基于含Carbopol～（？）的超多孔水凝胶复合物（SPHCc）载体的胰岛素新型口服给药系统研究

【作者】 唐翠；

【导师】 裴元英； 印春华；

【作者基本信息】 复旦大学 ， 药剂学， 2005， 博士

【摘要】 本文研究了新型给药载体—含Carbopol^?的超多孔水凝胶复合物（SPHCc）,并以胰岛素为模型药物研究基于该给药载体的蛋白质多肽类药物的口服给药系统及其胃肠道吸收机理。 1 胰岛素的胃肠道稳定性 根据圆二色谱图选择合适浓度的胰岛素溶液,考察不同pH值和蛋白酶（胃蛋白酶、胰蛋白酶、α-糜蛋白酶、胃肠道内容物中的酶和小肠黏膜中的酶）对胰岛素稳定性的影响。结果表明,当胰岛素溶液的浓度为100μg·ml^-1时,其主要以单体形式存在。在胃和小肠的生理pH值的条件下,3h内胰岛素的含量和降血糖作用基本不变。在各种酶作用下,胰岛素均发生降解,含量和降血糖效果均随作用时间的延长而下降。其中胃蛋白酶、胰蛋白酶、α-糜蛋白酶、胃肠道内容物中的酶和细胞液中的酶对胰岛素的降解作用较强,而小肠黏膜的刷状缘膜囊中的酶对胰岛素的降解作用较弱。降解产物的色谱行为不同,表明各种酶对胰岛素的作用位点不同。胰岛素降解后,含量和降血糖效果有相关性,说明胰岛素的酶解产物无生物活性。 2 胰岛素的小肠、结肠和Caco-2细胞的吸收和转运 采用在体肠回流法和肠襻法,研究胰岛素经小肠不同部位及结肠的吸收;采用Valia-Chien扩散池法研究胰岛素经小肠不同部位及结肠的渗透;采用Caco-2细胞模型研究胰岛素AP→BL和BL→AP方向的转运及转运途径。结果表明,大鼠的降血糖效果为结肠>回肠>空肠>十二指肠。离体各肠段的表观渗透系数为回肠>空肠>结肠>十二指肠。离体组织渗透和在体动物吸收试验结果存在差异。综合各试验结果,确定小肠后段为口服胰岛素较适宜的给药吸收部位。胰岛素通过Caeo-2细胞单层的表观渗透系数高于十二指肠,但低于空肠和回肠。AP→BL和BL→AP方向的转运无显著性差异,表明P-糖蛋白可能不参与胰岛素的转运。在Caco-2细胞单层中,FITC-胰岛素分布在细胞间,确证胰岛素同其他亲水性大分子一样,主要以胞间途径进行转运。 3 含Carbopol^?的超多孔水凝胶复合物的制备和表征 考察丙烯酸溶液的pH值、丙烯酸和丙烯酰胺溶液的比例、引发剂的浓度及Carbopol^?和水的用量对SPHCc制备的影响。采用SEM、密度和溶胀比等表征SPHCc。结果表明,采用溶液聚合法,以APS和TEMED为引发体系,调节丙烯酸溶液的pH值为5.0、丙烯酸和丙烯酰胺溶液的体积比为2:3、TEMED的浓度为16.7%及合适的Carbopol^?和水的用量,得到均相的SPHCc。SEM表明SPHCc含有大量的相互连接的孔隙。SPHCc的密度较小,能快速溶胀且溶胀比较高。更多还原

