胰岛素的海藻酸盐—壳聚糖微球载体制备和口服给药研究
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摘要
目前，注射几乎是胰岛素临床应用的惟一给药途径，存在突出问题是，药物在血液中半衰期短，必须重复注射给药。对于必须长期甚至终身依赖胰岛素治疗患者，这种特性将造成患者在身体、精神、经济上的巨大负担。因此，发展胰岛素非注射给药途径是国内外医药界关注的重大课题。可能的非注射给药途径中，口服是理想的给药方式，具有服用方便安全、患者治疗依从性高等优势。但是，胰岛素直接口服易被胃肠道消化酶降解并丧失疗效。同时，作为两性分子，其亲水特性使之不能跨越小肠上皮吸收屏障。
针对胰岛素口服给药面临的主要问题，本文采用脉冲电场微球制备工艺，以海藻酸盐、壳聚糖为载体材料，开展了载胰岛素口服微球的制备工艺研究。采用真空冷冻干燥技术对载药微球干燥处理后，进行了微球口服给药的药效学、药动学、以及药理相对生物利用度的研究。主要研究结果如下：
1．采用脉冲电场工艺制备了载胰岛素壳聚糖-海藻酸钙微球。以微球粒径和球形度为指标，采用正交实验设计法优化工艺条件；利用倒置生物显微镜和扫描电镜测定微球粒径、观察微球的表面形态以及内部结构。采用考马斯亮蓝法测定微球中胰岛素药物的包封率。结果表明，锐孔孔径是影响微球粒径的最显著的因素。优化工艺条件为450μm锐孔直径、2cm液面距、10mg／mL海藻酸钠浓度、8mL／h推进速度、金属皿作凝胶浴容器。
2．采用真空冷冻干燥法制备了载胰岛素微球的冻干剂，考察了微球冷冻干燥前后粒径变化以及微球中胰岛素相对活性的变化。结果表明，冻干对微球中药物的活性影响较小，但能够大大减小微球的粒径，冻干后，微球平均粒径由123μm减小为48μm，粒径减小率为61％。
3．建立糖尿病动物模型，为进行微球口服的药效学和药动学研究提供稳定可靠的动物模型。采用体重为18～22g的昆明种小白鼠腹腔注射200mg／kg四氧嘧啶生理盐水溶液，建立了类似人类Ⅰ型糖尿病的动物模型。
4．胰岛素微球口服给药的药效学实验表明：微球不仅对正常小鼠有降血糖作用，而且对四氧嘧啶致糖尿病小鼠也有降血糖作用，且血糖降低水平与剂量之间存在一定量效的关系。正常小鼠口服平均粒径48μm载胰岛素微球，6h时血糖降低率最大，10h内具有降血糖作用。给予正常小鼠不同剂量的胰岛素微球，口服3.2IU／kg剂量6h时使小鼠血糖下降27.2％，6IU／kg剂量使小鼠血糖降低40％。对于糖尿病小鼠而言，降血糖作用更为明显。口服粒径为48μm、剂量为4.8IU／kg载胰岛素微球，在6h～10h内维持血糖值处于较低水平，6h达到最低值，10h血糖值恢复为原有的46.01％，整个降血糖过程缓慢而有效。与皮下注射给药方式相比，口服胰岛素的药理相对生物利用度为41.20％。糖尿病小鼠口服高(4.8IU／kg)、中(3.6IU／kg)、低(2.4IU／kg)三个剂量的载胰岛素微球，高剂量的降血糖作用明显优于中低剂量，最大降血糖百分率分别为57.31％、40.16％、23.87％。
5．药动学实验表明：采用糖尿病小鼠模型作为研究对象，口服4.8IU／kg载胰岛素微球，血清胰岛素呈现出缓慢而平稳的上升过程，血清胰岛素含量在4h为最高值。与皮下注射给药方式相比，口服胰岛素的药理相对生物利用度为41.22％。对糖尿病小鼠口服不同剂量载胰岛素微球，高、中、低三种剂量均体现出良好的升高血清胰岛素含量的作用，体现良好的量效关系。药理相对生物利用度分别为41.22％，37.32％，35.37％。
At present, injection is the only means of administration of insulin in clinic. The problem is the short half-life and repeated injection administration, resulting physical and spiritual burdens for long-term or life-long insulin-dependent patients. Therefore, the development of non-injection administration of insulin is very important. Oral delivery is the perfect administration of the non-injection because of security and higher advantage of the compliance of patients. However, insulin, must first be broken down by digestive enzyme and loss of efficacy. Meanwhile, as amphoteric element, insulin with hydrophilic properties are not absorbed by intestinal epithelial barrier.
