【作者】 田莉；

【导师】 高晓黎；

【作者基本信息】 新疆医科大学 ， 药理学， 2008， 博士

【摘要】 复方甘草酸苷片(compound glycyrrhizin tablets)是由甘草酸单铵盐(glycyrrhizin,GL),甘氨酸(glycine,GLY),DL-蛋氨酸(DL-methionine,MET)组方的复方制剂,疗效确切,临床应用广泛,在肝病领域主要用于治疗慢性肝病,改善肝功能,在非肝病领域用于过敏性反应(如皮疹、荨麻疹、药物疹)、小儿异位性皮炎、扁平苔藓、系统性红斑狼疮、肾综合征出血热及传染性非典型肺炎(SAS)等。本课题临床前研究已完成复方甘草酸苷片的处方工艺研究,质量标准研究,稳定性考察及家兔体内生物等效性评价等工作,并获得国家食品药品监督管理局的临床批件(批件号:2006L01472)。在此基础上,拟进行复方甘草酸苷片工业化生产的处方工艺优化和质量标准修订,试生产和质量评价,人体生物等效性评价,以及GL的体外代谢研究等。以期通过本复方制剂的开发成功,为慢性肝炎的临床治疗提供国产新制剂;同时,从代谢途径研究复方制剂减毒增效的作用机理,为GL的体内代谢研究和临床安全用药评价奠定实验基础。第一部分、复方甘草酸苷片工业化生产的处方工艺参数优化和质量评价。首次建立了在同一色谱条件下检测三个成分的含量,溶出度和有关物质的HPLC法(PDA检测器),该法简便、灵敏、专属性强,重复性好,可将各破坏条件下的降解产物与主成分峰完全分离;优化了复方甘草酸苷片工业化生产的处方工艺参数,完成了试生产;深入进行了有关物质研究,三批试生产产品中的有关物质与原料药,加速试验放置6个月样品以及市售产品美能进行对比,经统计学分析,均有P>0.05,说明制剂过程中和加速放置6个月过程中没有使药物间、药物与辅料间产生明显的未知物,与美能相比也未增加新的有关物质,为有关物质限度的确定提供了翔实的依据;按临床批件要求和2005版《中国药典》规范修订质量标准,与日本进口产品美能(Stronger Neo-minophagen C,SNMC)的质量标准相比,分析方法更具先进性和可靠性,提升了质量标准,可有效控制复方甘草酸苷片的质量;对试生产产品进行了质量评价和稳定性考察,结果表明,复方甘草酸苷片工业化生产的处方合理,工艺稳定。第二部分、按《药品注册管理办法》和临床研究规范的有关要求,评价复方甘草酸苷片在健康人体的生物等效性。采用液相色谱-串联质谱法(LC/MS/MS)测定血浆中GL的代谢产物甘草次酸(Glycyrrhetinic Acid; GA)的浓度,美能为参比制剂,22名健康男性受试者自身交叉单剂量口服治疗剂量试验制剂与参比制剂,血浆样品经液-液萃取,内标为人参皂苷Rh2,经方法确证表明建立的检测方法快速,灵敏、重现性好、专属性强,内源性物质不干扰待测物的测定,样品前处理简便。采用DAS2.0药动学统计软件计算药动学参数,受试制剂和参比制剂的主要药动学参数为:AUC0-∞分别为(5794±3129)ng·h·ml-1和(5621±2752)ng·h·ml-1,AUC0-t分别为(5084±2191)ng·h·ml-1和(5187±2367)ng·h·ml-1,Cmax分别为(288±249)ng·ml-1和(287±160)ng·ml-1,tmax分别为(13.5±6.0)h和(16.7±7.2)h,t1/2分别为(13.1±8.5)h和(10.3±5.65)h,受试制剂相对生物利用度为(95.8±20.8)%。主要药动学参数AUC0-∞、AUC0-t、Cmax对数转换后进行方差分析,双单侧t检验和(1-2α)置信区间法进行生物等效性评价,其中tmax采用非参数检验法,结果均显示试验制剂与参比制剂间无统计学意义(P>0.05),试验制剂与参比制剂AUC0-t,Cmax比值的90%可信区间分别为85.4%～122.3%,80.9%～106.0%,均在AUC0-t,Cmax90%可信区间的80%～125%,70～143%范围之内。结果表明试验制剂与参比制剂具有人体生物等效性。第三部分、GL和GA在大鼠肝微粒体的体外代谢研究。本文建立了Sprague-Dawley大鼠肝微粒体体外代谢模型,苯巴比妥为诱导剂,按80mg·kg-1剂量腹腔注射3天,超速离心法制备肝微粒体,Bradford法测定蛋白浓度,分别以Phenacetin,Clivorine,Propofol作为探针底物进行肝微粒体的CYP450酶,酯酶和肝微粒体II相酶(UGT)的活性表征,证实了肝微粒体代谢模型和酶活性的有效性;本文首次建立了超高效液相色谱-质谱联用技术(UPLC/MS)同时测定GL,GA及其代谢产物,该法快速,灵敏、重现性好、专属性强,并通过质谱峰对代谢产物峰进行了定性和结构推证;在肝微粒体CYP450酶,酯酶和肝微粒体II相酶(UGT)孵育体系中以GL为底物进行温孵,没有检测到3-单葡萄糖醛酸甘草次酸(18β-glycyrrheticacid-3-O-β-D-monoglucuronide,GAMG)或其它任何代谢产物,即β-NADPH,UDPGA对GL代谢没有催化作用,该结果首次提供了CYP450酶,酯酶和肝微粒体II相酶(UGT)对GL无代谢的直接证据,国内外未见报道;在肝微粒体酯酶孵育体系中以GA为底物进行温孵,发现酯酶对GA无代谢;在肝微粒体CYP450酶孵育体系中以GA为底物进行温孵,检测到GA的五个羟化代谢产物(M1,M2,M3,M4,M5,m/z487,485)和一个未知代谢产物(M6,m/z501),NADPH对GA的代谢有催化作用,为NADPH依赖性的氧化代谢机理,该结果发现了在CYP450酶孵育体系中GA代谢生成M1,M2,M3,M4,M5,M6的代谢途径,比现有文献报道的两个羟化代谢产物有更深入的发现;在大鼠肝微粒体II相酶(UGT)孵育体系中以GA为底物进行温孵,发现GA与葡萄糖醛酸结合后生成GAMG和GL,即UGPDA对GA的代谢有催化作用,为UGPDA依赖性的代谢机理,首次发现GA在大鼠肝微粒体温孵体系中与葡萄糖醛酸结合,此现象国内外尚未见报道;同时,在研究中还观察到UGT能使GA经过CYP450代谢的代谢产物进行II相酶代谢,产生3个结合产物M1’,M2’,M3’(m/z 664)的新代谢途径;在肝微粒体孵育体系中加CYP450酶化学抑制剂以鉴定参与GA代谢的CYP450亚酶,选择在药物代谢中起主要作用的CYP1A2(Furafylline)、CYP3A4(Ketoconazole)、CYP2C19(Ticlopidine)、CYP2D6(Quinidin)、CYP2E(4-Methlpyrazole)五种CYP450亚酶和黄素单氧化酶(FMO,Methimazole)为研究对象,本研究发现,GA的代谢主要由CYP3A参与,对M1,M2,M3,M4和M6的生成均有明显影响,代谢产物的抑制与Ketoconazole呈剂量依赖性,比现有文献报道的CYP3A1/2只参与了GA的C-22α羟化代谢,有更深入的发现,同时,首次观察到FMO参与了GA的部分羟化代谢(M1,M2,M3),而CYP1A2、CYP2C19、CYP2D6、CYP2E1对GA的代谢没有影响;在大鼠肝微粒体CYP450孵育体系中以各拆方组作为底物进行孵育,考察对GA代谢的影响,发现GLY增加了GA的转化,同时,羟化产物M1,M2,M3的生成量减少,未知代谢产物M6的生成量增加,而GLY,MET与GA共同孵育时对GA的转化和羟化代谢物的生成影响不大;在大鼠肝微粒体II相酶(UGT)孵育体系中以各拆方组作为底物进行孵育,考察对GA代谢的影响,各拆方组均发现GA的剩余量以及GAMG和GL的生成量增加的现象,其中,GLY,MET与GA共同孵育时可使GA的剩余量增加约90%,即活性产物量的增加,推测对GA有一定的增效作用,GAMG和GL的生成量增加约30%,对代谢产物的排泄可能有促进作用。本文进行了复方甘草酸苷片工业化生产的处方工艺优化和质量标准修订,完成了试生产及其质量评价,完成了人体生物等效性评价,采用UPLC/MS法,在Sprague-Dawley大鼠肝微粒体孵育液中系统的研究了GL,GA的体外代谢过程,通过拆方试验研究了复方甘草酸苷片中GLY和MET对GA代谢的影响,为GL类制剂的临床安全用药评价、进一步进行体内代谢研究奠定了实验基础,并为其他复方制剂的代谢研究提供了思路和方法。更多还原

