【作者】 张杰；

【导师】 黄桂华； 李洁；

【作者基本信息】 山东大学 ， 药学（专业学位）， 2015， 硕士

【摘要】 近年来心脑血管疾病发病率逐年攀升,因高致死率和致残率而备受瞩目,给广大患者特别是老年患者带来了极大的恐惧和担忧。诱发心脑血管疾病的主要因素就是血栓,血栓是一种影响全身机能的疾病,分为动脉、静脉和微血管血栓。常用于抗血栓药物有抗血小板药、抗凝血药和溶栓药,其中抑制血小板聚集的药物是临床上预防和治疗血栓的常用药物,这类药物中阿司匹林应用较早,属于经典老药,以其低廉的价格和较好的疗效而受到广大患者的青睐,指南推荐低剂量阿司匹林作为心血管疾病预防的一线用药。阿司匹林在应用过程中常见的副作用是对胃肠道的刺激,长期应用易造成胃溃疡和胃出血,因此,临床上经常采用联合质子泵抑制剂类药物抑制胃酸分泌,避免阿司匹林带来的副作用。但是,分别使用两种单方药物联合应用治疗该疾病时,患者容易出现漏服或缺服现象,用药顺应性较差。因此本课题设计了阿司匹林和新一代质子泵抑制剂埃索美拉唑组成复方制剂,提高患者的顺应性,为临床用药提供更多的选择。本课题首先建立了阿司匹林、埃索美拉唑镁单方肠溶微丸及阿司匹林和埃索美拉唑镁复方肠溶微丸的含量及释放度测定方法；其次采用两种设计方案制备阿司匹林和埃索美拉唑镁复方肠溶微丸胶囊剂,并对处方及工艺进行了系统的筛选与优化；最后通过动物实验,对两种方案研制的复方肠溶微丸胶囊剂体内释药行为进行了研究,通过体内外释药性评价复方微丸胶囊剂的质量和稳定性。1.复方肠溶微丸胶囊剂含量及释放度测定方法的建立采用紫外分光光度法分别测定单方阿司匹林肠溶微丸、埃索美拉唑镁肠溶微丸的含量及释放度；采用高效液相色谱法测定复方肠溶微丸胶囊剂的含量及释放度。通过专属性、线性和范围、精密度、准确度等试验对紫外分光光度法和高效液相色谱法进行了方法学考察。结果表明,分别以蒸馏水和pH6.8 PBS为介质,阿司匹林的线性关系良好、精密度和准确度高,辅料不干扰主药测定；以pH11.0PBS为介质,埃索美拉唑镁的线性关系良好、精密度高、准确度高,辅料不干扰测定。结果证明,所建立的紫外分光光度法分别测定阿司匹林肠溶微丸和埃索美拉唑镁肠溶微丸的含量和释放度方法可行；所建立的高效液相色谱法同时测定复方肠溶微丸胶囊剂中阿司匹林和埃索美拉唑镁两种药物的含量,分离度好,辅料不干扰测定,在pH6.8和pH11.0 PBS两种介质中线性关系良好,精密度高,准确度高,符合复方肠溶微丸胶囊剂含量及释放度的测定要求。2.复方肠溶微丸胶囊剂的制备及质量标准研究本课题设计了两种方案制备复方肠溶微丸胶囊剂,第一种方案是将阿司匹林和埃索美拉唑镁两种药物分别制成肠溶微丸后灌装胶囊；第二种方案是采用层层包衣工艺,将阿司匹林和埃索美拉唑镁两种药物共载于同一微丸中再灌装胶囊。2.1第一种方案首先,通过挤出-滚圆法制备阿司匹林含药丸芯；其次,通过离心造粒法制备空白丸芯,再采用流化床包衣上药法制备埃索美拉唑镁含药微丸；最后,采用流化床包衣法,将两种含药微丸分别外包肠溶层制得肠溶微丸,通过含量测定确定装量,将其灌装于胶囊中即得复方肠溶微丸胶囊剂。采用挤出滚圆法制备阿司匹林含药丸芯,以圆整度、收率和溶出度为评价指标,对丸芯中填充剂、粘合剂和稳定剂的种类与用量等处方因素；筛板孔径、挤出速度、滚圆速度和滚圆时间等工艺因素进行了筛选与优化,通过重现性试验证明处方组成的合理性和制备工艺的稳定性。结果表明,确定的最优处方为：阿司匹林：微晶纤维素：低取代羟丙基纤维素：酒石酸比例为50：50：3：3；最佳制备工艺为：筛板孔径为0.5mm,挤出速度为12Hz,滚圆速度为20Hz,滚圆时间为3min；按最优处方和工艺制备三批微丸,圆整度为0.96±0.01(短径/长径),收率为(96.4±0.66)%,45min溶出度为(85.6±1.1)%,处方和工艺稳定,重现性好。采用离心造粒法制备微晶纤维素空白丸芯,以圆整度和收率做考察指标,对喷液速度、鼓风流量和转盘速度等工艺因素进行了筛选与优化,通过重现性试验证明制备工艺的稳定性。采用流化床包衣技术在空白丸芯外包埃索美拉唑镁药物层和隔离层,以收率和含量均匀度做考察指标,对含药层包衣液中粘合剂的浓度、药物浓度等处方因素和喷液压力、喷液速度、风机频率等工艺因素进行了筛选与优化；以收率做评价指标,对隔离层包衣液中聚合物的浓度、抗粘剂和增塑剂的种类与用量等处方因素和流化床包衣工艺因素进行了筛选与优化。结果表明,微晶纤维素空白丸芯以最佳工艺：喷液速度15rpm,鼓风流量15Hz,底盘转速450rpm,制备三批,收率为81.5±0.78%,圆整度为0.91±0.02；空白丸芯包裹含药层以处方：埃索美拉唑镁7.5%,HPMC E53%,NaHCO35%和工艺：喷液压力0.15MPa,喷液速度1.5-2.0rpm,风机频率35Hz,进行包衣,收率大于90%,含量均匀度RSD<2.0%；隔离层包衣液以处方：HPMC E57.5%,单硬脂酸甘油脂10%,柠檬酸三乙酯20%和工艺：喷液压力0.15MPa,喷液速度1.2rpm,风机频率35Hz,进行包衣,收率大于90%。因此,所制备的埃索美拉唑镁含药微丸,处方组成合理,工艺稳定,重现性好。采用流化床包衣技术分别对两种药物微丸外包肠溶层,以商品化的尤特奇L30D-55水分散体做包衣材料,包衣液处方中添加5%单硬脂酸甘油脂和10%柠檬酸三乙酯。以收率和体外释放度作评价指标,对包衣工艺和肠溶层的厚度进行考察。结果表明,确定的包衣工艺为喷液速度2.0rpm、喷液压力为0.12MPa和风机频率35-40Hz,对阿司匹林含药丸芯外包增重30%的肠溶层,对埃索美拉唑镁含药微丸外包增重40%的肠溶层,能使药物定位于肠道释放,分别制备三批阿司匹林肠溶微丸和埃索美拉唑镁肠溶微丸收率分别为95.5±0.8%和90.6±1.66%,工艺稳定,重现性好。分别测定阿司匹林肠溶微丸和埃索美拉唑镁肠溶微丸的含量,按预定剂量(每粒胶囊中含阿司匹林81mg,埃索美拉唑镁20mg)灌装于1号胶囊中,即得复方肠溶微丸胶囊剂(第一设计方案)。体外释放试验结果表明,肠溶微丸耐酸度好,在酸中药物累积释放百分率均小于5%,在pH6.8 PBS中阿司匹林45min内累积释放百分率为(72.8±2.6)%,埃索美拉唑镁30min内累积释放百分率为(89.8±4.6)%,均符合药典规定的要求,三批胶囊剂不同批次间释放曲线相似因子f2均大于70,处方和工艺重现性好。2.2第二种方案首先,通过挤出-滚圆法制备阿司匹林含药丸芯；然后,采用流化床包衣法,在阿司匹林含药丸芯外包隔离层Ⅰ；再包埃索美拉唑镁药物层；再包隔离层Ⅱ,最后包肠溶层。通过层层包衣工艺将阿司匹林和埃索美拉唑镁共载于同一微丸中,通过含量测定确定装量,灌装于胶囊中即得复方微丸胶囊剂。固定阿司匹林含药丸芯的处方和工艺,以体外释放度作评价指标,首先对隔离层Ⅰ、埃索美拉唑镁药物层、隔离层Ⅱ及肠溶层的处方和工艺进行筛选与优化；然后以最佳处方工艺进行重现性试验；最后采用相似因子f2计算批间释药曲线的相似度,考察处方和工艺的稳定性。结果表明,隔离层Ⅰ的主要成分是丙烯酸树脂和HPMC,其中丙烯酸树脂层增重40%,HPMC层增重10%；埃索美拉唑镁药物层由7.5%埃索美拉唑镁、3%HPMC E5和5%NaHCO3组成；隔离层Ⅱ主要成分是HPMC,其增重为10%；最外层为肠溶层,主要成分是丙烯酸树脂,增重为40%。隔离层Ⅰ和隔离层Ⅱ能充分隔离,保证两种药物的稳定性；最外层肠溶层增重40%,能使药物定位于肠道释放。