )， 第三组给予胰岛素药物微球混悬剂 （500 IU.kg-l )。 实 验前禁食 16 h， 可自由饮水。 腹腔注射戊巴比妥钠麻醉大鼠， 将大鼠腹部 朝上固定在平板上， 沿大鼠腹中线打开腹腔， 十二指肠给药， 具体操作如 下： 在距幽门约 5 cm处注射给药。 为了避免溶液外溢， 应尽量采用小号 针头， 且顺着肠道方向向下注射给药。 随后将大鼠腹部进行手术缝合， 分 别于 0.5、 1、 2、 3、 4、 5、 6、 8 h大鼠尾静脉取血， 用血糖仪测定血糖值 (图 6)。 由图可知， 胰岛素结晶微球的降血糖幅度呈剂量依赖性， 随给药 剂量的升高， 动物血糖的降低幅度增加， 提示随给药剂量的增加， 药物的 吸收也相应增加。 实施例 10 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
采用正常大鼠考察磷脂用量对胰岛素结晶微球 （200 IU.kg-l ) 体内生 物效应的影响。 健康雄性 SD大鼠 20只， 体重 180-200 g， 随机分成 4组， 第一组给予不含磷脂的胰岛素药 微球混悬剂， 第二组给予含 1%磷脂的 胰岛素药物微球混悬剂，第三组给予含 5%磷脂的胰岛素药物微球混悬剂， 第四组给予含 10%磷脂的胰岛素药物微球混悬剂。 实验前禁食 16 h， 可自 由饮水。 腹腔注射戊巴比妥钠麻醉大鼠， 将大鼠腹部朝上固定在平板上， 沿大鼠腹中线打开腹腔， 十二指肠给药， 具体操作如下： 在距幽门约 5 cm 处注射给药。 为了避免溶液外溢， 应尽量采用小号针头， 且顺着肠道方向 向下注射给药。 随后将大鼠腹部进行手术缝合， 分别于 0.5、 1、 2、 3、 4、 5、 6、 8 h大鼠尾静脉取血， 用血糖仪测定血糖值 （图 7)。 由图可知， 胰 岛素药物结晶微球混悬剂中磷脂的有无至关重要， 当体系中未加入磷脂 时， 胰岛素药物结晶微球几乎未表现出降血糖活性。 体系中磷脂含量的多 少对胰岛素药物结晶微球的生物效应几乎无影响， 且当含量增加至 10% 时， 降血糖活性有减弱的趋势。 . 实施例 11  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
采用正常大鼠考察 PEG链长 （PEG 4000 是指 PEG 400 中含 25% PEG4000) 对胰岛素结晶微球 （200 IU.kg-l ) 体内生物效应的影响。 健康 雄性 SD大鼠 15只， 体重 180-200 g， 随机分成 3组。 实验前禁食 16 h， 可自由饮水。 腹腔注射戊巴比妥钠麻醉大鼠， 将大鼠腹部朝上固定在平板 上， 沿大鼠腹中线打开腹腔； 十二指肠给药， 具体操作如下： 在距幽门约 5 cm处注射给药。 为了避免溶液外溢， 应尽量采用小号针头， 且顺着肠道 方向向下注射给药。 随后将大鼠腹部进行手术缝合， 分别于 0.5、 1、 2、 3、 4、 5、 6、 8 h大鼠尾静脉取血， 用血糖仪测定血糖值 （图 8 )。 由图可知， 在一定程度上 PEG链长对胰岛素药物结晶微球的降血糖活性无明显影响。 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

