【作者】 陶颖；

【导师】 金拓；

【作者基本信息】 上海交通大学 ， 药剂学， 2016， 硕士

【摘要】 重组促红细胞生成素(EPO)是一种多效细胞因子,对于肾衰性贫血、心衰性供氧不足、缺血造成的心脏损伤等具有治疗或修复作用。长效缓释注射能够降低EPO的注射频率以及降低EPO的血药浓度峰值,但也面临着EPO聚集、变性、产生有害免疫反应以及缓释制剂失败造成严重突释的困难。过去数十年来,EPO的长效缓释微球剂型的研究曾多有报道,但上述问题没有得到克服,相关产品至今没有问世。针对蛋白药物极其脆弱易变的高级结构,本实验室开发了一系列针对生物大分子的药物输送系统,诸如水相-水相乳化蛋白预处理法、膜乳化沉降微球制备法、相导向蛋白微包封法等一系列新颖而实用的技术,全面而系统地解决了上述难题。EPO的缓释制剂涉及到对EPO自然构象的保护、有害抗体的预防、以及EPO的突释现象的解决。本课题在实验室已有的微球制剂的基础上,针对EPO治疗心衰性供氧不足这一新适应症,开发了接近零级释放的微球制剂,保护蛋白药物的自然形态的同时,维持体内体外小剂量恒定释放的要求。本研究首先采用高效液相色谱法考察了EPO在柠檬酸缓冲液中的稳定性,检测了EPO柠檬酸缓冲液在不同时间不同温度条件下的含量变化,确定了p H6.8的柠檬酸缓冲液可以作为EPO的释放介质,且至少保存EPO达半个月之久。接下来进行EPO微球制剂的处方摸索。先利用低温冷冻相分离法制备了蛋白多糖颗粒来保护蛋白,防止其在包封或者释放过程中变性失活。再将EPO蛋白多糖颗粒包封进PLGA微球中,通过调节微球制备材料的分子量以及释放添加剂的含量,筛选出拥有最佳体外释放曲线的处方。EPO微球制剂的处方确定后,采用SPG膜乳化沉降法制备微球,考察了不同压力不同膜孔径下制备的微球粒径大小及均一性,优化了SPG膜乳化法的制备工艺,提高微球粒径均一性,排除小粒径的微球对释放行为的干扰。此外,我们采取了分段控释技术解决微球三段式释放的问题,即对于前期的突释,采用回温实验,利用PLGA的自身修复功能关闭微球表面孔洞,减少表面药物残留而引起的突释。对于后期释放不完全的特点,通过添加释放调节剂氢氧化镁并调节其含量来获得平稳的释放曲线。经过处方优化包括释放调节剂的干预,回温处理以及SPG膜乳化法制备的微球,体外释放曲线显示突释小,累积释放达80%以上,且持续释放达30天。大鼠药动学的考察结果显示,EPO微球可以在大鼠体内维持释放达1个月。更多还原

【Abstract】 Recombinant erythropoietin is a kind of pleiotropic cytokine to treat anemia caused by renal failure or heart failure,cardiac trauma by anemia and so on.Sustained-release therapeutics can reduce the frequency of injection and decrease the concentration peak.However,it may bring the problem of aggregation,denature,immunoreaction and the risk of failure of sustained release preparation.In the past decades,there were many research about EPO-loaded microspheres.But none of the problems have been conquered.None of the products have been launched in the market successfully yet.Our lab have developed some delivery systems of biomolecules to protect the advanced structure of proteins which is fragile and changeable.A lot of innovative and practical technologies have been studied by us to solve the problems comprehensively,for example,aqueous-aqueous emulsion method,SPG membrane emulsification method,phase orientation microencapsulation method and so on.The sustained release preparation of EPO can protect their natural structure,prevent adverse antibodies and reduce the burst release.On the basis of the technologies we had,long-acting sustained-release microspheres loading erythropoietin with closely zero-release behavior have been studied in this article to treat the new indication,heart failure by insufficient of oxygen.High performance liquid chromatography has been used to study the stability of EPO in citric acid solution.As a release medium,citric acid can protect EPO at least for half a month in 4℃,25℃,and 37℃.Dextran particles loaded with EPO were prepared by phase separation method before encapsulated into PLGA microspheres in case of denature during encapsulation and release.A better prescription was selected according to the release curve.Then SPG membrane method was used to produce uniform and spherical microspheres without small-sized particles which would have adverse effect on release kinetics.Some controlled release technologies were applied to solve the particular problems of microspheres’ release.In order to decrease the burst release,annealing heal was used to make the microspheres smooth so as to make the pores in the surface of the microspheres closed.The theory of annealing is that PLGA can restore by itself when the temperature gets its Tg.To modify the incomplete release of microspheres especially in later period,magnesium hydroxide was added into the prescription to neutralize the degradation products of PLGA and the content was studied to make the release more sustained.Formulation optimization including magnesium hydroxide,annealing combined with SPG membrane method,long-acting sustained-release microspheres loading erythropoietin with closely zero-release behavior can be achieved.In the last part of the thesis,the pharmacokinetic was studied by mice with subcutaneous injection.An in-vivo release curve with low initial burst,smoothly sustained released over 30 days and released more than 80% of the total loading amount.更多还原