【作者】 陈睿；

【导师】 郭瑞臣；

【作者基本信息】 山东大学 ， 药理学， 2013， 硕士

【摘要】 目的：评价国产仿制的硝苯地平缓释片和普通片与拜耳硝苯地平缓释片(原研药)的一致性,包括体外一致性(硝苯地平特殊杂质、溶出度、晶型和含量测定)评价和体内一致性(体内生物等效性)评价。考察国产硝苯地平口服制剂质量,探讨影响硝苯地平口服制剂质量的因素,为药品的生产及研发提供实验依据。方法：1硝苯地平特殊杂质检查：建立专属性强,灵敏度高的HPLC-MS法,色谱柱为Phenomenex C18(2)柱(150×4.6mm,5μm),流动相为乙腈：水(55：45,v/v),等梯度洗脱,流速为0.8mL·min-1,进样量10μL。在规定条件下同时测定硝苯地平、硝苯地平杂质I和硝苯地平杂质II的浓度,计算硝苯地平口服制剂的杂质含量。2硝苯地平不同制剂溶出度测定：建立专属性强的HPLC法,色谱柱为Phenomenex C18(2)柱(150x4.6mm,5『m),流动相为乙腈：0.3%三乙胺(60：40,v/v),检测波长为333nm,流速为0.8mL·min-1,进样量为20μL。在规定条件下测定三种硝苯地平口服制剂的溶出度浓度,并评价其溶出相似度。3硝苯地平制剂及原料药晶型测定：采用X-射线衍射法及差示扫描量热法分别测定三种硝苯地平口服制剂及国产硝苯地平缓释片剂原料药晶型。4硝苯地平口服制剂含量测定：采用HPLC-MS/MS法测定三种硝苯地平口服制剂的含量及标示含量百分含量。色谱柱为Diamonsil C18(2)柱(150x4.6mm,5μm);流动相为乙腈:5mmol·L-1乙酸铵(60：40,v/v)；流速为0.8mL·min-1;进样量为10μL；柱温为25℃。5硝苯地平人血浆浓度测定方法的建立与评价：建立HPLC-MS/MS测定人体血浆中硝苯地平方法,色谱柱为Diamonsil C18(2)柱(150×4.6mm,5μm);流动相为乙腈:5mmol·L-1乙酸铵(60：40,v/v)；流速为0.8mL·min-1;进样量为10μL；柱温为25℃；离子源为ESI离子源,负离子模式检测；多级反应监测(MRM)模式,345.1→222.2(硝苯地平),150.1→107.1(对乙酰氮基酚,IS), EMV为400V。碎片电压均为100V,碰撞能分别为5eV和20eV。考察方法的专一性、灵敏度、线性、精密度、准确度、稳定性和稀释效应。6.硝苯地平口服制剂人体生物等效性研究：20名男性健康志愿者随机分为两组,双交叉双周期单次口服拜耳缓释片和国产硝苯地平缓释片20mg,第三周期,20名志愿者均服用硝苯地平国产普通片20mg,于设定时间采血0.5mL,经液-液萃取法处理,采用HPLC-MS/MS法分析血浆中硝苯地平浓度,采用DAS2.0软件分析服用国产缓释片组与拜耳缓释片组、服用国产普通片组与拜耳缓释片组的生物等效性。结果：1.三种硝苯地平片剂特殊杂质检查均符合2010版中国药典质量要求,原研药杂质含量明显小于国产硝苯地平缓释片与普通片剂。2.三种片剂体外溶出度考察结果显示,两种缓释片均有明显缓释效果,国产缓释片释放特点接近控释片,拜耳缓释片释放特点为典型缓释片。硝苯地平普通片剂在1h累计溶出百分含量为82.19%,符合普通制剂溶出度规定。3.三种硝苯地平口服制剂晶型结果显示,原料药与国产硝苯地平缓释片和拜耳缓释片主要衍射峰角度相似,提示国产硝苯地平与原研药的晶型相似。国产硝苯地平普通片剂与其他两种片剂的主要衍射峰角度差别较大,晶型存在差异。DSC法分析晶型结果与X-射线粉末衍射结果一致。4.三种片剂的含量测定结果显示,标示量百分含量均在90%-110%范围内,符合中国药典2010版的要求。5.本课题建立测定人血浆硝苯地平浓度的HPLC-MS/MS法灵敏度高,选择性强,精密度、准确度和稳定性良好,适用于硝苯地平人体内血药浓度检测。6.20名健康志愿者分别单次口服拜耳缓释片(原研药)、国产缓释片和国产普通片后,主要药代动力学参数：t1/2分别为(6.70±2.22)h、(9.63±8.12)h和(5.43±1.31)h,Tmax分别为(2.80±0.50)h、(2.70±0.47)h和(1.87±1.21)h,max分别为(76.69±19.50) ng·mL-1、(75.56±17.60)ng·mL-1和(277.22±93.26) ng·mL-1, AUCo-36分别为(534.13±159.94) ng·mL-1·h、(526.68±154.83)ng·mL-1·h和(1292±602.86) ng·mL-1-h,AUC0-∞分别为(551.68±174.34) ng·mL-1·h、(610.83±353.90) ng·mL-1·h和(1303.71±611.54)ng·mL-1·h。国产缓释片和拜耳缓释片具有体内生物等效性,拜耳缓释片和国产普通片体内生物不等效,国产普通片的相对生物利用度过高。结论：硝苯地平两种缓释片剂体外一致性评价结果显示,杂质检查及含量测定均符合2010版中国药典相关规定,溶出度和晶型相似,与体内生物等效性结果一致。硝苯地平普通片体外结果显示,杂质检查、溶出度测定和含量测定均符合2010版中国药典相关规定,但晶型与拜耳缓释片差异较大,体内生物不等效,提示硝苯地平的晶型与剂型影响硝苯地平体内动力过程的重要因素。更多还原

【Abstract】 Objective:The purpose of this work was to study the consistency of domestic nifedipine sustained release tablets and the ordinary nifedipine tablets with the Bayer nifedipine sustained release tablets (original drug), the study was composed of the consistency in vitro:nifedipine impurities, dissolution, crystal forms, content, the consistency in vivo:bioequivalence and bioavailability, the evaluation of the gap between domestic nifedipine tablets and the original nifedipine tablets, the exploration of the main indicators which value the consistency of generics and the original drug, to the study to provide experimental evidence for the production and development of drugs.Method:1. nifedipine impurities were checked:a specificity, high sensitivity HPLC-MS analysis method was established for checking nifedipine impurities Ⅰ and Ⅱ, which were separated on a Phenomenex C18(2) column (150×4.6mm,5μm) at