胰岛素的聚乙二醇化和聚乙二醇化胰岛素降血糖作用的研究

【作者】 张敏；

【导师】 印春华；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 以胰岛素为模型药物,研究了胰岛素的PEG化以及PEG化胰岛素的物理化学性质和生物活性,并以互穿网络超多孔水凝胶（SPH-IPN）为药物载体,研究了口服PEG化胰岛素的降血糖作用。1 PEG的活化采用N-羟基琥珀酰亚胺活化酯法对单甲氧基聚乙二醇（MPEG5000）进行活化,分别制备了单甲氧基聚乙二醇琥珀酸琥珀酰亚胺酯（MPEG5000-SS）和单甲氧基聚乙二醇羧甲基琥珀酰亚胺酯（MPEG5000-SCM）两种活性酯衍生物。比色法测定两种衍生物的活化度均超过90%。MPEG-SS和MPEG-SCM的红外光谱都具有以下特征峰:1740 cm^-1处为活性酯羰基的伸缩振动峰,1785 cm^-1附近和1813 cm^-1处分别为琥珀酰亚胺分子中两个羰基的伸缩振动峰。核磁共振分析结果表明两种衍生物均具备各自的特征基团。2 PEG化胰岛素的制备采用合成的MPEG5000-SS和MPEG5000-SCM对胰岛素进行随机修饰。以MPEG5000-SS为例,用三硝基苯磺酸（TNBS）比色法分析PEG化胰岛素混合物的平均修饰程度,SDS-PAGE分析联结不同数目PEG链的胰岛素组分分布,以平均修饰度和混合物的组分分布情况为考察指标,分别研究了反应溶剂、原料配比和反应时间对胰岛素PEG化合成的影响,确定了本文胰岛素PEG化的合成工艺:将适量胰岛素溶解于DMF/0.1 mol·l^-1pH 7.4硼酸盐缓冲液（60:40,v/v）混合溶剂中,制得浓度在1-10 mg·ml^-1范围内的胰岛素溶液。按照胰岛素/MPEG-NHS活性酯为1:5（mol/mol）的配比投入MPEG-NHS活性酯固体粉末,室温下搅拌反应4 h。加入相当于MPEG5000-NHS活性酯2倍摩尔量的6-氨基正己酸溶液终止反应。反应混合液对0.01%的NH₄HCO₃溶液进行彻底的透析,冷冻干燥,制得PEG化胰岛素混合物。采用Sephadex G75凝胶过滤色谱,Protein-Pak^TM SP 8HR阳离子交换色谱和C18反相高效液相色谱分离PEG化胰岛素。结果表明:Sephadex G75凝胶过滤色谱可以有效去除PEG化胰岛素混合物中未反应的胰岛素和小分子杂质,但不能将PEG化胰岛素按修饰度不同而逐一分离。而SP阳离子交换色谱和C18反相高效液相色谱都能够将PEG化胰岛素按修饰度不同而逐一分离。阳离子交换色谱各组分的洗脱顺序为,较高修饰度PEG化胰岛素先于较低修饰度PEG化胰岛素洗脱出来,胰岛素最后洗脱出来。而在C18反相高效液相色谱中,胰岛素最先洗脱出来,而较高修饰度PEG化胰岛素后于较低修饰度PEG化胰岛素洗脱出来,刚好与PEG化胰岛素经阳离子交换色谱分离的洗脱顺序相反。在上述分离工作的基础上,先后采用SP Sepharose阳离子交换色谱和QSepharose阴离子交换色谱分离纯化PEG化胰岛素,得到三种不同修饰度的PEG化胰岛素。三种PEG化胰岛素的纯度经RP-HPLC检测均在90%以上。3 PEG化胰岛素的表征采用非还原SDS-PAGE法和MALDI-TOF MS法测定单,双和三修饰PEG化胰岛素的分子量。SDS-PAGE测出三种PEG化胰岛素的表观分子量分别为11.1,23.5和35.0 KDa,除了单修饰PEG化胰岛素的表观分子量比较接近其实际分子量（10.8 KDa）外,双、三修饰PEG化胰岛素的表观分子量都大于其实际分子量（15.8和20.8 KDa）。MALDI-TOF MS测得的单、双修饰PEG化胰岛素的分子量分别为10.8 KDa和15.9 KDa,与实际分子量相符。MALDI-TOF MS未能检出三修饰PEG化胰岛素的分子量。三种PEG化胰岛素的紫外吸收光谱和胰岛素相比差异较小,在220 nm和275 nm附近均有吸收峰。通过测定275 nm处的吸光度值计算三种PEG化胰岛素溶液的浓度,将PEG化胰岛素的浓度以胰岛素浓度计。PEG化胰岛素的圆二色谱分析表明,胰岛素的PEG化不会对胰岛素的高级结构造成影响。PEG化胰岛素的自缔合受到一定程度的抑制,其自缔合状态随PEG化程度的提高而减少。采用加速振荡实验研究了胰岛素和三种PEG化胰岛素的物理稳定性。在37℃,200 rpm的加速振荡条件下,胰岛素分子发生纤维化反应,在12 h内基本沉淀完全。PEG化胰岛素的纤维化反应受到抑制,1PEG-insulin的物理稳定性至少是胰岛素的17倍。而2PEG-insulin和3PEG-insulin经过加速振荡24天时,溶液中样品含量仍保持在初始值的80%以上,物理稳定性远大于胰岛素和1PEG-insulin。胃蛋白酶、胰蛋白酶和糜蛋白酶对胰岛素和三种PEG化胰岛素的体外酶解试验表明,胰岛素的PEG化能够在一定程度上抑制三种蛋白酶对胰岛素的降解,而且抑制的程度随PEG化程度的提高而增加。4 PEG化胰岛素体内降血糖作用的研究比较了正常小鼠皮下注射(0.5 U·kg^-1)胰岛素和PEG化胰岛素的降血糖作用。胰岛素给药后30 min左右血糖降至低谷,为初始值的26%左右;给药后3 h,血糖回复到初始值的90%以上。1PEG-insulin给药后90 min左右血糖降至低谷,为初始值的52%左右;5 h左右血糖回复到初始水平的90%左右。2PEG-insulin给药后120 min左右血糖降至低谷,为初始值的76%左右;3 h至4 h左右血糖回复到初始水平的90%左右。3PEG-insulin给药后90 min左右血糖降至低谷,仅为初始值的87%左右;2 h内血糖迅速回复到初始血糖水平。以胰岛素为基准,通过比较各给药组降血糖曲线上面积,计算得1PEG-insulin,2PEG-insulin和3PEG-insulin的生物活性分别为天然胰岛素的93%,46%和24%。正常大鼠静脉注射给药降血糖试验结果表明,1PEG-insulin具有明显的降血糖作用,其降血糖作用时间较胰岛素有所延长。2PEG-insulin和3PEG-insulin的活性损失较多,其体内降血糖作用不明显。采用吸附法将1PEG-insulin载入SPH-IPN,载药量达（10.52±0.87）%（wt）。