【Abstract】 Carbopol?-containing superporous hydrogel composites （SPHCc） as novel vehicles for drug administration were investigated. Insulin being used as model drug, the peroral delivery systems based on SPHCc for protein and peptide drugs and their absorption mechanism in the GI tract were studied.1 Stability of insulin in gastrointestinal （GI） tractThe concentration of insulin solution was chosen according to the circular dichroism spectra. Effects of various pH and proteolytic enzymes on the stability of insulin, including pepsin, trypsin, chymotrypsin, enzymes in the content of GI tract, as well as in the intestinal mucosa, were investigated. The results demonstrated that most of the insulin stayed in the presence of monomer when the concentration of solution was 100μg.ml^-1. No obvious change of the contents and hypoglycemic effects of insulin took place within 3h in the physiological pH environment of GI tract. Insulin was degraded when incubated with any kind of proteolytic enzymes, thus the contents and hypoglycemic effects reduced with the increase of the incubation time. Insulin was strongly degraded by pepsin, trypsin, chymotrypsin, proteases in the gastrointestinal lumen, and enzymes in the cytosol in comparison with weak degradation by proteases in the brush border membrane vesicles. The different chromatographic behavior of degraded products revealed that various proteolytic enzymes cleaved insulin molecule at different sites. The correlation between the contents and hypoglycemic effects of degraded products by the proteases primarily indicated that they were loss of bioactivities.2 Absorption and transport of insulin through the intestine, colon and Caco-2 cell monolayersWith the in situ perfusion and closed loop method, the absorption of insulin in the duodenum, jejunum, ileum, and colon was studied. With the use of the Valia-Chien diffusion chambers, the permeability of insulin across various intestinal sites and colon was investigated. Caco-2 cell monolayer model being used, the transport of insulin between AP to BL and BL to AP and transport pathway were investigated. The order of hypoglycemic effects was colon > ileum > jejunum > duodenum in in vivo absorption test. The rank of in vitro apparent permeability coefficients was ileum > jejunum > colon >duodenum. Under the grounds of abovementioned results, the distal intestine may be an advantageous region for oral delivery of insulin. The apparent permeability coefficient of insulin across Caco-2cell monolayer model was higher than that in the duodenum and lower than that in the jejunum and ileum. The transport of insulin between AP to BL and BL to AP showed no significant difference, which indicated that the p-glycoprotein may be not involve in the insulin transport. The FITC-insulin being dispersed in the paracellular localization confirmed that like other hydrophilic macromolecules, the transport of insulin was mainly in the paracellular route.3 Preparation and characterization of SPHCcThe effects of pH of acrylic acid solution, acrylic acid/acrylamide ratio, concentration of initiator, and amount of Carbopol? and water on the preparation of Carbopol?-containing superporous hydrogel composites （SPHCc） were respectively investigated. SPHCc were characterized by SEM, density, and swelling ratio. The results demonstrated that the pH 5.0 of acrylic acid solution, 2:3 of the acrylic acid/acrylamide ratio （v/v）, 16.7% of TEMED, and suitable amount of Carbopol? and water help to obtain homogeneous SPHCc from free radical copolymerization using APS and TEMED as initiator system. It was approved by SEM that the SPHCc possessed lots of pores interconnected with each other. The low density SPHCc could swell rapidly and possess higher swelling ratio.4 Toxicity of SPHCcThe acute toxicity of SPHCc and the damage of intestinal mucosa and Caco-2 cell monolayer after application of the SPHCc were studied. The maximum tolerance amount was high. The sections of the intestinal mucosa demonstrated that the rat intestinal villi of the control and refresh group was intact and that of experiment group was intact as well. The damage after application of SPHCc was little. The cells were able to exclude trypan blue as well as propidium iodide after incubation with SPHCc. The damage of Caco-2 cell monolayer was little5 Swelling kinetics of SPHCc and in vitro drug releaseThe influences of the Carbopol? content in the SPHCc, the pH of swelling medium, the ion strength of swelling medium, and the apparent surface area on the swelling kinetics were respectively studied. The swelling ratios decreased with the increase of the Carbopol? content. As the pH of swelling medium was between 3.0 and 7.4, it had no influence on the swelling ratios. When the pH of swelling medium was lower than 2.0, SPHCc shrank and the swelling ratios reduced with the decrease of pH. When the ion strength was O.OOOlmoH’1 and O.OOlmoH"1, it had no effect on the swelling ratios and the maximum swelling ratios of SPHCc were obtained. Whilethe ion strength of swelling medium surpassed 0.00lmoH"1, the swelling ratio enhanced with the decrease of ion strength. The time to equilibrium swelling ratio was shortened with the increase of ion strength. The effect of apparent surface area on swelling kinetics was insignificant.Insulin was loaded into SPH polymers by the immersion method. The results indicated that the amount loading insulin reached （4.78 + 0.13） % （wt） . The cumulative release amount of insulin from SPHCc with 0.072 of Carbopol?/monomer reached 90% during the first 15min. The FTIR spectra demonstrated no new absorption peak in FTIR spectrum after loading SPHCc with insulin. Insulin did not covalently bind to the SPHCc and interacted only physically with SPHCc.6 Insulin delivery via GI tract with SPHCcThe enteric-coat capsules containing the SPHCc loaded with insulin were administrated via intrajejunum in the anaesthetized rats and via ig in the conscious rats, respectively. In the anaesthetized rats, the hypoglycemic effect was significant and enhanced with the increase of the dose. When the dose was 35u-kg’] and 70ukg"1, the blood glucose level decreased down to about 60% of their initial values between 2h and 4h and 40% of their initial values between 2h and 5h, respectively. Intrajejunal administration of insulin solution and capsules containing only SPHCc as controls did not result in a significant decrease of blood glucose concentrations. In the conscious rats, the hypoglycemic effect was observed. When the dose was 35ukg’’ and 70u-kg"1, the lowest blood glucose level decreased down to about 80% and 60% of their initial values, respectively. Intragastric administration of capsules containing insulin or only SPHCc as controls did not result in a significant hypoglycemic effect.7 Enhancement mechanism of SPHCc for the intestinal absorption of insulinSPHCc had in vitro adhesive capacity and an increase in Carbopol? content gave a rise of adhesive force in every polymer tested. SPHCc with fast swelling mechanically fixed to the absorption sites in the intestine. In the presence of SPHCc, the degradation of insulin decreased significantly. This indicated that SPHCc were able to inhibit to some extent the activity of trypsin and a-chymotrypsin and their inhibition was stronger as compared to SPH. After SPHCc was put in situ closed loops pre-washed with saline, the insulin solution was administrated and the blood glucose levels decresed down to about 30% of their initial values at 30min after更多还原