column temperature of25℃by acetonitrile:water (55:45, v/v), and the flow rate was0.8mL·min-1, and10μL solvent was injected into HPLC for analysis. The impurities of these three nifedipine tablets were analysed and determinationed by the established method.2. Dissolution of domestic nifedipine sustained release tablets and the Bayer nifedipine sustained release tablets during24h, and ordinary nifedipine tablets during2h were detected by HPLC method. The chromatographic conditions were:column was Phenomenex C18(2) column (150x4.6mm,5μm), mobile phase was acetonitrile:0.3%triethylamine (60:40, v/v), detection wavelength was333nm, the flow rate was0.8mL·min-1, and20μL was injected into HPLC for analysis.3. Crystal forms of bulk drug and the three tablets were studied by X-ray diffraction method and differential scanning calorimetry method to compare the different crystal forms of the four substances. 4. Content determination of three nifedipine oral formulations were studied, which the chromatographic conditions were described as below. The analytical column was the Diamonsil C18(2) column (150×4.6mm ID,5μm), mobile phase was acetonitrile:water with5mmol·L-1acetic acid ammonium (60:40, v/v), flow rate was0.8mL·min-1,10μL solvent was injected into the HPLC-MS/MS for analysis at column temperature25℃.5. High performance liquid chromatography methods were developed and validated for the quantification of nifedipine in human plasma. Nifedipine was separated on Diamonsil C18(2) column (150×4.6mm,5μm) by acetonitrile:5mmol·L-1ammonium acetate (60:40, v/v), the flow rate was0.8mL·min-1, and the injection volume was10μL. Mass spectrometer was operated using electrospay ionization (ESI) with negative ionization mode at the following parameters. The voltage of EMV was400V. The fragment voltages were all100V, collision gas with energy of5eV for nifedipine and20eV for acetaminophen. The following ion transitions (m/z) were345.1→222.2for nifedipine and150.1→107.1for acetaminophen. Specificity, sensitivity, linearity, precision, accuracy, stability and dilution effect were validated.6. The bioequivalence of nifedipine oral formulations was studied,20healthy male volunteers were randomly divided into two groups. A single dose, double-cross test was designed in the first and the second period, while20mg domestic nifedipine sustained release tablets or Bayer nifedipine sustained release tablets were took, ordinary nifedipine tablets were took in third period for all volunteers. The blood samples (5mL) were collected at the given time points. The samples were deposed by a liquid-liquid extraction method and10μL was injected into HPLC for analysis. Bioequivalence of groups between administrate domestic nifedipine sustained release tablets and Bayer nifedipine sustained release tablets, ordinary nifedipine tablets and Bayer nifedipine sustained release tablets were analyzed by Drug and statistic software (DAS2.0).Results:1. Nifedipine impurities of three Nifedipine tablets met the quality requirements of2010edition of Chinese Pharmacopoeia, However, the content of the impurities of the original nifedipine tablets were significantly