In order to sovle problems of oral insulin delivery, impulsive electrostatic technique was used with chitosan and sodium alginate as microsphere material, studies on preparation of insulin microspheres.The pharmacodynamic, pharmacokinetic and the relative pharmacological bioavailability of oral freeze-drying insulin-loaded Alginate/ chitosan microspheres, were mainly studied in this article.The research were made as follow:
1. Insulin-loaded alginate/chitosan microspheres were preparede by impulsive electrostatic technique. The interrelated factors influencing the diameter and sphericity were studied through orthogonal experiments,and finally the statistic analysis made sure the optimum conditions to prepare microspheres. The scanning electron microscope and biological inverted microscope were used to observe diameter, the surface morphology and internal structure of microspheres. The encapsulation efficiency of insulin-loaded microspheres were determined by CBBG.The results showed that the diameter of needle was the most significant factor to the diameter of microspheres. The optimum conditions for the least diameter of microspheres were 450μm diameter of needle, 2cm from needle tips to the gelation surface, 10mg/mL alginate concentration,8mL/h speed of flowing-liquid and metal containers.
2. Microspheres were prepared by Vacuum freeze-drying, then inspected the diameter of microsphere before freeze-drying, and changes in the relative activity of insulin. The results showed that the freeze-dried microspheres were slight effect on the drug activity, but the diameter of the microspheres were greatly reduced. The average diameter of microspheres decreased from 123 urn to 48 urn , the rate of decreasing is 61%.
3. Diabetic animal model provided stable and reliable animal model for following oral pharmacokinetic and pharmacodynamic. In this paper, the weight of 18～22g Kunming-rats were subcutaneous injected 200mg/kg alloxan-saline solution, to establish a similar animal model of human type 1 diabetes.
4. Studies of pharmacodynamic on insulin-loaded microspheres showed that microspheres had hypoglycemic effect not only on the normal rats, but also on alloxan-induced diabetic rats, Also there was a certain relationship between dose and hypoglycemic effect. Hypoglycemic effect showed the maximum rate at 6h, and maintained within 10h after oral 48μm microspheres on normal rats. Blood glucose level decreased to 27.2% , 40% respectively after 6h with oral 3.2IU/kg, 6IU/kg on normal rats. For diabetic rats, the hypoglycemic effect became more apparent, and blood glucose level maintained lower during 6～10h after oral 4.8IU/kg microspheres.The lowest level was at 6h, then cameback to 46.01% of original level .The whole process showed a steady and effective hepoglycemic effect after oral. Compared to the administration of subcutaneous injection method, the relative pharmacological bioavailability of oral insulin-loaded microspheres reached 41.20%.The hypoglycemic effect of the high dose (4.8IU/kg)was better than lower dose after oral different dose of insulin-loaded microspheres to diabetic rats,, the largest of hypoglycemic effect reached was 57.31%, 40.16%, 23.87%.