以最佳处方和工艺重现三批所制得复方肠溶微丸胶囊剂在0.1mol/L盐酸溶液中2h阿司匹林和埃索美拉唑镁的累积释放百分率均小于10%；在pH6.8 PBS中15min阿司匹林和埃索美拉唑镁的累积释放百分率分别为(5.9±1.1)%和(78.5±1.4)%,60min累积释放百分率分别为(77.4±3.3)%,和(83.5±1.9)%,且批间释药曲线相似因子f2均大于50,处方和工艺重现性好。隔离层Ⅰ中采用了肠溶材料丙烯酸树脂,目的是既能充分隔离两种药物,避免相互接触而降解,又能通过包衣膜控制药物释药行为。这种设计形式的复方肠溶微丸胶囊剂中阿司匹林和埃索美拉唑镁存在短暂的释药时滞,埃索美拉唑镁优先快速释放,抑制胃酸分泌,拮抗阿司匹林胃肠道副作用,然后阿司匹林大量释放,发挥抗血栓的作用,释药机制更先进、更合理,能更好的发挥复方制剂协同作用的优势。3.两种方案研制的复方肠溶微丸胶囊剂的体内药动学的研究首先通过标准曲线的建立、专属性试验、精密度试验和回收率试验,建立了高效液相色谱法测定血浆样品中两种药物浓度的方法。然后选择大鼠作为实验动物,分别口服给药后在0.5,1,1.5,2,2.5,3,3.5,4,5,6,8,10,12,24h于大鼠颈静脉窦取血,样品处理后进行血药浓度的测定,分别绘制阿司匹林代谢物水杨酸和埃索美拉唑镁的血药浓度随时间的变化曲线。最后通过DAS2.0处理数据,计算主要药动学参数,对两种形式的复方肠溶微丸胶囊剂的体内释药行为进行比较。结果表明,所建方法线性相关性良好、专属性强、样品测定的精密度高、准确度高,能用于血浆中药物浓度的测定；两种方案制备的复方胶囊剂给药后埃索美拉唑镁的血药浓度达峰时间基本相同,而以第二种方案制备的复方肠溶微丸胶囊剂给药后水杨酸的血药浓度达峰时间比第一种方案的慢一小时。因此可以得出,第二种方案设计的复方肠溶微丸胶囊中阿司匹林在体内释药较慢,更有利于埃索美拉唑镁优先释放抑制胃酸分泌,与体外释药差别一致,这种方案制备的复方肠溶微丸胶囊剂通过控制隔离层Ⅰ和肠溶层的材料和厚度,能够有效的控制药物的释放行为,能更好的发挥复方制剂的配伍优势。综上所述,本课题采用两种方案制备复方肠溶微丸胶囊剂,将阿司匹林与埃索美拉唑镁联用,既能充分发挥阿司匹林抗血栓的临床应用,又能避免其胃肠道的副作用,为临床用药增加了新选择,能提高患者顺应性；制备工艺稳定,重现性好,均能满足两种药物的释药要求,其中采用第二种方案制备的复方肠溶微丸胶囊剂,将两种药物共载于同一微丸中,既避免了两种药物微丸灌装胶囊的复杂灌装工序,又通过适当的控释膜材料,控制两种药物的释药时滞,更好的发挥了复方制剂的优势。相信在目前国内外比较精良的包衣设备和成熟的包衣技术的条件下,本课题具有一定的开发应用前景,为临床用药和复方制剂的开发提供了新选择、新思路。更多还原

【Abstract】 The incidence of cardiovascular disease trends to rise year by year around the world, which have brought great fear and worry to patient due to its high probability of death and disability. Thrombus is one of the major factors to cause cardiovascular disease that could affect our whole body capacity including artery, vein and microvascular thrombosis. Antiplatelet, anticoagulation and thrombolytic agents are commonly used drugs and one of the most classical and widely used antiplatelet agents is aspirin which has advantages of low cost and good effects. Therefore, low-dosage aspirin has been regarded as first-line drug to prevent cardiovascular diseases.However, aspirin has the common side effects that it can stimulate the gastrointestinal tract even cause stomach bleeding after long-term use. Therefore, the proton pump inhibitors are usually chosen to protect gastrointestinal mucosa against side effects brought by aspirin. However, drug combination has some deficiency that patients need take multiple drugs with different dosages and times, which has bad patient compliance. Therefore, the compound preparation of aspirin and esomeprazole magnesium was developed to promote patient compliance and provide more choices for clinic treatment.Firstly, the methods of content assay and in vitro release tests of aspirin (ASA) and esomeprazole magnesium (EMZ-Mg) single enteric-coated pellets and compound enteric-coated pellets were respectively carried out. Secondly, the aspirin and esomeprazole magnesium compound enteric-coated pellet capsules were prepared by two kinds of schemes and the formulation and process were systematically studied. Thirdly, the drugs release in vivo of compound enteric-coated pellet capsules prepared by two kinds of schemes was studied by animal experiments in order to evaluate the quality and stability of compound enteric-coated pellet capsules.1. Establishment of determination method for drug content and release of compound enteric-coated pellet capsulesThe content assay and in vitro release test of aspirin and esomeprazole