权 利 要 求 Rights request
1. 一种胰岛素结晶微球的制备方法， 其特征在于包括如下步骤：A method for preparing insulin crystalline microspheres, comprising the steps of:
( 1 ) 胰岛素加入盐溶液中， 滴加冰醋酸并搅拌至胰岛素完全溶解， 加入适量的聚乙二醇，搅拌溶解；胰岛素与聚乙二醇的重量比为 1:20-200; (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) 调节步骤 （1 ) 中的溶液 pH至 3.5-4.5， 析出沉淀；  (2) adjusting the pH of the solution in step (1) to 3.5-4.5, and precipitating the precipitate;
(3 ) 加热并搅拌步骤 （2) 中的溶液至沉淀完全溶解；  (3) heating and stirring the solution in step (2) until the precipitate is completely dissolved;
(4)将溶液放置室温后调 pH至 5.0-6.0， 1-10°C下放置， 再次析出沉 淀；  (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 ) 离心、 洗涤、 分离沉淀， 得胰岛素结晶微球。  (5) Centrifugation, washing, and separation of the precipitate to obtain insulin crystal microspheres.
2. 根据权利要求 1所述的胰岛素结晶微球的制备方法，其特征在于还 进一步包含如下步骤：  2. The method for preparing insulin crystalline microspheres according to claim 1, further comprising the steps of:
( 6) 将所得胰岛素结晶微球混悬于聚乙二醇溶液中， 冷冻干燥， 得 到胰岛素结晶微球的冻干粉形态。  (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.
3. 根据权利要求 1所述的胰岛素结晶微球的制备方法，其特征在于步 骤 （1 ) 所述的盐溶液为 NaCl溶液。  The method for preparing insulin crystalline microspheres according to claim 1, wherein the salt solution in the step (1) is a NaCl solution.
4. 根据权利要求 1所述的胰岛素结晶微 ¾^的制备方法，其特征在于步 骤 （1 ) 所述的聚乙二醇为聚乙二醇 2000或聚乙二醇 4000。  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.
5. 根据权利要求 1所述的胰岛素结晶微球的制备方法，其特征在于步 骤 （3 ) 所述的加热步骤的温度为 55-65°C。  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.
6. 根据权利要求 1所述的胰岛素结晶微球的制备方法，其特征在于步 骤（5 )所述的洗涤过程为用 0.5% NaCl洗涤 3次， 然后采用 0.5% 聚乙二 醇 2000溶液洗涤 3次。  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.
7. 根据权利要求 2所述的胰岛素结晶微球的制备方法，其特征在于所 述的聚乙二醇溶液为聚乙二醇 2000或聚乙二醇 4000。  The method for producing insulin crystalline microspheres according to claim 2, wherein the polyethylene glycol solution is polyethylene glycol 2000 or polyethylene glycol 4000.
8. 一种胰岛素结晶微球，其特征在于所述胰岛素药物结晶微球由权利 要求 1-7任一项所述方法制备获得。  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.
9. 一种胰岛素结晶微球混悬剂，其特征在于由权利要求 8所述的胰岛 素结晶微球， 极性有机溶媒与亲脂性成份组成。  An insulin crystalline microsphere suspension comprising the insulin crystalline microsphere of claim 8, a polar organic solvent and a lipophilic component.
10. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在于含有 少于 10%的水。 10. The insulin crystalline microsphere suspension according to claim 9, characterized by comprising Less than 10% water.
11. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在.于所述 的极性有机溶媒为聚乙二醇类。  The insulin crystalline microsphere suspension according to claim 9, wherein the polar organic solvent is a polyethylene glycol.
12. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在于所述 的亲脂性成份为磷脂。  The insulin crystalline microsphere suspension according to claim 9, wherein the lipophilic component is a phospholipid.
13. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在于所述 胰岛素结晶微球与聚乙二醇、磷脂的重量 /体积比 (g/ml)为 1 :1-100， 磷脂与 聚乙二醇中的重量 /体积比 (g/ml)为 0.001-0.05:1。  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.
14. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在于所述 聚乙二醇的分子量为 200-600。  The insulin crystalline microsphere suspension according to claim 9, wherein the polyethylene glycol has a molecular weight of from 200 to 600.
15. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在于所述 磷脂为磷脂酰胆碱、 磷脂酰乙醇胺、 磷脂酰肌醇或磷脂酰丝氨酸。  The insulin crystalline microsphere suspension according to claim 9, wherein the phospholipid is phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol or phosphatidylserine.
16. 根据权利要求 9所述的胰岛素结晶微球混悬剂， 其特征在于混悬 剂可灌封于软胶囊或硬胶囊中。  16. The insulin crystalline microsphere suspension of claim 9, wherein the suspension is potable in a soft or hard capsule.
17. 一种如权利要求 9-16任一项所述的胰岛素药物结晶微球混悬剂的 制备方法， 其特征在于将权利要求 8所述胰岛素药物结晶微球混悬于含磷 脂的 PEG溶液中， 得到胰岛素微球混悬剂。  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. 权利要求 8所述的胰岛素药物结晶微球或权利要求 9-16任一项所 述的胰岛素药物结晶微球混悬剂在制备口服降血糖药物中的应用。  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.
PCT/CN2011/001560 2010-09-15 2011-09-14 Insulin crystal microsphere, suspension and preparation method thereof WO2012034360A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2010102847832A CN102397535B (en) 2010-09-15 2010-09-15 Insulin crystal microsphere, insulin crystal microsphere suspension, and preparation method thereof
CN201010284783.2 2010-09-15

Publications (1)

Publication Number Publication Date
WO2012034360A1 true WO2012034360A1 (en) 2012-03-22

Family

ID=45830958

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/001560 WO2012034360A1 (en) 2010-09-15 2011-09-14 Insulin crystal microsphere, suspension and preparation method thereof