5. Study of pharmacokinetic showed : With diabetic rats as animal model, the serum insulin level were determined by Radioimmunoassay(RIA) after oral insulin-loaded microspheres. the serum insulin level showed a slow and steady increase after oral 4.8IU/kg insulin-loaded microspheres, and the serum insulin level was the highest value at 4h. Compared to the administration of subcutaneous injection method, the relative pharmacological bioavailability of oral insulin-loaded microsphere reached 41.22%. It was a good dose-response relationship after oral different doses of insulin-loaded microspheres in diabetic rats, the relative pharmacological bioavailability were 41.22% , 37.32% and 35.37% respectively.
针对胰岛素口服给药面临的主要问题，本文采用脉冲电场微球制备工艺，以海藻酸盐、壳聚糖为载体材料，开展了载胰岛素口服微球的制备工艺研究。采用真空冷冻干燥技术对载药微球干燥处理后，进行了微球口服给药的药效学、药动学、以及药理相对生物利用度的研究。主要研究结果如下：
1．采用脉冲电场工艺制备了载胰岛素壳聚糖-海藻酸钙微球。以微球粒径和球形度为指标，采用正交实验设计法优化工艺条件；利用倒置生物显微镜和扫描电镜测定微球粒径、观察微球的表面形态以及内部结构。采用考马斯亮蓝法测定微球中胰岛素药物的包封率。结果表明，锐孔孔径是影响微球粒径的最显著的因素。优化工艺条件为450μm锐孔直径、2cm液面距、10mg／mL海藻酸钠浓度、8mL／h推进速度、金属皿作凝胶浴容器。
2．采用真空冷冻干燥法制备了载胰岛素微球的冻干剂，考察了微球冷冻干燥前后粒径变化以及微球中胰岛素相对活性的变化。结果表明，冻干对微球中药物的活性影响较小，但能够大大减小微球的粒径，冻干后，微球平均粒径由123μm减小为48μm，粒径减小率为61％。
3．建立糖尿病动物模型，为进行微球口服的药效学和药动学研究提供稳定可靠的动物模型。采用体重为18～22g的昆明种小白鼠腹腔注射200mg／kg四氧嘧啶生理盐水溶液，建立了类似人类Ⅰ型糖尿病的动物模型。
4．胰岛素微球口服给药的药效学实验表明：微球不仅对正常小鼠有降血糖作用，而且对四氧嘧啶致糖尿病小鼠也有降血糖作用，且血糖降低水平与剂量之间存在一定量效的关系。正常小鼠口服平均粒径48μm载胰岛素微球，6h时血糖降低率最大，10h内具有降血糖作用。给予正常小鼠不同剂量的胰岛素微球，口服3.2IU／kg剂量6h时使小鼠血糖下降27.2％，6IU／kg剂量使小鼠血糖降低40％。对于糖尿病小鼠而言，降血糖作用更为明显。口服粒径为48μm、剂量为4.8IU／kg载胰岛素微球，在6h～10h内维持血糖值处于较低水平，6h达到最低值，10h血糖值恢复为原有的46.01％，整个降血糖过程缓慢而有效。与皮下注射给药方式相比，口服胰岛素的药理相对生物利用度为41.20％。糖尿病小鼠口服高(4.8IU／kg)、中(3.6IU／kg)、低(2.4IU／kg)三个剂量的载胰岛素微球，高剂量的降血糖作用明显优于中低剂量，最大降血糖百分率分别为57.31％、40.16％、23.87％。
5．药动学实验表明：采用糖尿病小鼠模型作为研究对象，口服4.8IU／kg载胰岛素微球，血清胰岛素呈现出缓慢而平稳的上升过程，血清胰岛素含量在4h为最高值。与皮下注射给药方式相比，口服胰岛素的药理相对生物利用度为41.22％。对糖尿病小鼠口服不同剂量载胰岛素微球，高、中、低三种剂量均体现出良好的升高血清胰岛素含量的作用，体现良好的量效关系。药理相对生物利用度分别为41.22％，37.32％，35.37％。
At present, injection is the only means of administration of insulin in clinic. The problem is the short half-life and repeated injection administration, resulting physical and spiritual burdens for long-term or life-long insulin-dependent patients. Therefore, the development of non-injection administration of insulin is very important. Oral delivery is the perfect administration of the non-injection because of security and higher advantage of the compliance of patients. However, insulin, must first be broken down by digestive enzyme and loss of efficacy. Meanwhile, as amphoteric element, insulin with hydrophilic properties are not absorbed by intestinal epithelial barrier.