【Abstract】 Compound glycyrrhizin tablets is compose of glycyrrhizinate (GL), glycine (GLY) and DL-methionine (MET). It is used clinically for the treatment of allergic disorder, inflammation and several virus diseases including chronic hepatitis, anaphylactic reaction,systemic lupus erythematosus,hemorrhagic fever with renal syndrome and atypical pneumonia (SAS) and so on. Preclinical studies finished the study of prescription and preparation technology, quality criterion studies, stability and bioequivalence evaluation in rabbits. As well as we obtained the clinical ratify document from the State Food and Drug Administration (No: 2006 L01472). On the basis of preclinical studies, the aim of this work was, therefore, to development of compound glycyrrhizin tablets for industrial production, revise the quality criterion, evaluate the bioequivalence of self-made compound glycyrrhizin tablets and SNMC in healthy Chinese male volunteers and studies on in vitro metabolism in rat liver microsomes. This study was divided into three parts:Part one The purpose of the research is to obtain optimizing parameters of prescription and technology for industrial production and quality evaluation. A HPLC (PDA dectator) method was developed to simultaneously determinate content, related substances and dissolution of the three principal compositions in compound glycyrrhizin tablet at the first. After method investigation and destroyed test by acid, alkali, oxidation, high temperature showed that the method was sensitive, accurate, reproducible, simple and specificity. It can be used as a reliable, effective method for the quality control of compound glycyrrhizin tablet. The analysis method is more advanced and reliable than the analysis method of SNMC (Stronger Neo-Minophagen C tablet, SNMC), which is imported from Janpan as market product. Trial production of compound glycyrrhizin tablets were finished and optimizing parameters of prescription and technology, which is suitable for industrialized production, were be obtained. According to requirements of clinical ratify document and Chinese Pharmacopoeia (2005 version) to revise quality criterion, quality and stability of three batch sample of trial production were investigated and compared with SNMC. The results shows that the prescription and technology of industrial production are reasonable and stable.Part two According to the formulation of requirements of“Drug Registration and Management Method”and GCP (good clinical practice), to evaluate the bioequivalence of self-made compound glycyrrhizin tablets and SNMC in healthy Chinese male volunteers. The concentration of glycyrrhetic acid (GA) in plasma was determined by LC/MS/MS. SNMC as reference