体外释放试验结果表明,1PEG-insulin从SPH-IPN中释放较快,10 min时超过80%的药物被释放。将载有1PEG-insulin的SPH-IPN装于大鼠用普通肠溶胶囊中,大鼠口服给药,产生明显的降血糖作用,8-10 h左右血糖值降至最低,为初始值的65%左右。对照组口服给予胰岛素溶液,1PEG-insulin胶囊或空白SPH-IPN胶囊,均无明显的降血糖作用。以皮下注射(1 U·kg^-1)胰岛素的PBS溶液为对照,计算得大鼠口服(40 U·kg^-1)载1PEG5000-insulin的SPH-IPN胶囊的相对药理生物利用度为5.1±2.0%。胰岛素的PEG化在尽量不影响胰岛素生物活性的前提下,能够抑制胰岛素单体的自缔合,提高胰岛素的物理稳定性和抵抗蛋白酶降解的能力,延长胰岛素体内降血糖作用的时间,结合SPH-IPN具有的抑酶、促渗及黏膜黏附的作用,能够提高胰岛素的口服生物利用度,是一种较有前景的蛋白质多肽类药物口服给药技术。更多还原

【Abstract】 Insulin was chosen as a model drug for the study of PEGylation of protein drugs. The physicochemical properties and bioactivity of PEGylated insulin were studied. And the hypoglycemic effect following the oral administration of mono-PEGylated insulin was studied with superporous hydrogel containing interpenetrating polymer networks（SPH-IPN）as a drug carrier.1 Activation of PEGMonomethoxy-poly（ethylene glycol） 5000（MPEG5000） was activated to produce MPEG-succinimidyl succinate（MPEG-SS） and succinimidyl ester of carboxymethyl MPEG（MPEG-SCM） with activity degree both over 90%.The characteristic absorption band at 1740 cm^-1（ester carbonyl）,1785 cm^-1 and 1813 cm^-1 （carbonyl on succinimidyl） of the infrared spectrum of MPEG-SS and MPEG-SCM indicated the existence of MPEG succinimidyl active ester.The ¹H-NMR analysis demonstrated that each MPEG derivative contained the corresponding characteristic groups.2 Preparation of PEGylated insulinThe resultant MPEG5000-SS and MPEG5000-SCM were used to modify insulin randomly.Taken MPEG5000-SS as an example,effects of reaction solvents,initial molar ratio of MPEG derivative to insulin and reaction time on synthesis of PEGylated insulin were investigated with TNBS spectrometry and SDS-PAGE analysis.And the optimal synthesis scheme was as follows:insulin was dissolved in the solvent of DMF/0.1 M borate buffer pH 7.4（60:40,v/v）,and the concentration of insulin was in the range from 1 mg·ml^-1 to 10 mg·ml^-1.MPEG-NHS active ester powder was added at the ratio of 1:5（mol/mol） of insulin to MPEG-NHS active ester. After stirring at ambient temperature for 4 h,6-Aminocaproic acid about 2 molar times of MPEG-NHS active ester was added to quench the reaction.Then the reaction mixture was dialyzed against 0.01%NH₄HCO₃ solution and lyophilized.Gel filtration chromatography using Sephadex G75,cation exchange chromatography on Protein-Pak^TM SP 8H,C18 reversed-phase high performance liquid chromatography were employed to separate the PEGylated insulin.The results showed that SEC was able to separate PEGylated insulin from intact insulin and impurity,but did not exhibit heterogeneity of MPEG-insulin conjugates or remove the inactive