magnesium single enteric-coated pellets were carried out by UV, and compound enteric-coated pellet capsules was determined by HPLC. The methodology of UV and HPLC spectrophotometry was validated by specificity, linearity and range, precision, accuracy, etc. The results showed that methods for content assay and in vitro release tests of aspirin in water and pH6.8 PBS and esomeprazole magnesium in pH11.0 PBS was specific with no interference of excipients, showing good linearity within the appropriate concentration range, good precision of the reference or test solutions and high accuracy on the basis of recovery determination meeting with the requirements. Therefore, the method of UV spectrophotometry for content assay and in vitro release tests of aspirin and esomeprazole magnesium single enteric-coated pellets were feasible and the method of HPLC spectrophotometry for content assay and in vitro release tests of compound enteric-coated pellet capsules showed good system suitability, linearity, precision and high accuracy in pH6.8 and pH11.0 PBS, meeting with the requirements.2. Preparation and Quality specification study of compound enteric-coated pellet capsulesThe compound enteric-coated pellet capsules were designed with two schemes. The first scheme was that aspirin and esomeprazole magnesium single enteric-coated pellets were prepared respectively and then were filled into hard capsules based on dosages. The second scheme was that aspirin and esomeprazole magnesium compound enteric-coated pellets were prepared by films coating technology and then were filled into hard capsules based on dosages.2.1 The first scheme:The ASA pellets were prepared by extrusion-spheronization equipment and EMZ-Mg were layered on the MCC pellets which were prepared by centrifugal granulation. Then ASA pellets and EMZ-Mg pellets were coated by enteric-coated films to prepare enteric-coated pellets that were filled into hard capsules based on dosages.ASA pellets were prepared by extrusion-spheronization. The roundness, yield and dissolution were employed to select and optimize formulations including filler, adhesive and stabilizer, and process including diameter of sieve plate, extrusion rate, spheronization rate and time. Finally, reproducibility test was conducted to verify the rationality of formulation and stability of process. The results indicated that the optimal formulation included ASA(47%), MCC(47%), L-HPC(3%) and tartaric acid(3%), and the optimal process was 0.5mm of sieve plate,12Hz of extrusion rate and 20Hz of spheronization rate in 3min. Three batches of pellets were prepared by the optimal formulation and process, which performed high roundness that the ratio of long radius and short radius was 0.96±0.01, high yield of (96.4±0.66)% and rapid dissolution above 80% in 45min.MCC pellets were prepared by centrifugal granulation. The roundness and yield were employed to select and optimize the process parameters including rotor rotation speed, slit air flow rate and spray air rate. Then the MCC pellets were coated by EMZ-Mg and isolated layers. The yield and content uniformity were employed to select and optimize formulation and process parameters of drug and