preparation, twenty two volunteers were randomly divided into two groups (test and reference), with double cross-over design and single-dose oral administration. The sample was prepared for injection using a liquid-liquid extraction method, with ginsenoside Rh2 as the internal standard (IS). The method was proved to be suitable for clinical investigation and bioequivalence evaluation of different formulations containing GL, which offered advantages of convenience, accurate, sensitivity and specificity. The main pharmacokinetic parameters were calculated and the bioequivalence was evaluated with DAS2.0 practical pharmacokinetic program. The main pharmacokinetic parameters of test and reference were as follows: AUC0-∞(5794±3129)ng·h·ml-1and (5621±2752)ng·h·ml-1, AUC0-t (5084±2191) ng·h·ml-1 and (5187±2367)ng·h·ml-1, Cmax (288±249)ng·ml-1and (287±160)ng·ml-1, tmax (13.5±6.0) h and (16.7±7.2) h, t1/2 (13.1±8.5)h and (10.3±5.65)h, respectively. The two-one side t-test analysis showed that the confidence intervals of Cmax, AUC0-t and AUC0-∞were (80.9%-106.0%), (85.4%-122.3%) and (86.8%-127.3%), respectively. The relative bioavailahilitv of the test preparation was (95.8±20.8)%. Results of variance analysis and two one-side t-test showed that there were no statistical significant difference between the two preparations in the AUC and Cmax. The two-one side t-test analysis showed that the confidence intervals of Cmax, AUC0-t and AUC0-∞were (80.9%～106.0%), (85.4%～122.3%) and (86.8%～127.3%), respectively. In the case of Tmax was carried out by the Wiloxon-ranked sign test for the matched pairs, and the statistical result showed that there was no significant difference between the two preparations. The test and reference preparations were bioequivalentPart three Studies on in vitro mtabolism of GL and GA in rat liver microsomes. The Sprague-Dawley rats were induced for 3d with Phenobarbital (ip, 80mg/kg) and sacrificed after fast 12h. The liver microsomes were prepared by applying ultracentrifuga- tion approach. The protein concentration was determined by Bradford method. The in vitro metabolism study model of liver microsomes was established for the investigation of the enzyme activites. Phenacetin, Clivorine and Propofol as probe substrate of Cytochrome p450 (CYP450) isozymes, esterase and phase II transferase (UGT), respectively. The results verified the in vitro metabolism study model of Sprague-Dawley rat liver microsomes and enzyme activites are available. A UPLC/MS method was developed and validated for the simultaneous determination of GL, GA and its metabolites for the first time. The method is convenience, accurate, sensitivity and specificity. Metabolites were identified by UPLC/MS spectrum in rat liver microsomes incubates. GL, as probe substrate is in incubation with rat liver microsomes. The results showed that no metabolites of GL, such as 18β-glycyrrhetic acid-3-O-β-D- monoglucuronide (GAMG, 3-MGA), were detected in rat liver microsomes incubations. CYP450, esterase and phase II transferase (UGT ) had no effect on the metabolism of GL. The results provided direct evidence for the