MPEG from the mixture of PEGylated insulin.IEC and RP-HPLC could resolve heterogeneous composition or PEGylated insulin mixture.The elution sequence of PEGylated insulin in IEC was just the opposite in RP-HPLC.On the basis of the above research,SP Sepharose cation exchange chromatography and Q Sepharose anion exchange chromatography were used to separate PEGylated insulin in two steps,and three species of PEGylated insulin, namely mono-,di-,and tri-PEGylated insulin were purified,and the purity were above 90%according to the results of RP-HPLC.3 Characterization of PEGylated insulinThe molecular weights of mono-,di- and tri-PEGylated insulin were determined through SDS-PAGE and MALDI-TOF MS.The apparent molecular weights of mono-, di-,and tri-PEGylated insulin were 11.1,23.5,35.0 KDa through SDS-PAGE,and the apparent molecular weight of mono-PEGylated insulin is closed to its’ calculated molecular weight（10.8 KDa）.The molecular weights of mono-,di-PEGylated insulin determined by MALDI-TOF MS were 10.8 KDa and 15.9 KDa,which were corresponding to the calculated molecular weights of them.The ultraviolet absorption spectra of three species of PEGylated insulin were similar to that of insulin,which had absorption peaks at about 220 nm and 275 nm.So the concentration of PEGylated insulin could be calculated as the concentration of insulin solution that could be determined by measuring OD₂₇₅.The circular dichroic spectra analysis of PEGylated insulin demonstrated that the attachment of PEG to insulin does not alter the tertiary structure of conjugates as compared to native insulin. The self-association of PEGylated insulin was inhibited and the inhibition correlated with the degree of PEGylation.The physical stability of insulin and three species of PEGylated insulin were studied with accelerated shake test under the condition of 200 rpm of shaking speed at 37℃,the fibrillation of mono-PEGylated insulin was inhibited,and it gave at least 17-fold increase of physical stability as compared to insulin while the physical stability of di- and tri-PEGylated insulin were greatly enhanced more than insulin and mono-PEGylated insulin.The in vitro enzymatic degradation experiment showed that the degradation of insulin by pepsin,trypsin and chymotrypsin could be inhibited through PEGylation, and the inhibition correlated with the degree of PEGylation.4 The hypoglycemic effect of PEGylated insulinThe hypoglycemic effect of PEGylated insulin was compared with insulin after subcutaneous injection(0.5 U·kg^-1) in healthy mouse.In the group of insulin,the blood glucose level reduced to 26%of the control value within 30 