In order to sovle problems of oral insulin delivery, impulsive electrostatic technique was used with chitosan and sodium alginate as microsphere material, studies on preparation of insulin microspheres.The pharmacodynamic, pharmacokinetic and the relative pharmacological bioavailability of oral freeze-drying insulin-loaded Alginate/ chitosan microspheres, were mainly studied in this article.The research were made as follow:
1. Insulin-loaded alginate/chitosan microspheres were preparede by impulsive electrostatic technique. The interrelated factors influencing the diameter and sphericity were studied through orthogonal experiments,and finally the statistic analysis made sure the optimum conditions to prepare microspheres. The scanning electron microscope and biological inverted microscope were used to observe diameter, the surface morphology and internal structure of microspheres. The encapsulation efficiency of insulin-loaded microspheres were determined by CBBG.The results showed that the diameter of needle was the most significant factor to the diameter of microspheres. The optimum conditions for the least diameter of microspheres were 450μm diameter of needle, 2cm from needle tips to the gelation surface, 10mg/mL alginate concentration,8mL/h speed of flowing-liquid and metal containers.
2. Microspheres were prepared by Vacuum freeze-drying, then inspected the diameter of microsphere before freeze-drying, and changes in the relative activity of insulin. The results showed that the freeze-dried microspheres were slight effect on the drug activity, but the diameter of the microspheres were greatly reduced. The average diameter of microspheres decreased from 123 urn to 48 urn , the rate of decreasing is 61%.
3. Diabetic animal model provided stable and reliable animal model for following oral pharmacokinetic and pharmacodynamic. In this paper, the weight of 18～22g Kunming-rats were subcutaneous injected 200mg/kg alloxan-saline solution, to establish a similar animal model of human type 1 diabetes.
4. Studies of pharmacodynamic on insulin-loaded microspheres showed that microspheres had hypoglycemic effect not only on the normal rats, but also on alloxan-induced diabetic rats, Also there was a certain relationship between dose and hypoglycemic effect. Hypoglycemic effect showed the maximum rate at 6h, and maintained within 10h after oral 48μm microspheres on normal rats. Blood glucose level decreased to 27.2% , 40% respectively after 6h with oral 3.2IU/kg, 6IU/kg on normal rats. For diabetic rats, the hypoglycemic effect became more apparent, and blood glucose level maintained lower during 6～10h after oral 4.8IU/kg microspheres.The lowest level was at 6h, then cameback to 46.01% of original level .The whole process showed a steady and effective hepoglycemic effect after oral. Compared to the administration of subcutaneous injection method, the relative pharmacological bioavailability of oral insulin-loaded microspheres reached 41.20%.The hypoglycemic effect of the high dose (4.8IU/kg)was better than lower dose after oral different dose of insulin-loaded microspheres to diabetic rats,, the largest of hypoglycemic effect reached was 57.31%, 40.16%, 23.87%.
5. Study of pharmacokinetic showed : With diabetic rats as animal model, the serum insulin level were determined by Radioimmunoassay(RIA) after oral insulin-loaded microspheres. the serum insulin level showed a slow and steady increase after oral 4.8IU/kg insulin-loaded microspheres, and the serum insulin level was the highest value at 4h. Compared to the administration of subcutaneous injection method, the relative pharmacological bioavailability of oral insulin-loaded microsphere reached 41.22%. It was a good dose-response relationship after oral different doses of insulin-loaded microspheres in diabetic rats, the relative pharmacological bioavailability were 41.22% , 37.32% and 35.37% respectively.
引文
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