Country Status (2)

Country Link
CN (1) CN102397535B (en)
WO (1) WO2012034360A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220339114A1 (en) * 2021-04-17 2022-10-27 Bhami's Research Laboratory, Pvt. Ltd. Polypeptide formulations

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2014120626A (en) * 2014-05-22 2015-11-27 Владимир Андреевич Сабецкий INSULIN-CONTAINING MEDICINE OF LONG-TERM ACTION

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1425464A (en) * 2002-12-26 2003-06-25 西安交通大学 Process for preparing oral insulin nano material
CN1562356A (en) * 2004-03-19 2005-01-12 中国科学院长春应用化学研究所 Biological degradable macromolecule microspheres of carrying insulin and preparation method
CN101199482A (en) * 2007-12-20 2008-06-18 中国科学院长春应用化学研究所 Process for preparing degradable polyester microsphere wrapping

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100508695B1 (en) * 2001-02-13 2005-08-17 한국과학기술연구원 Formulation for oral delivery of insulin and preparation method thereof
CN1160122C (en) * 2001-04-20 2004-08-04 清华大学 Method of preparing oil-phase oral insulin preparation
CN101618023B (en) * 2008-06-30 2011-11-30 江苏先声药物研究有限公司 Method for preparing micronized protein

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1425464A (en) * 2002-12-26 2003-06-25 西安交通大学 Process for preparing oral insulin nano material
CN1562356A (en) * 2004-03-19 2005-01-12 中国科学院长春应用化学研究所 Biological degradable macromolecule microspheres of carrying insulin and preparation method
CN101199482A (en) * 2007-12-20 2008-06-18 中国科学院长春应用化学研究所 Process for preparing degradable polyester microsphere wrapping

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220339114A1 (en) * 2021-04-17 2022-10-27 Bhami's Research Laboratory, Pvt. Ltd. Polypeptide formulations

Also Published As

Publication number Publication date
CN102397535A (en) 2012-04-04
CN102397535B (en) 2013-08-28

Similar Documents

Publication Publication Date Title
US8974819B2 (en) Sustained-release chitosan capsules comprising chitosan and phytic acid
JP3903061B2 (en) Nanoparticles containing drug, method for producing the same, and preparation for parenteral administration comprising the nanoparticles
KR101306384B1 (en) Diketopiperazine salts, diketomorpholine salts or diketodioxane salts for drug delivery
Chen et al. Cp1-11 peptide/insulin complex loaded pH-responsive nanoparticles with enhanced oral bioactivity
CA2435455A1 (en) Therapeutic agents complexed with calcium phosphate and encased by casein
CN105056212B (en) A kind of chitosan nano and preparation method for improving oral insulin colonic absorption
CN111569082B (en) Oral delivery system for protein-loaded polypeptide drug exosomes
KR20040037016A (en) A composition for an enteric coating of natural product containing lectin
EP3416628A1 (en) Two-stage microparticle-based therapeutic delivery system and method
Ma et al. Crosslinked zwitterionic microcapsules to overcome gastrointestinal barriers for oral insulin delivery
CN107638388B (en) Asiatic acid chitosan deoxycholic acid graft micelle and preparation method thereof
VP et al. Novel approaches for oral delivery of insulin and current status of oral insulin products
WO2012113116A1 (en) Emulsion containing hydrophilic biological macromolecule, preparation method and application thereof
WO2012113117A1 (en) Oral preparation comprising protein or polypeptide, preparation method thereof, and use thereof
CN104739806B (en) oral insulin composite microcapsule and preparation method
Liu et al. Nanoencapsulation of chitooligosaccharides enhances its oral bioavailability and anti-liver fibrotic effects
WO2012034360A1 (en) Insulin crystal microsphere, suspension and preparation method thereof
AlSalem et al. Enhance the oral insulin delivery route using a modified chitosan-based formulation fabricated by microwave
CN110464835B (en) Insulin flexible particles and preparation thereof
Paul et al. Tricalcium phosphate delayed release formulation for oral delivery of insulin: a proof-of-concept study
CN106421807A (en) Preparation method of insulin-supporting carboxymethyl chitosan/chitosan nanometer preparation
TW200410729A (en) Multilayered delivery system and method for producing the same
CN109675020B (en) Oral GLP-1 polypeptide nano preparation and preparation method and application thereof
CN113116818B (en) Encapsulated bodies and their use for producing medicaments or foodstuffs
CN112999194A (en) Preparation method and application of oral insulin polypeptide nanocapsule

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11824431

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11824431

Country of ref document: EP

Kind code of ref document: A1