min then started rising and approach to 90%of the control value within 3 h.In the groups of mono-, di-,and tri-PEGylated insulin,the blood glucose level reduced to 52%,76%,and 87% of the control value within 90min,120min,and 90min,respectively.After that,the blood glucose level rose to 90%of the control value within 5 h in mono-PEGylated insulin group that is a little slower than di-（3-4 h） and tri-（2 h） PEGylated insulin. Using insulin as a standard,the hypoglycemic effects of mono-PEGylated insulin, di-PEGylated insulin and tri-PEGylated insulin were calculated by comparing the AAC of serum glucose level-time course curve between different groups,which were 93%,46%and 24%of native insulin respectively.The hypoglycemic effect of intravenous administration of insulin and three species of PEGylated insulin in healthy rats showed that mono-PEGylated insulin had an evident hypoglycemic effect and a longer hypoglycemic time comparing to insulin.The activity of di-PEGylated insulin and tri-PEGylated insulin had a great loss and their hypoglycemic effects in vivo were not obvious.A drug loading of（10.52±0.87）%（wt） was achieved by loading the SPH-IPN with mono-PEGylated insulin by adsorption.The result of drug release experiment in vitro showed that mono-PEGylated insulin was released from the SPH-IPN rapidly and the amount of released drug exceeded 80%in 10 min.The enteric-coated capsules for rat were filled with mono-PEGylated insulin loaded SPH-IPN.After oral administration of these capsules for rats,obvious hypoglycemic effect was observed.The blood glucose level declined to the nadir in 8-10 h that was approximately 65%of the initial value.The control groups were orally administered with insulin solution,enteric-coated capsules filled with mono-PEGylated insulin or enteric-coated capsules filled with SPH-IPN respectively and notable hypoglycemic effect failed to be found out in these condition.And a relative pharmacological availability（PA） of 5.1±2.0%was achieved after oral administration of mono-PEGylated insulin loaded SPH-IPN compared with subcutaneous administration of insulin solution.PEGylation of insulin could inhibit the self-association tendency of insulin,and increase the physical stability as well as resistance to proteolysis while retaining most bioactivity of native insulin.And with the functions of enzyme inhibition,permeation enhancing and adhesion to mucous membrane,SPH-IPN could increase the pharmacological availability of PEGylated insulin after oral administration. Combination of PEGylation and SPH-IPN would be a promising technique for oral delivery of protein and peptide drugs.更多还原