less than of domestic nifedipine sustained release tablets and ordinary nifedipine tablets.2. The results of dissolution showed that the two sustained-release tablets had significantly sustained-release effect, as the release characteristics of domestic nifedipine sustained-release tablets were close to the controlled-release characteristics, Bayer nifedipine sustained-release tablets were typical sustained-release tablets. Cumulative dissolution percentages of the ordinary nifedipine tablets were82.19%in one hour, which met the requirement of dissolution.3. The polymorph test results of three nifedipine oral formulations showed that the positions of the main diffraction peaks of bulk drugs were similar with domestic nifedipine sustained release tablets and Bayer nifedipine sustained release tablets, which indicated that there were similar crystal forms between the domestic nifedipine sustained release tablets and Bayer nifedipine sustained release tablets. However, there were significantly different crystal forms between ordinary nifedipine tablets and other two nifedipine sustained release tablets. The results of DSC and X-ray power diffraction were consistent.4. The content of the three nifedipine tablets met the requirements of the2010edition of Chinese Pharmacopoeia, which the percentage of the labeled amount met the range of90%-110%.5. The established HPLC-MS/MS method was sensitive, selectivity, accurate and stability in the study, which was applied for the determination of nifedipine level in human plasma.6.20healthy volunteers separately took a single oral dose of Bayer nifedipine sustained release tablets, domestic nifedipine sustained release tablets and ordinary nifedipine tablets, the main pharmacokinetic parameters of them were that t1/2were (6.70±2.22) h,(9.63±8.12) h and (5.43±1.31) h; Tmax were (2.80±0.50) h、(2.70±0.47) h and (1.87±1.21) h; Cmax were (76.69±19.50) ng·mL-1,(75.56±17.60) ng·mL-1and (277.22±93.26) ng·mL-1; AUC0-36were (534.13±159.94) ng·mL-1·h,(526.68±154.83) ng·mL-1·h and (1292±602.86) ng·mL-1·h; AUC0-∞were (551.68±174.34) ng·mL-1·h,(610.83±353.90) ng·mL-1·h and (1303.71±611.54) ng·mL-1·h, separately. The two kinds of nifedipine sustained release tablets were bioequivalent. Bayer sustained release nifedipine tablets and ordinary nifedipine tablets were not bioequivalent, the relative bioavailability of ordinary nifedipine tablets was higher.Conclusion:The results of consistency of two nifedipine sustained-release tablets in vitro showed that nifedipine impurity and content all met the relevant provisions of the2010Chinese Pharmacopoeia. Both of the results of dissolutions and crystal forms were similar, and were bioequivalent in vivo. The results of consistency of domestic nifedipine tablets in vitro showed that nifedipine impurities and dissolution determination and determination all met the relevant provisions of the2010Chinese Pharmacopoeia, while the crystal forms were different with the other two sustained-release tablets, and were not bioequivalent in vivo, which indicated that polymorphs and formulations of nifedipine were the critical factors for the nifedipine dynamics in vivo.更多还原