metabolic pathway of GL in liver microsomes at first time. GA as probe substrate is in incubation with rat liver microsomes. It was found that no metabolites of GA were detected in rat liver microsomes esterase incubations. NADPH as cofactor had catalytic effect on the metabolism of GA. Six metabolites (M1, M2, M3, M4, M5 were hyhroxy-GA, m/z 487; M6 was unknown metabolite, m/z 501) of GA were detected in rat liver microsomes CYP450 incubations inβ-NADPH presence at first time. It was shown that GA metabolism and M1, M2, M3 formation exhibited NADPH-dependent oxidation mechanism. GAMG and GL were detected in rat liver microsomes phase II transferase incubations in UGT presence at first time. It was shown that GAMG formation exhibited UGT-oxidation mechanism.At the same time, Three metabolites (M1’, M2’, M3’) of hydroxy-GA were detected in rat liver microsomes phase II transferase incubations in UGT presence at first time. Selective chemical inhibitors to various CYP450 isoforms (CYP1A2?CYP3A?CYP2C19?CYP2D6?CYP2E1 and FMO ) were employed to conduct inhibition experiments. Ketoconazole reduced hydroxylation of GA in a dose-dependent manner. The kinetic behaviors of M1, M2, M3 were described well by a single-enzyme Michaelis-Menten equation, respectively. Methimazole, as FMO inhibitors, displayed partly inhibition for hydroxylation of GA.Inhibition experiments showed that Furafylline, Ticlopidine, Quinidine and 4-Methlpyrazole Methimazole, which were inhibitors as CYP1A2?CYP2C19?CYP2D6 and CYP2E1, respectively, did not display significant inhibition. The present study indicates that hydroxylation of GA in rat liver microsomes is mediated by CYP3A. To observe the effects of GLY and MET on the metabolism of GA. GA combine with GLY or MET as substrate is in incubation with rat liver microsomes, respectively. GLY can enhance metabolism of GA.The formation of M1, M2, M3 were decrease, while The formation of unknomn metabolite M6 were increase. GLY and MET on the metabolism of GA in CYP450 incubation had no significant effects. At same time, GLY and MET can both increase active component GA 90% and increase formation of GAMG and GL 35% in II transferase incubation. The present study indicates that GLY and MET probably enhance the effects of GA and accelerate the excrete of metabolites of GA.In this thesis, the prescription and technology of compound glycyrrhizin tablet for industrial production was optimized. We finished three batch trial production and revised quality criterion to investigate production quality. The study of bioequivalence evaluation in heathy volunteers was finished and the result showed the test and reference preparations were bioequivalence. In vitro mtabolism of GL and GA in Sprague-Dawley rat liver microsomes was systemic studied. To observe the effects of GLY and MET on the metabolism pathway of GA. The metabolism studies of the GL and GA in vitro will laid a experiments foundation for safety evaluation and in vivo metabolism studies of GA and GL in further. Meanwhile, it will provide some ideas and methods to investigate metabolism of other compound preparation.更多还原