isolated layers. The results indicated that the optimal process is:spray air rate is 15rpm, slit air flow rate is 15Hz and rotor rotation speed is 450rpm. Three batches of MCC pellets were prepared by optimal process with high yield of (81.5±0.78)% and roundness of 0.91± 0.02. The drug layer with formulation including EMZ-Mg(7.5%), HPMC E5(3%) and NaHCO3 (5%) was prepared with the process parameters:0.15MPa of spray pressure, 1.5 to 2.0rpm of spray air rate and 35Hz of air flow rate, which performed high yield above 90% and high content uniformity(RSD<2.0%). The isolate layer with formulation including HPMC E5(7.5%), GMS(10%) and TEC(20%) was prepared with process parameters:0.15MPa of spray pressure,1.2rpm of spray air rate and 35Hz of air flow ratewhich performed high yield above 90%. Therefore, the formulation and process of EMZ-Mg pellets were suitable and stable with highly reproducibility.The enteric-coated layers with Eudragit L30D-55 including GMS(5%) and TEC(10%) were coated on ASA and EMZ-Mg pellets. The release profiles in vitro and yield were employed to screen the coating process and thickness of layer. The results indicated that ASA pellets and EMZ-Mg pellets were coated by enteric-coated layers with 30% and 40% weight gain respectively in the process:0.12MPa of spray pressure,2.0rpm of spray air rate and 35 to 40Hz of air flow rate. Three batches of enteric-coated pellets were prepared by optimal formulation and process, drug in which released at intestinal tract and had high yield of (95.5±0.8%) and (90.6±1.66)%.The drug content of ASA and EMZ-Mg enteric-coated pellets were determined to ensure the amount of capsule (size 1) filled corresponding to ASA 81mg and EMZ-Mg 20mg. Three batches of compound enteric-coated pellet capsules were prepared, of which drug release percentage in artificial gastric fluid in 2 h was less than 5%, while drug release percentages of ASA and EMZ-Mg in artificial intestinal fluid in 45min was 72.8% and in 30min was 89.8%. The release profiles of compound enteric-coated pellet capsules were quite well and similarity of which was high with the factor of f2 above 70.2.2 The second scheme:ASA pellets were also prepared by extrusion-spheronization and then isolated layer Ⅰ, drug layer of EMZ-Mg, isolated layer Ⅱ and enteric-coated layer were coated to prepare compound enteric-coated pellet capsules, the amount filled in which was ensured by drug content determination.The formulation and process of ASA pellets were unaltered. The release profiles in vitro were employed to screen the formulations and processes of isolated layer Ⅰ, drug layer, isolated layer Ⅱ and enteric-coated layer. Finally, reproducibility of three batches of compound enteric-coated pellet capsules was evaluated with similarity factor method. The result indicated that isolated layer Ⅰ with 40% weight gain of eudragit and 10% weight gain of HPMC were applied to isolate two drugs; the drug layerconsisted of EMZ-Mg(7.5%), HPMC E5(3%) and NaHCO3(5%); the isolated layer Ⅱ mainly consisted of HPMC; the outermost layer of compound enteric-coated pellets mainly consisted of eudragit with 40% weight gain. This designed scheme not only isolated two drugs sufficiently but also made the drug release at intestinal tract. Three batches of compound enteric-coated pellet capsules were prepared, and drug release percentages of ASA and EMZ-Mg in artificial intestinal fluid was(5.9±1.1)% and (78.5±1.4)% in 15min, and (77.4±3.3)% and (83.5±1.9)% in 60min. The release profile of compound enteric-coated pellet capsules was quite well and similarity factor between two of three batches was above 50, which demonstrated the high stability of formulations and process.Isolated layer Ⅰ with eudragit can not only avoid drugs degradation causing by interaction, but also control drug release profiles. The lag-time between ASA and EMZ-Mg release had an advantage that gastrointestinal tract mucosa was protected by rapid drug release of EMZ-Mg and then ASA released largely to play an efficient role of antithrombosis. This design scheme was more reasonable and advanced, which could exploit the advantages nearly to the full of synergism of compound preparation.3. The pharmacokinetic study of two kinds of compound enteric-coated pellet capsulesFirstly, the methodology of HPLC spectrophotometry which was used to determine the plasma drug concentrations was validated by specificity, linearity and range, precision, accuracy, etc. The capsules were orally administered to the rats and blood samples were collected from jugular vein at 0.5,1,1.5,2,2.5,3,3.5,4,5,6,8, 10,12,24h after administration. The plasma was processed and the drug content was determined by HPLC. The diagrams with time and plasma drug concentration of SA and EMZ-Mg were drawn. And then the pharmacokinetic parameters were calculated by non-compartmental analyses using the software program DAS2.0. The drug release in vivo of two kinds of compound pellet capsules was compared. The result indicated that the method was specific, good linearity within the appropriate concentration range, good precision of the reference or test solutions and high accuracy, meeting with the requirements. After orally administrated, the peak time of EMZ-Mg was same but the peak time of SA was one hour apart between two kinds of compound pellet capsules. Therefore, the drug release of ASA from the second scheme of compound pellet capsules in vivo was slower, which could protect gastrointestinal tract mucosa by EMZ-Mg.In conclusion, two schemes had been applied to prepare compound pellet capsules, which can make ASA play an efficient role of antithrombosis and avoid side effect of stimulating gastrointestinal tract. This compound formulation would increase patient compliance and provide more choices for clinic treatment. It could be transformed into large scale of manufacture because of stabilized fomulations and processes. Especially, the second scheme of compound enteric-coated pellet capsules combined two drugs in one pellet, which can not only avoid the complicated process of capsules filling but also control the lag-time of two drug release to exploit the advantages nearly to the full of synergism of compound preparation. Since the film coating equipment was excellent and the process was proficient, this subject has application prospects to pharmaceuticals industry and clinical applications.更多还原